CENTER FOR DRUG EVALUATION AND RESEARCH APPLICATION NUMBER: 205718Orig1s000 CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS REVIEW(S)   NDA 205-718 Office of Clinical Pharmacology Review_PART2 INDIVIDUAL STUDY RESULTS and REVIEWER’s COMMENTS   Study NETU-09-21: Oral ADME Study   Title of the Study An Open Label, Single Dose Study in Healthy Male Subjects Designed to Assess the Mass Balance Recovery, Pharmacokinetics, Metabolite Profile and Metabolite Identification of 300 mg [14C]-Netupitant   Methodology This was a single dose study in 6 healthy male subjects. For all subjects, the mean total radioactivity recovered by 336 h was approximately 70%; therefore, subjects were required to collect feces samples for a 24 h period at home (456 to 480 h, Days 20 to 21), and both feces and urine samples for an additional 24 h period in the clinic (672 to 696 h, Days 29 to 30) to better characterize the terminal excretion of the total radioactivity. Blood, urine and feces samples were collected throughout the study for the analysis of total radioactivity, netupitant and metabolites M1, M2 and M3, characterization of metabolites and [14C]-netupitant binding to plasma proteins.   PK Results After oral administration of [14C]-netupitant to healthy male subjects, netupitant was rapidly absorbed. Individual peak plasma netupitant concentrations, ranging from 99.2 to 517 ng/mL, were observed at 2 to 5.5 h post-dose (Tmax). The actual dose of netupitant received ranged from approximately 187 to 264 mg.   The total drug-related material in plasma was higher than that of whole blood, as few subjects had detectable radioactivity levels measurable in whole blood. Mean plasma netupitant/plasma radioactivity ratios ranged from 0.13 to 0.49 over 96 h post-dose. The ratios were time dependent with values decreasing gradually beyond 24 h post-dose, indicating that the drug is being rapidly metabolized.   Exposure (AUC0-t) and Cmax geometric mean values for netupitant were approximately 34% and 41% of the exposure and Cmax geometric mean values for total radioactivity; this difference is attributed to metabolite species. However, it should be noted that a full comparison of the netupitant and plasma radioactivity data cannot be made because of the difference between the lower limit of detection of total radioactivity and the limit of quantification of netupitant; this difference resulted in netupitant concentrations that were measurable at later sampling time points compared to total radioactivity, potentially skewing these comparisons. Further analysis of the plasma samples for the metabolites M1, M2, and M3 indicated that, on average, exposure to these metabolites was equivalent to 29%, 14%, and 33%, respectively, of the systemic exposure to netupitant; thus, these results confirm that M1, M2 and M3 are all major metabolites of 1    Reference ID: 3537650 netupitant and account for >10% of parent drug-related exposure. Exposure to the additional metabolite M4, based on Cmax, accounts for approximately 7% of parent drug exposure.   Approximately half the administered dose of radioactivity was recovered within 120 h of dosing.   Based on the total radioactivity recovered in all samples, including the additional collection periods, total radioactivity from the urine accounted for 3.95% (range 2.2% to 4.6%) of the dose and total radioactivity from the feces accounted for 70.7% (range 62.1% to 75.2%) of the dose at 696 h post-dose. These data indicate that the hepatic/biliary route, rather than renal clearance, is the major elimination route for drug- related entities. Reviewer’s comment: This recovery over 696 h post-dose is underestimated due to missing samples.   Subsequently including the extrapolated values for the periods 336 to 456 h and 480 to 672 h, the total drug-related material to have been excreted by 696 h post-dose via the feces and the urine was estimated to be 86.49% and 4.75%, respectively. Based on this the time estimated to reach 80%, 90% and 95% of excretion was 465 h, 665 h, and 866h, respectively. Figure 1. Recover pattern of radioactivity after administration of [14C] netupitant*  Submitted in an amendment dated 5/22/14  Values during the time from 336 to 456 h and from480-672 h were obtained by extrapolation taking the mean value of recoveries estimated in the collection intervals just prior to and just after the missing collection period as below. 2    Reference ID: 3537650 Figure 2: Radioactivity excretion rates as a function of the mid-time of the sample collection intervals (A) urine data, (B) fecal data   Metabolite Identification Results   Netupitant was shown to undergo extensive metabolism, forming both phase I and phase II metabolites. Phase I metabolites observed included those formed through N- demethylation (mono and bis), mono and di-hydroxylation, N-oxidation, desaturation, N- formylation, oxidation and reduction to a keto group, and oxidation to an acid (including oxidation of the toluene methyl group to an acid). Intermediate metabolites in the 1- methylpiperazine 3    Reference ID: 3537650 degradation pathway to the further oxidised 6-amino-pyridinyl derivatives were also observed. Phase II metabolites included those formed by glucuronidation and conjugation to a hexose (C6 sugar) group. A glucuronic acid conjugate of the acid ½ molecule of netupitant was also observed in urine.   Safety One subject reported a total of 5 AEs during the study, of which 4 events (abdominal pain, diarrhea, dyspepsia and nausea) were considered IMP-related. All events were mild in severity and had resolved by the end of the study. There were no clinically significant findings in clinical laboratory assessments, vital signs parameters, ECG measurements or physical examinations.     Reviewer’s comments: The actual administered dose was 200 mg although the dose of 300 was to be administered. This study shows that upon oral absorption netupitant is distributed extensively to the tissues, slowly released and metabolized over a long-period of time. Study NP16603: Single Ascending Doses of Netupitant   Title of the Study   Double-blind, placebo controlled single ascending oral dose study of RO0673189 (netupitant) in healthy volunteers   Methodology This was a single center, randomized double-blind, placebo-controlled single ascending dose study conducted in healthy males. Five dose levels of netupitant were investigated: 10, 30, 100, 300 and 450 mg. For each dose group, six subjects were randomly assigned to netupitant (4 subjects) or placebo (2 subjects).   Blood samples for pharmacokinetic analysis were collected pre-dose and at 15 and 45 min and 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 h post-dose. Blood and urine samples for laboratory safety tests were collected at screening, pre-dose and at 24, 72 and 168 h post-dose.   PK Results Following a lag time of up to 3 h, netupitant was absorbed in a first order fashion, with maximum plasma concentrations being reached at approximately 5 h post-dose. The terminal t1/2 was estimated to be 30 to 60 h. The mean plasma concentration-time profiles for each dose group are shown below (Figure 1). 4    Reference ID: 3537650   Figure 1 Mean Netupitant Plasma Concentration versus Time Curves       For doses up to 300 mg, there was a statistically significant over-proportional increase with dose in Cmax, AUClast and AUC0-’ for netupitant. Dose-proportionality was observed between the 300 mg and 450 mg doses, with ratios being close to one. The 3 metabolites detected in animal studies, metabolites RO0681133 (M1), RO0713001 (M2) and RO0731519 (M3) were measurable with maximum metabolite plasma concentrations reaching one tenth to one fifth of parent levels, and AUC values of between one twentieth and one third of parent. Reviewer’s comment: In this study additional blood samples were taken pre-dose and 24 h post- dose for exploratory EM analysis of lamellar inclusion bodies, as a screen for phospholipidosis. This evaluation is considered only exploratory.   5    Reference ID: 3537650 Study NP16601: Multiple Ascending Doses of Netupitant   Title of the Study   A Double-Blind, Randomized, Placebo-Controlled Evaluation of the Clinical Pharmacology of RO0673189 (Netupitant) Following Multiple Oral Dosing to Healthy Young And Elderly Volunteers   Methodology Subjects fasted overnight for approximately 10 hours prior to each dose. They then received a standard breakfast which was to be consumed within 30 minutes and the study medication was administered within 5 minutes of completing the breakfast.   PK blood samples were taken at 15, 30 and 45 minutes and 1, 1.5, 2, 3, 5, 8, 12 and 24 h after dosing on Day 1 and 7. Additional samples were taken after the final dose at 36, 48, 60, 72, 96, 120, 144 and 168 h post-last dose.   Originally, the effect of age on the pharmacokinetics of netupitant was also planned to be investigated in this trial but the elderly portion of the study was discontinued and only the ascending dose portion of the study results in young healthy volunteers are presented.   PK Results The PK data showed an increase in netupitant exposure of approximately 3-fold after 7 days of dosing consistently with the long t1/2 of the compound. Mean maximum plasma concentrations and AUC(0-23.5) values recorded on Days 1 and 7 of dosing with 100, 300 or 450 mg of netupitant are shown in Table 1. Exposure to netupitant showed a slightly greater than proportional increase with dose. Low levels of the major metabolite RO068133 (M1) were detected, with levels not exceeding 30% of the parent.   Table 1 Mean Exposures on Day 1 and Day 7 following Daily Dosing with Netupitant for Seven Days   Dose Day 1 (n=8) Day 7 (n=8) Cmax AUC(0-23.5) Cmax AUC(0-23.5)         (ng/mL) (h.ng/mL) (ng/mL) (h.ng/mL) 100mg 111 (23.1) 1360 (21.6) 269 (19.4) 4160 (24.0)           300mg 599 (38.0) 6400 (26.5) 1060 (19.0) 17100 (16.6)           720 (35.4) 9670 (34.9) 1790 (43.1) 28800(45.1) 450mg Values are arithmetic means and coefficient of variation (CV%)     Safety Daily doses of up to 450 mg of RO0673189 for 7 days were well tolerated in this study. The most frequent adverse event was headache. The majority of adverse events were of mild intensity and most were considered unrelated or remotely related to trial treatment. There were 6    Reference ID: 3537650 no deaths or serious adverse events during the study, and no subject was withdrawn as a result of an adverse event. Reviewer’s comments: There was a greater than dose-proportional increase in the systemic exposure as the dose increases from 100 mg to 300 mg after single and multiple doses while the dose-proportional increase in the systemic exposure was observed as the dose increases from 300 mg to 450 mg. These results suggest that doses higher than 300 mg the mechanisms that may limit the oral bioavailability are saturated. This study provides safety information, although limited at the high systemic exposure. The mean Cmax for netupitant after multiple doses of 450 mg netupitant was about 3-fold higher than the mean Cmax after single dose administration of 300 mg netupitant. The mean AUC for netupitant after multiple doses of 450 mg netupitant was > 5-fold higher than the mean AUC observed in other PK studies.   7    Reference ID: 3537650 PK in Special Populations   Study NP16600: Effects of Food and Age on Netupitant   Title of the Study   Evaluation of the Effects of Food and Age on the Pharmacokinetics of RO0673189 (Netupitant) in Healthy Volunteers   Methodology   The food effect portion of the study was an open-label, randomized cross-over study, where 12 healthy volunteers (aged 18 to 45 years) received a single oral dose of 300 mg of netupitant on 2 occasions, once under fasted conditions and once under fed conditions, with a 2-week (minimum) wash-out period between treatments.   The age effect portion of the study was a double-blind, randomized study, where 6 healthy elderly volunteers (aged 65 to 85 years) received a single oral dose of either 100 mg netupitant (4 subjects) or placebo (2 subjects).   Subjects were fasted overnight (except for water) for at least 10 hours. Subjects in the fed treatment phase of the food effect part of the study and all subjects in the age effect study received a standardized breakfast prior to dosing. Subjects in the fasting treatment phase of the food effect study were administered the study medication after the overnight fast.   Blood samples for PK analysis of netupitant and its 3 main metabolites were collected pre-dose, and at 15 and 45 min and 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 h postdose.   PK Results Netupitant was absorbed in a first-order fashion following oral administration. The mean plasma concentration-time profiles for fed and fasted administration are shown below (Figure 2). The peak plasma concentration and AUC, increased by between 69% (Cmax) and 47% (AUClast) on average on administration with breakfast compared to fasted administration. The bioavailability estimates and 95% confidence intervals (CIs) for treatment under fed conditions relative to fasted were 153% [122, 192] for AUC0-’, 147% [117, 185] for AUClast and 169% [102, 279] for Cmax. The effect of food on the bioavailability of netupitant was highly variable between subjects, ranging from no effect to a greater than 3-fold increase. Exposure to the metabolites M1, M2 and M3 was also increased by between 5% (Cmax, M2) and 57% (AUC0-’, M1), on average, on administration with food.   8    Reference ID: 3537650 Figure 2 Mean Netupitant Plasma Concentration Profiles Following Fasted and Fed Conditions in Healthy Volunteers     A single dose of 100 mg administered after food to elderly subjects (aged 66 to 70 years) resulted in similar exposure to that observed following administration of the same dose to younger volunteers (aged 23 to 44 years) in the single ascending dose study (Study NP16603). Given the wide inter-subject variability seen with this compound, there was no significant difference between elderly subjects (in this study) and younger subjects in mean (min-max) Cmax [elderly subjects: 136 (92 – 185) ng/mL; younger subjects (Study NP16603): 185 (144 – 224) ng/mL] or in AUC0-’ [4009 (3167 – 5046) h.ng/mL; younger subjects 4795 (3413 – 6284) h.ng/mL] for netupitant. These results suggest that age is unlikely to have any significant effect of the PK of netupitant. However, the number of subjects investigated was small (N=4).   Reviewer’s comment: In a definitive study with AKYNZEO, no significant food effect was observed (NETU-10-20). The effect of age on PK of netupitant is considered could not be adequately evaluated in this study due to the small number of study and the dose difference i.e.100 mg versus the proposed dose of 300 mg.   9    Reference ID: 3537650 Study NETU-10-12: Effect of Food and Age on Netupitant/Palonosetron FDC Formulation   Title of Study An Open-Label Trial to Investigate the Effect of Food and Age on the Pharmacokinetics of a Single Dose Administration of Oral Netupitant and Palonosetron (300 mg/0.5 mg) in Healthy Subjects and In Healthy Elderly Subjects     Methodology For the investigation of the effect of food, a crossover was performed in 24 healthy male and female subjects, where drug administration in the fed state was compared to drug administration in the fasted state.   The effect of age was evaluated in a parallel group of 12 healthy elderly male and female subjects under fasted state and was compared to PK in all younger subjects from the crossover portion under fasted state.   In 1 period, the subjects received the investigational product in fasted state, and in the other period, the same subjects received the investigational product in the fed state. The parallel group of elderly subjects underwent 1 treatment period of 12 days with a single administration of the investigational product on Day 1. The subjects received the investigational product in the fasted state only.   In each treatment period, blood samples for PK analysis of netupitant and its metabolites M1, M2, and M3 were collected until 240 h after administration and blood samples for palonosetron were collected until 192 h after administration.   PK Results   Food effect. Under fed condition, the Cmax and AUC was 15-17% higher than under fasted condition. (Table 1) Table 1 Mean ± SD Netupitant Pharmacokinetic Parameters after Oral Dose Administration of Netupitant/Palonosetron FDC (300 mg/0.5 mg) in Fed (T) and Fasted Conditions (R) and Results of Analysis of Variance               Parameter Cmax [μg/L] AUC0- [h·μg/L] T 649.8±141.6 22391±8650 R PE%* 596.4±233.0 117.74 20039±8396 115.96   90%CI   100.65 - 137.74   104.54 - 128.62   AUC0-tz [h·μg/L] 19406±4919 17150±6122 117.88 Values are arithmetic means ±SD; *Point estimate (PE): ratio of geometric means (T/R) CI: confidence interval, SD: standard deviation T: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fed state (Test) 10    Reference ID: 3537650 106.66 - 130.27 R: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state (Reference)   For palonosetron, no relevant differences were observed between the fasted and fed condition (Table 2). Table 2 Mean ± SD Palonosetron Pharmacokinetic Parameters after Oral Dose Administration of Netupitant/Palonosetron FDC (300 mg/0.5 mg) in Fed (T) and Fasted Conditions (R) and Results of Analysis of Variance               Parameter Cmax [ng/L] AUC0-’ [h·ng/L] T 767.9±159.2 33199±6945 R PE%* 785.6±223.5 99.00 33645±8974 99.99   90%CI   93.05 - 105.33   95.41 - 104.79   AUC0-tz [h·ng/L] 29760±6539 30371±8416 99.29 Values are arithmetic means ±SD; * Point estimate (PE): ratio of geometric means (T/R) CI: confidence interval, SD: standard deviation T: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fed state (Test) R: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state (Reference)   Age effect. In healthy elderly subjects, Cmax and AUC0young adults (Table 3). ∞ 94.51 - 104.30 was 36% and 25% higher, respectively than in   Table 3 Mean ± SD Netupitant Pharmacokinetic Parameters after Oral Dose Administration of Netupitant/Palonosetron FDC (300 mg/0.5 mg) in Fasted Conditions in Elderly Subjects (R+) and in Adult Subjects (R) and Results of Analysis of Variance               AUC0- [h·μg/L] 20039±8396 Parameter Cmax [μg/L] R 596.4±233.0 R+ PE%* 880.8±479.2 136.36 24739±9390 124.91   90%CI   95.87 - 193.96   95.29 - 163.75 17150±6122 19604±6747 113.42 87.66 - 146.75 AUC0-tz [h·μg/L] Values are arithmetic means ±SD; *Point estimate (PE): ratio of geometric means (R+/R) CI: confidence interval, SD: standard deviation R+: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state to elderly subjects R: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state to younger adults (Reference)   Comparison of the primary pharmacokinetic parameters Cmax and AUC0-’ of palonosetron showed a 10% higher mean Cmax in adult subjects, (90% CI from 95.96% to 127.11%) and a 37% higher mean AUC (90% CI from 117.44% to 159.56%) in elderly subjects compared to adult subjects. (Table 4).   11    Reference ID: 3537650 Table 4 Mean ± SD Palonosetron Pharmacokinetic Parameters after Oral Dose Administration of Netupitant/Palonosetron FDC (300 mg/0.5 mg) in Fasted Conditions in Elderly Subjects (R+) and in Adult Subjects (R) and Results of Analysis of Variance         Parameter Cmax [ng/L] AUC0-’ [h·ng/L]       R 785.6±223.5 33645±8974 R+ 851.2±146.3 45047±7903 PE%* 110.44 136.89       90%CI 95.96 - 127.11 117.44 - 159.56   AUC0-tz [h·ng/L] 30371±8416 39577±6617 133.81 114.28 - 156.68 Values are arithmetic means ±SD; *Point estimate (PE): ratio of geometric means (R+/R) CI: confidence interval, SD: standard deviation R+: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state to elderly subjects R: one capsule of 300 mg netupitant and 0.5 mg palonosetron in fasted state to younger adults (Reference)   Conclusions   Food-effect. A high fat, high-caloric breakfast led to a delay in absorption of netupitant with an increase in exposure of about 16% for AUC0-’ and about 18% for AUC0-tz and Cmax; however, the increase in netupitant exposure to this degree is considered clinically insignificant.   For palonosetron, the exposure was not affected by food. Based on the slight increase in netupitant exposure following food administration and the lack of effect of food on palonosetron concentrations, the FDC can be administered without regard to food.   Age effect. The effect of age on the pharmacokinetic parameters of netupitant and palonosetron was compared between 22 healthy adult subjects (22 and 45 years old) and 2 healthy elderly subjects (66 and 79 years old).   The exposure to netupitant and palonosetron was higher in elderly subjects with an increase of about 25% and 37% for AUC0-∞, about 13% and 34% for AUC0-tz, and about 36% and 10% for Cmax. An increase in exposure to both netupitant and palonosetron is not expected to be clinically relevant; therefore, no dosage adjustment is indicated for elderly subjects.     Reviewer’s comments: The study results and conclusions are acceptable. In the tQT study, a single dose combination of 600 mg netupitant and 0.5 mg palonosetron was well tolerated. 12    Reference ID: 3537650     Study NETU-10-10: Patients with Hepatic Impairment   Title of the Study   Pharmacokinetics of a Single Dose of Netupitant and Palonosetron Fixed-Dose Combination Capsules in Patients with Different Stages of Hepatic Impairment Based on Liver Cirrhosis Classified By Child-Pugh Score in Comparison to Healthy Volunteers   Methodology   This study was conducted in a single center according to an open label, 1-period, nonrandomized study design. A maximum of 48 subjects were planned to be enrolled: 24 subjects (8 per group) with hepatic impairment classified by Child-Pugh scoring system as mild (Child-Pugh 5-6), moderate (Child Pugh 7-9) and severe (Child Pugh 10-15) and 24 healthy subjects (8 per group) matched to the subjects with hepatic impairment by age, weight and gender.   A single dose of netupitant/palonosetron FDC (300 mg/0.5 mg) was administered on Day 1 after an overnight fast of at least 10 hours. Blood samples for PK of netupitant and its metabolites M1, M2 and M3 were collected from pre-dose through 240 hours post-dose and blood samples for palonosetron PK were collected from pre-dose through 192 hours post-dose.   Subjects were discharged on Day 5 after blood sampling and returned on Days 7, 9 and 11 for PK sampling and safety measurements. Final check was performed on Day 11.   PK Results   In subjects with mild hepatic impairment, exposure to netupitant was slightly higher compared to matching healthy subjects with an increase of 11% for Cmax, 28% for AUC0-tz, and 19% for AUC0∞. The mean coefficient of variation of Cmax was 65.7% in the group of subjects with mild hepatic impairment and 22.7% in the group of matching healthy subjects. The observed increase in exposure of netupitant was not statistically significant.   The formation of metabolite M1 was slightly delayed in mild hepatic impaired subjects compared to the matched healthy cohort (median Tmax of 10 h versus 8 h). Mild hepatic impairment did not, however, have an impact on the time of appearance of the other netupitant metabolites M2 and M3 in plasma. In mild hepatic impaired subjects, exposure to metabolite M1 was reduced, and exposure to metabolite M2 was increased. For metabolite M3, a reduced maximum concentration and an increased total exposure was observed.   In subjects with mild hepatic impairment, maximum concentrations of palonosetron were slightly higher compared to matching healthy subjects with an increase of 14% for Cmax. The increase was not statistically significant. Total exposure was significantly higher in mild hepatic impaired subjects compared to matching healthy subjects with an increase of 35% for AUC0-tz and 33% for AUC0-∞， the respective 90% CIs were 109% to 169% for AUC0-tz, and 107% to 167% for AUC0∞. (Table 1 and Table 2) 13    Reference ID: 3537650     Table 1 Overview of the Pharmacokinetic Characteristics of Netupitant and Results of Statistical Analysis - Mild Hepatic Impairment   Point Estimate Mild / Normal Ratio [%] Lower Limit of 90% CI2[%]   111.12   69.57     12486±5294 (42.4) 128.43   86.44   Mild Hepatic Impairment1 N=8 Normal Hepatic Function1 N=8       Cmax .0±304.9 (65.7)             AUC0-tz 687±8683 (52.0)     344.9±78.3 (22.7)   177.50     190.82     119.14 68±11824 58±21398 (54.8) (101.6) 1 Values are arithmetic mean±standard deviation (coefficient of variation %) 2 Pre-specified no-effect limit for the confidence interval (CI): 80% to 125% AUC0- Upper Limit of 90% CI2 %] ’   70.87   200.29     Table 2 Overview of the Pharmacokinetic Characteristics of Palonosetron and Results of Statistical Analysis- Mild Hepatic Impairment   Mild Hepatic Impairment1 N=8 Normal Hepatic Function1 N=8       AUC0-tz     65±11669 (32.4) 26787±9273 (34.6)           Cmax   AUC0’ 5.1±270.4 (34.0)   673.5±137.2 (20.4)   63±12739 (31.3) 30627±9923 (32.4) Point Estimate   Mild / Normal Lower Limit of 90% CI [%] Ratio [%]   Upper Limit of 90% CI %]     113.86   92.30   135.45   108.66   133.48   106.82   140.46     168.84     166.79 1 Values are arithmetic mean±standard deviation (coefficient of variation %)   In subjects with moderate hepatic impairment, exposure to netupitant was significantly higher compared to matching healthy subjects with an increase of 70% for Cmax, 88% for AUC0-tz, and 143% for AUC0-’, the respective 90% CIs were 106% to 271% for Cmax, 127% to 280% for AUC0-tz, and 145% to 409% for AUC0-’. The netupitant exposure parameters exhibited high variability in subjects with moderate hepatic impairment and moderate variability in matching healthy subjects (Table 3).   In moderate hepatic impaired subjects, the formation of metabolite M1 was delayed (median Tmax of 10 h vs. 7 h), and the formation of metabolites M2 and M3 was accelerated (median Tmax: 3 h vs. 5 h and 6 h vs. 12 h, respectively) compared to matching healthy subjects. In subjects with moderate hepatic impairment, maximum concentration and exposure (Cmax, AUC0tz) to metabolite M1 was reduced, whereas for metabolite M2 and M3, Cmax and AUC0-tz were increased. 14    Reference ID: 3537650     In subjects with moderate hepatic impairment, maximum concentration of palonosetron was similar to that of matching healthy subjects. Total exposure was significantly higher in moderate hepatic impaired subjects compared to matching healthy subjects with an increase of 60% for AUC0-tz and 62% for AUC0- ， the respective 90% CIs were 129% to 200% for AUC0-tz, and 129% to 202% for AUC0-∞. (Table 4)   Table 3 Overview of the Pharmacokinetic Characteristics of Netupitant and Results of Statistical Analysis- Moderate Hepatic Impairment     Moderate Hepatic Impairment1 N=8             Cmax AUC0-tz   AUC0- .9±304.3 (68.9) Normal Hepatic 1 Function N=8 239.0±100.0 (41.8) 169.93   106.38 188.32 126.75 10312±2881 (27.9) 243.15 144.64 88±9794 (53.0) 83±2896 (31.5)     81±15495 (55.2) Point Estimate Moderate / Lower Limit of 90% CI2[%] Normal Ratio [%]   Upper Limit of 90% CI2 %]   271.43   279.81   408.77 ’ 1 Values are arithmetic mean±standard deviation (coefficient of variation %) 2 Pre-specified no-effect limit for the confidence interval (CI): 80% to 125%   Table 4 Overview of the Pharmacokinetic Characteristics of Palonosetron and Results of Statistical Analysis- Moderate Hepatic Impairment       Cmax   AUC0-tz   AUC0- Moderate Hepatic Impairment1 N=8       7.6±124.0 (17.8)   81±12066 (27.2)   48±15030 (29.0) Normal Hepatic 1 Function N=8 Point Estimate Moderate / Normal Ratio [%] 712.6±163.2 (22.9) 98.59   35±12401 (43.3)   53±13230 (40.4)   Lower Limit of 90% CI[%] 79.91 160.18 128.50 161.80 129.48 Upper Limit of 90% CI %]   121.62   199.67   202.18 ’ 1 Values are arithmetic mean±standard deviation (coefficient of variation %)   In subjects with severe hepatic impairment, maximum concentration and exposure to netupitant were higher compared to matching healthy subjects with an increase of 81% for Cmax, 101% for AUC0-tz, and 144% for AUC0-’.   The observed increase in exposure of netupitant was not statistically significant. It should be noted, however, that the small sample size and variability in the data may preclude a definitive 15    Reference ID: 3537650   conclusion on these data (Table 5).   In severely hepatic impaired subjects, the formation of metabolite M2 was slightly accelerated compared to matching healthy subjects (2 and 3 h vs. 4.5 h). An impact of severe hepatic impairment on the formation of metabolites M1 and M3 could not be observed due to the high variability of data and the small sample size. No clear trend was observed for the effect of hepatic impairment on the exposure of metabolite M1, M2, and M3 due to the high variability of data and the small sample size.   In subjects with severe hepatic impairment, maximum concentration and exposure to palonosetron were lower compared to matching healthy subjects with a decrease of 31% for Cmax, 9% for AUC0-tz, and 18% for AUC0-’ .The decrease in exposure of palonosetron was not statistically significant. It should be noted, however, that the small sample size and variability in the data may preclude a definitive conclusion on these data (Table 6).   Table 5 Overview of the Pharmacokinetic Characteristics of Netupitant and Results of Statistical Analysis- Severe Hepatic Impairment     Severe Hepatic Impairment1 N=8 Normal Hepatic Function1 N=8                   Cmax AUC0-tz 469.6-1336 21179-44845 266.1-720.4 10953-21506 Point Estimate Severe / Normal Ratio [%] Lower Limit of 2 90% CI [%] 180.90 70.90 200.80 90.95   26857-70952 13176-24180 244.57 86.54 AUC01 Values are arithmetic mean±standard deviation (coefficient of variation %) 2 Pre-specified no-effect limit for the confidence interval (CI): 80% to 125%   Upper Limit of 90% CI2 %]   461.55   443.28   691.18 Table 6 Overview of the Pharmacokinetic Characteristics of Palonosetron and Results of Statistical Analysis- Severe Hepatic Impairment     Severe Hepatic Impairment1 N=8 Normal Hepatic Function1 N=8                   Cmax AUC0-tz 399.1-1019 28142-65203 829.6-1030 29423-75525 Point Estimate Severe / Normal Ratio [%] Lower Limit of 2 90% CI [%] 68.97 45.32 90.87 58.48   32029-69539 34116-96841 82.11 52.58 AUC01 Values are arithmetic mean±standard deviation (coefficient of variation %) 2 Pre-specified no-effect limit for the confidence interval (CI): 80% to 125% 16    Reference ID: 3537650 Upper Limit of 90% CI2 %]   104.97   141.19   128.20     Reviewer’s comments The sponsor calculated the ratio of PK parameters between subjects with hepatic impairment and matching control group i.e. one group for mild hepatic impairment and another group for moderate hepatic impairment. Upon review of the data, the demographic information such as age and gender was similar across control groups to different degree of hepatic impairment while the PK parameters for netupitant showed differences among controls due to the variability among control groups. This variability between control groups confounded the evaluation of the effect of hepatic impairment on the PK of netupitant. Therefore PK parameters from patients with hepatic impairment were compared to the pooled control group. One healthy subject had a substantially high AUC for netupitant, that was similar to the highest AUC observed in a patient with severe hepatic impairment. The AUC was not considered reliable due to ~75% extrapolation for AUCi and excluded from the control group. The mean AUC of netupitant was 58% and 101% higher in patients with mild and moderate hepatic impairment, respectively than in healthy subjects. The Cmax of netupitant was about 30% higher in patients with mild and moderate hepatic impairment. Only two patients with severe hepatic impairment provided PK data. In one patient with severe hepatic impairment, Cmax and AUC of netupitant were about 2- and 6-fold higher, respectively while Cmax and AUC of palonosetron were about 2-fold higher than the mean for control group. The 2-fold higher AUC for netupitant was within the 2-fold higher AUC at 600 mg netupitant compared to that of 300 mg netupitant in the tQT study. Table 7 Geometric mean and ratio of PK parameters for netupitant in subjects with hepatic impairment and healthy subjects PK blood samples for netupitant were collected up to 240 hours post-dose Table 8 Geometric mean of PK parameters for palonosetron in subjects with hepatic impairment and healthy subjects PK blood samples for palonosetron were collected up to 192 hours post-dose.   17    Reference ID: 3537650   Figure  1  Individual  AUCinf  for  (A)  netupitant  and  (B)  palonosetron  in  patients  with  hepatic  impairment and in healthy subjects  (A) netupitant    (B) palonosetron        18    Reference ID: 3537650   Drug-Drug Interaction Studies   Study NP16599: Netupitant with Midazolam and Erythromycin   Title of the Study   Impact of RO0673189 (Netupitant) on the Pharmacokinetics of Midazolam and Erythromycin, Two CYP3A4 Substrates, in Healthy Volunteers     Objectives This study was designed to assess the impact of netupitant on the PK of midazolam and erythromycin, and the impact of these agents on netupitant.   Methodology   In every period, subjects fasted overnight for approximately 10 hours and received a standard breakfast prior to drug administration (days 1, 22 and 43). Study medication was administered with approximately 200 mL of water within 5 minutes of completing breakfast.   For subjects taking erythromycin or midazolam alone (period I for groups A and C, and period II for groups B and D), blood samples for pharmacokinetic analysis were taken pre-dose and at 15, 30 and 45 min and 1,1.5, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h post-dose.   For subjects taking netupitant alone and netupitant in combination with erythromycin or midazolam (period I for groups B and D, period II for groups A and C and period III for all subjects), blood samples for pharmacokinetic analysis were taken as above with additional samples at 72, 96, 120, 144 and 168 h post-dose.   PK Results   The systemic exposure to the CYP3A4 substrate midazolam was significantly increased then taken in combination with netupitant compared to administration of midazolam alone, with Cmax increasing by approximately 40% and AUC0-’ by approximately 144% (Table 1).   Similar results were seen for erythromycin, with exposure as judged by Cmax and AUC0-’ approximately 30% higher following administration with netupitant compared to erythromycin taken alone. The mean (± SD) exposure parameters Cmax and AUC0-’ for each of the treatments are shown in Table 2.   19    Reference ID: 3537650   Table 1 Mean (± SD) Pharmacokinetic Parameters Showing Exposure to Netupitant and Erythromycin Taken Alone and in Combination     Reviewer’s comments: These results indicate that netupitant is a moderate inhibitor of CYP3A4 in vivo.   20    Reference ID: 3537650   Study NETU-06-27: Netupitant with Palonosetron   Title of Study   Evaluation of Pharmacokinetic Interaction between Netupitant (450 mg, PO) and Palonosetron (0.75 mg, PO): a Randomized 3-way Crossover Study in Healthy Males and Females    Methodology   This was a randomized, open-label, single-dose, 3-period crossover study investigating 3 treatments:   Treatment A: oral netupitant 450 mg administered as single dose   Treatment B: oral palonosetron 0.75 mg and oral netupitant 450 mg administered simultaneously   Treatment C: oral palonosetron 0.75 mg administered as single dose   A total of 18 subjects (9 males and 9 females) were included in the study and randomized to treatment sequence. Each subject was to receive 1 of the 3 treatments during each of the 3 treatment periods.   The subjects fasted overnight (for approximately 10 hours) before dose administration on Day 1 in each treatment period. Fasting continued for 4 hours after dose administration. Water was allowed during fasting, except for 1 hour before and after dose administration.   In addition, during Treatment A only (netupitant single dose), fractional urine collection was performed. The subjects were discharged after collection of the 24 hour post-dose PK sample(s). The subjects then returned to the investigational site for PK blood sampling and delivery of collected urine every 24 hours until 240 hours post-dose (Day 11). There was a minimum washout of 14 days between Day 1 of any 2 consecutive treatment periods.   PK Results   Netupitant PK. The exposure to netupitant, in terms of Cmax and AUC, was similar after administration of netupitant alone and in combination with palonosetron to healthy male and female volunteers. In addition, the 90% confidence intervals for the treatment geometric mean ratios of Cmax and AUC for netupitant were contained within the equivalence range of 80-125%. Median Tmax, reflecting rate of exposure, and median apparent T½,z of netupitant were not affected by palonosetron.     The extent of exposure to M3, which is pharmacologically equipotent to netupitant, was about 33% of the exposure to netupitant in terms of AUC0-’, while it was 32% and 12% for M1 and M2, respectively. The pharmacokinetic parameters of the netupitant metabolites M1, M2 and M3 were similar after administration of netupitant alone and in combination with palonosetron. There were 21    Reference ID: 3537650   neither any consistent nor relevant gender effects for M1, M2 or M3. Overall, palonosetron had no relevant impact on the pharmacokinetics of netupitant metabolites.   A very low fraction of the oral dose of netupitant was excreted unchanged into urine (mean fe was 0.03%). There were no consistent indications of any gender effects for the pharmacokinetics of netupitant.   Palonosetron PK. The exposure to palonosetron, in terms of Cmax and AUC, was similar after administration of palonosetron alone and in combination with netupitant to healthy male and female volunteers. In general, the ratios indicate that the exposure to palonosetron was slightly higher in subjects treated with combination therapy compared to palonosetron alone, but not clinically relevant according to bioequivalence standards. The pharmacokinetic parameters obtained for palonosetron in this study (e.g. mean apparent T1/2,z and median Tmax), were comparable to those obtained in previous oral single dose studies.   Consistent with other studies for palonosetron, mean Cmax and mean AUC of palonosetron were 35-65% higher and median apparent T½,z was 15-47% longer in female subjects (35 hours after palonosetron alone and 47 hours after palonosetron + netupitant) compared to male subjects (30 hours after palonosetron alone and 32 hours after palonosetron + netupitant).   The extent of exposure to palonosteron metabolites M4 and M9, which have negligible pharmacologic activity, was 9 and 6%, respectively, of the exposure to palonosetron in terms of AUC0-’. Overall, the pharmacokinetics of M4 were similar after administration of palonosetron alone and in combination with netupitant. For M9, mean Cmax was 28% higher after the concomitant administration of palonosetron and netupitant. There were no apparent gender effects for M4 whereas mean AUC0-t of M9 was 58-64% higher and median apparent T1/2z was 52-194% longer in females compared to males.     Table 1 Summary of Netupitant and Palonosetron Pharmacokinetic Parameters by Treatment             Treatment       Netu450 mg (N=18)   Cmax (μg/L)   AUC0(h*μg/L) Palonosetron T1/2 (h)   Geo. Mean Mean (SD) (CV%) Reference ID: 3537650 Cmax (ng/L) AUC0-’ (h*ng/L)     22        T1/2 (h)         Geo. Geo. Geo. Mean Mean Mean Mean Mean Median Mean Median (SD) (SD) (SD) (CV%) (CV%) (CV%)                             650.2 575.1 25927 24000 71.81 (257.8) (39.6) (10156) (39.2)     Netupitant           Palo 0.75 mg +Netu 450 mg (N=18)                       Palo 0.75 mg (N=17)   659.7 560.0 26241 23182 (325.7) (49.4) (13219) (50.4) - - -   -                 78.31 1863.1 1799.9 77254 72596 (487.1) (26.1) (25402) (32.9) - 1638.4 1587.2 70813 67593 (415.5) (25.4) (20415) (28.8)         36.91 34.73   Reviewer’s comment  The results of this study indicated that no relevant pharmacokinetic interaction is expected when 300 mg netupitant and 0.5 mg palonosetron are administered as a combination therapy. The fraction of an oral dose of netupitant excreted unchanged in urine was very low (less than 1%). While it is unknown if the outpatient based collection of urine samples may have affected the results, the observed negligible excretion to urine is consistent with the known elimination pathway and the findings from the ADME study.   Study NETU-06-07: Netupitant with Oral Dexamethasone   Title of Study   Evaluation of Pharmacokinetic Interaction Between Three Doses of Oral Netupitant and Oral Dexamethasone Regimen: a Randomized Three Period Crossover Study in Healthy Males and Females   Methodology   This was a randomized, open, 3-period crossover study utilizing an incomplete Latin Square design. A total of 30 male and female subjects were to be randomized to 1 of the 3 treatment sequences ABC, BDA or CAD, corresponding to the following treatments:   Treatment A: dexamethasone regimen alone (20 mg on Day 1, followed by 8 mg b.i.d. [every 12 hours] from Day 2 to Day 4)   Treatment B: dexamethasone regimen (20 mg on Day 1, followed by 8 mg b.i.d. [every 12 hours] from Day 2 to Day 4) plus oral netupitant 100 mg on Day 1 only.   Treatment C: dexamethasone regimen (20 mg on Day 1, followed by 8 mg b.i.d. [every 12 hours] from Day 2 to Day 4) plus oral netupitant 300 mg on Day 1 only.   Treatment D: dexamethasone regimen (20 mg on Day 1, followed by 8 mg b.i.d. [every 12 hours] from Day 2 to Day 4) plus oral netupitant 450 mg on Day 1 only.   Ten subjects (5 males and 5 females) were to be randomized to each treatment sequence. In each treatment period the subjects fasted overnight (for approximately 10 hours before dose administration) and up to 4 hours after dose administration on Day 1.   Repeated PK blood sampling (determination of netupitant and dexamethasone) was performed 23    Reference ID: 3537650   up to the 120 hour post-dose. There was a wash-out of no less than 14 days between Day 1 of 2 consecutive treatment periods.   PK Results   Dexamethasone. Co-administration of netupitant significantly increased the exposure to dexamethasone in a dose- and time-dependent manner. The mean plasma concentrations of dexamethasone when coadministered with netupitant are shown in Figure 1.   Figure 1 Arithmetic Mean Plasma Concentration of Dexamethasone versus Planned Time, by Treatment       Pharmacokinetic parameters for dexamethasone alone and after 100, 300 and 450 mg of netupitant are shown below in Table 1. The AUC0-24 (Day 1) of dexamethasone increased 1.5, 1.7 and 1.8-fold with co-administration of 100, 300 and 450 mg netupitant, respectively. The AUC24-36 (Day 2) and of dexamethasone increased 2.1, 2.4 and 2.6-fold and AUC84-108 and AUC84-’ (Day 4) increased 1.7, 2.4 and 2.7-fold, with coadministration of 100, 300 and 450 mg netupitant, respectively. Dexamethasone Cmax on Day 1 was only slightly affected by co-administration of netupitant (1.1–1.2-fold increase during co- administration with 100–450 mg netupitant) while Cmax on Day 2 and Day 4 was increased approximately 1.7-fold in subjects administered netupitant. Dexamethasone Cmin on Days 2–4 was increased approximately 2.8, 4.3 and 4.6-fold with coadministration of 100, 300 and 450 mg netupitant, respectively. The T½,z of dexamethasone was increased by 1.9–3.2 hours on Day 1 and by 2.0–2.4 hours on Day 4.   There was no relevant change in Tmax for dexamethasone when administered in 24    Reference ID: 3537650   combination with netupitant. There was no relevant gender effect for AUC or Cmin but Cmax was slightly higher in female subjects.       Table 1 Summary of the Pharmacokinetic Parameters for Dexamethasone           Parameter AUC0-24 [h* μg/L] AUC24-36 [h*μg/L] AUC84-108 [h*μg/L] AUC84-∞ [h*μg/L] Cmax (0-24h) (μg/L) Cmax (24-36h) (μg/L) Cmax (84-108h) (μg/L) Cmin (24-36h) (μg/L) Cmin (36-48h) (μg/L) Cmin (48-60h) (μg/L) Cmin (60-72h) (μg/L) Cmin (72-84h) (μg/L) Tmax (0-24h) [h] Tmax (24-36h) [h] Tmax (84-108h) [h] T½,z (Day 1) [h] T½,z (Day 4) [h]   Dexamethasone Alone (N=22) 1089 (352) 330 (126) 364 (157) 390 (174) 156.5 (38.6) 62.7 (19.6) 58.2 (18.6) 8.4 (6.7) 10.2 (7.1) 8.2 (6.8) 10.5 (7.1) 16.8 (41.3) 3.00 (1.00 ; 5.00) 1.00 (0.98 ; 4.00) 1.01 (1.00 ; 5.00) 3.66 (2.67 ; 6.99) 4.42 (3.21 ; 7.30) Dexamethasone + Netu 100 mg (N=15) 1444 (320) 600 (90) 558 (137) 599 (166) 161.2 (32.0) 94.9 (16.4) 80.7 (29.0) 21.0 (6.1) 23.6 (7.0) 18.9 (6.4) 21.1 (5.3) 17.0 (7.0) 4.00 (2.00 ; 6.00) 2.00 (0.98 ; 3.00) 1.02 (1.00 ; 6.00) 5.50 (4.12 ; 7.72) 4.73 (3.81 ; 7.98) Dexamethasone + Netu 300 mg (N=13) 1782 (369) 760 (174) 836 (221) 913 (251) 169.9 (26.9) 100.3 (26.1) 96.2 (26.0) 35.7 (10.3) 38.3 (10.8) 36.3 (11.4) 35.0 (10.0) 29.2 (9.9) 4.00 (1.00 ; 5.08) 1.97 (0.97 ; 5.00) 2.00 (1.00 ; 3.00) 6.52 (4.70 ; 7.72) 6.45 (4.29 ; 7.63) Dexamethasone + Netu 450 mg (N=16) 1984 (430) 871 (159) 1005 (252) 1119 (308) 190.4 (35.5) 118.4 (27.4) 110.0 (29.8) 38.7 (8.9) 44.3 (10.6) 39.8 (12.5) 40.4 (11.7) 35.3 (11.9) 4.00 (1.00 ; 6.00) 1.01 (0.98 ; 5.00) 1.50 (0.98 ; 3.00) 7.50 (5.47 ; 8.31) 6.83 (5.19 ; 9.66) Mean and SD are shown, except for Tmax and T½, z where median and range are shown.   Netupitant. The extent of exposure to netupitant increased in a higher than proportional manner in the studied dose range of 100 to 450 mg. The dose normalized AUC0-t and AUC0-’ were increased higher than proportionally by 34 and 38%, respectively, after 450 mg netupitant. The increase in Cmax was approximately dose-proportional. PK parameters for netupitant were comparable to results obtained in previous studies: the PK profile of netupitant was not significantly altered in the presence of dexamethasone. There were no indications of any gender effect on the PK parameters for netupitant.   25    Reference ID: 3537650   Table 2. Summary of PK parameters for netupitant     Reviewer’s comments The mechanism of this drug interaction is most likely due to inhibition of CYP3A4. Reduction of the dexamethasone dose is therefore recommended when netupitant is coadministered. It was noted that the inhibitory effect was significant on Day 4 after single dose administration of netupitant and the extent of inhibition was similar between on Day2 and Day4 while the plasma concentrations for netupitant were decreased suggesting potential contribution of metabolites. Analyses based on [I]/Ki values over time suggested that the sum of [I]/Ki would be decreased to below 0.1 on Day 6 after single dose administration of 300 mg netupitant.   Study NETU-07-01: Netupitant with Oral Digoxin   Title of Study   Evaluation of Pharmacokinetic Interaction Between Netupitant (450 mg PO, Single Dose) and Digoxin (0.25 mg PO, Daily): An Open-Label, One-Way Study in Healthy Males and Females   Methodology   This was an open-label study in a total of 16 healthy subjects (8 males and 8 females). Each subject received a loading dose of 3 x 0.5 mg digoxin on Day 1, followed by a daily oral dose of 0.25 mg digoxin for 11 consecutive days and 450 mg netupitant on Day 8.   Subjects were admitted to the Clinical Unit twice: on Days 1 to 2 for safety reasons during digoxin loading phase and on Days 5 to 9 (4 overnight stays) for pharmacokinetic blood sampling.   PK Results   In this study, no influence on the extent of exposure of digoxin at steady-state after coadministration of netupitant was observed (Table 1)   26    Reference ID: 3537650   Table 1 Point Estimates of Digoxin PK Parameters   Pharmacokinetic Parameter AUC(0-24h,ss) Cmax,ss Cmin,ss Point Estimate Test/Ref. 104.13 108.97 96.65 90% Confidence Interval 95.86 - 113.11 90.30 - 131.49 88.84 - 105.14   Mean minimum concentrations of digoxin during the Day 6-8 study period did not fluctuate, indicating that digoxin was at steady state. After netupitant administration, on Days 8-12, mean minimum concentrations appeared stable, also confirming that there was no effect of netupitant on digoxin concentrations. The excretion of digoxin in urine was 55% without netupitant and 57% after netupitant co-administration, indicating the insignificant effects of netupitant on the P-gp mediated urinary excretion of digoxin.   Figure 1 Arithmetic mean concentrations-time profile of digoxin (mcg/L) in plasma Figure 2 Arithmetic mean concentration-time profile of netupitant, M1, M2, and M3 (mcg/L) administered with digoxin (n=16) There were no gender differences observed regarding extent of exposure to digoxin with or without netupitant. The digoxin pharmacokinetic parameters generated in this study are consistent with those described in published literature. Pharmacokinetic parameters generated 27    Reference ID: 3537650   in this study for netupitant and its metabolites were also consistent with previous data generated in the netupitant development program.   Digoxin was used in this study as a probe drug to assess the effect of netupitant on Pglycoprotein. If netupitant were to inhibit P-glycoprotein, the extent of digoxin bioavailability, as measured by systemic exposure (AUC), would be increased. This effect is typically quite dramatic when interactions are seen, with an example of the well-known interaction between itraconazole and digoxin producing a 68% increase in digoxin AUC. No influence on the extent of exposure of digoxin after coadministration of netupitant was observed.    Table 2 Summary of Digoxin PK Parameters   Pharmacokinetic Parameter AUC(0-24h,ss) [h*μg/L]                           Cmax,ss [μg/L] Tmax,ss [h] Statistic   Mean (SD) Geo.Mean (Geo.SD) Median (min - max) Mean (SD) Geo.Mean (Geo.SD) Median (min - max) Median (min - max) Digoxin Alone N=16 10.96 (2.39) 10.69 (1.27) 10.63 (5.72-15.01) 1.129 (0.334) 1.092 (1.29) 1.003 (0.848 - 2.057) 1.00 (0.50 - 1.50) Digoxin with Netupitant N=16   11.37 (2.38)   11.13 (1.24)   11.35 (6.80-16.43)   1.239 (0.285)   1.190 (1.40)   1.280 (0.365 - 1.700)   1.00 (0.50 - 1.50)   Reviewer’s comments One subject (Subject 007) did not have typical digoxin concentration-time profile in the period with netupitant co-administration. The plasma concentration of digoxin was above LLOQ but near (0.2 mcg/L) throughout the PK sampling period. It is unclear if the dosing of digoxin was done properly. As such the reviewer recalculated after excluding PK parameters from subject 007. The geometric mean ratio for Cmax and AUC excluding subject 007 was 117.9 and 107.6, respectively. Figure 3 Concentrations-time profile of digoxin (mcg/L) in Subject 007 28    Reference ID: 3537650   In this study the median Tmax of netupitant was 4 hours and the plasma concentration of netupitant when the maximum absorption of digoxin occurred. The inhibitory effects of itraconazole on P-gp in the kidney resulted in an increased in the systemic exposure and halflife of digoxin when itraconazole was administered an hour before digoxin administration. The median Tmax for itraconazole is 3-4 hours.   Study NETU-10-08: Netupitant with Oral Contraceptives   Title of Study An Open-Label, Randomized, Two-Way, Crossover Trial to Evaluate the Effect of a Single Dose Administration of Oral Netupitant and Palonosetron (300 mg/0.5 mg) on the Pharmacokinetics of Ethinylestradiol and Levonorgestrel after Single Dose Oral Administration in Healthy Female Subjects   Methodology This single-center study was conducted according to an open, randomized, two way, cross-over design in 24 healthy female subjects. The treatments were administered on Day 1 of each period according to the assigned treatment sequence. The Reference treatment (oral contraceptive alone) was administered in one period and the Test treatment (co-administration of oral contraceptive and FDC of netupitant and palonosetron) was administered in the other period. Administrations were separated by a washout phase of 28 days.   Blood samples for pharmacokinetics of ethinylestradiol and levonorgestrel were collected in both periods until 96 h after administration of the Test or Reference treatment. Blood samples for pharmacokinetics of netupitant (and its metabolites) and palonosetron were collected only in the period where the Test treatment was administered. The last blood sample for palonosetron and netupitant was collected 192 h and 240 h after administration of the Test treatment, respectively.   PK Results Effect on PK of ethinylestradiol 29    Reference ID: 3537650   The extent of absorption of ethinylestradiol based on AUC0-t and AUC0-∞ was 16% and 12% higher, respectively, after intake of the oral contraceptive together with netupitant and palonosetron compared to intake of the oral contraceptive alone.   The Cmax of ethinylestradiol was not significantly different after administration of the oral contraceptive together with netupitant and palonosetron compared to administration of the oral contraceptive alone. The point estimate of the Test/Reference ratio for Cmax was 105.09% (90% CIs: 98.33%; 112.32%) and AUC0-’ (102.80%; 122.22%).   Table 1 Mean Exposure Parameters for Ethinylestradiol after Oral Administration of Microgynon® with and without Netupitant/Palonosetron (300 mg/0.5 mg)       Parameter   T R PE*   115.6±30.9 105.09   98.33 - 112.32   1224±428.7 AUC0-∞ [h•pg/mL]   90%CI   120.6±28.3 Cmax [pg/mL]     1091±400.9 112.09   102.80 - 122.22   1071±397.0 928.3±383.2 116.05 106.21 - 126.79 AUC0-tz [h•pg/mL] Values are arithmetic means ±SD; SD: standard deviation. *Point estimate (PE): ratio of geometric means CI: confidence interval, SD: standard deviation R: two tablets each containing 30 μg ethinylestradiol and 150 μg levonorgestrel (Microgynon®) (Reference) T: two tablets each containing 30 μg ethinylestradiol and 150 μg levonorgestrel (Microgynon®) and one capsule containing netupitant and palonosetron 300 mg/0.5 mg (Test)   Effect on PK of levonorgestrel. The extent of absorption of levonorgestrel based on AUC0-t and AUC0-∞was 46% and 40% higher, respectively, after administration of the oral contraceptive together with netupitant and palonosetron compared to administration of the oral contraceptive alone. Mean Cmax of levonorgestrel was not significantly different after administration of the oral contraceptive together with netupitant and palonosetron compared to administration of the oral contraceptive alone.   Table 2 Mean Exposure Parameters for Levonorgestrel after Oral Dose with and without Administration of Microgynon® Netupitant/Palonosetron (300 mg/0.5 mg)     Parameter     Cmax [pg/mL]   AUC0-’ [h•ng/mL]   AUC0t-z [h•ng/mL]       T 8.11±2.93 87.4±54.1 R 8.23±2.79 60.0±37.0 PE* 98.06 146.21         90%CI 92.53 - 103.92 129.38 - 165.22 113.1±63.5 80.4±42.4 139.55 123.55 - 157.61 Values are arithmetic means ±SD; SD: standard deviation. *Point estimate (PE): ratio of geometric means CI: confidence interval, SD: standard deviation R: two tablets each containing 30 μg ethinylestradiol and 150 μg levonorgestrel (Microgynon®) (Reference) 30    Reference ID: 3537650   T: two tablets each containing 30 μg ethinylestradiol and 150 μg levonorgestrel (Microgynon®) and one capsule containing netupitant and palonosetron 300 mg/0.5 mg (Test)    PK of netupitant and palonosetron.   The effect of the oral contraceptive (ethinylestradiol and levonorgestrel) on the pharmacokinetics of netupitant, its metabolites M1, M2, and M3, and palonosetron was not investigated in this study. However, no marked effects on rate and extent of absorption of netupitant, its metabolites M1, M2, and M3, and palonosetron were observed when compared to the pharmacokinetic parameters shown in previous clinical studies.   Reviewer’s comments: The study results are acceptable.   Study NETU-10-11: Netupitant/Palonosetron FDC with Ketoconazole and Rifampicin   Title of Study An Open-Label, Randomized, Two-Groups, Two-Way, Cross-Over Trial to Evaluate a Possible Influence of Oral Ketoconazole, a CYP3A4 Inhibitor and Oral Rifampicin, a CYP3A4 Inducer, on the Pharmacokinetics of Netupitant and Palonosetron After Single Dose Administration as Fixed Dose Combination (300 mg/0.5 mg).     Methodology This single-center study was conducted according to an open-label, randomized, two- group, two-way cross-over design in 36 healthy male and female subjects to evaluate the influence of oral ketoconazole (Group 1, N=18 subjects) and oral rifampicin (Group 2, N=18 subjects) on the pharmacokinetics of netupitant and palonosetron. Each of the two groups underwent a screening phase lasting a maximum of 21 days, a treatment phase with two treatment periods separated by a washout of 28 days between the two FDC administrations and a final check (within 3 to 8 days after the end of the second period).   A single dose of netupitant/palonosetron FDC was administered alone in one period and either together with the CYP3A4 inhibitor ketoconazole (Group 1) or with the CYP3A4 inducer rifampicin (Group 2) in the other period. In both groups and both periods, blood samples for pharmacokinetics of netupitant and its metabolites were collected until 240 h after administration of the netupitant/palonosetron FDC and blood samples for palonosetron were collected until 192 h after administration of the FDC.   PK Results   Assessment of ketoconazole effect Administration of the CYP3A4 inhibitor ketoconazole with netupitant/palonosetron FDC increased the exposure of netupitant and resulted in an AUC0-tz of 1.8 fold, AUC0-’ of 2.4 fold, and Cmax of 1.3 fold when compared to the administration of netupitant/palonosetron FDC alone. Coadministation with ketoconazole did not affect the pharmacokinetics of palonosetron. 31    Reference ID: 3537650   The primary pharmacokinetic parameters Cmax and AUC0-’ of netupitant showed a higher maximum plasma concentration and a higher overall plasma exposure after intake of netupitant/palonosetron FDC with ketoconazole (T1) than after intake of netupitant/palonosetron FDC alone (R1) (Table 1).   Table 1 Mean ± SD Netupitant Pharmacokinetic Parameters after Oral Administration of Netupitant/Palonosetron (300 mg/0.5 mg) with and without Ketoconazole (400 mg q.d.)           Parameter Cmax [μg/L] AUC0- [h·μg/L]   T1   650.2±217.6   43459±16911 R1 546.0±241.0 17971±5618   PE%*   125.42   239.88   90%CI 101.27 - 155.33 205.60 - 279.89   28494±7703 16072±5132 180.42 159.51 - 204.06 AUC0-tz [h·μg/L] Values are arithmetic means ±standard deviation (SD); *Point estimate (PE): ratio of geometric means (T1/R1) and 90% confidence interval (CI) T1: One capsule of netupitant/palonosetron (300 mg/0.5 mg) in combination with 400 mg q.d. (2 tablets of 200 mg) ketoconazole (Test 1) R1: One capsule of netupitant/palonosetron (300 mg/0.5 mg) (Reference)   The primary pharmacokinetic parameters Cmax and AUC0-’ of palonosetron showed a higher maximum plasma concentration and a higher overall plasma exposure after intake of netupitant/palonosetron FDC with ketoconazole (T1) compared to intake of netupitant and palonosetron FDC alone (R1)(Table 2).   Table 2 Mean±SD Palonosetron Pharmacokinetic Parameters after Oral Administration of Netupitant/Palonosetron (300 mg/0.5 mg) with and without Ketoconazole (400 mg q.d.)           Parameter Cmax [ng/L] AUC0-’ [h·ng/L]       T1 898.7±220.1 40910±9261 R1 775.3±185.0 37524±9577   PE%* 115.35 110.09       90%CI 109.62 - 121.37 105.43 - 114.96   36899±8667 32564±7459 113.41 108.26 - 118.80 AUC0-tz [h·ng/L] Values are arithmetic means ±standard deviation (SD); *Point estimate: ratio of geometric means (T1/R1) and 90% confidence interval (CI) T1: One capsule of netupitant/palonosetron (300 mg/0.5 mg) in combination with 400 mg q.d. (2 tablets of 200 mg) ketoconazole (Test 1) R1: One capsule of netupitant/palonosetron (300 mg/0.5 mg) (Reference)   The formation of M1 and M3 was delayed when netupitant was co-administered with ketoconazole compared to intake of netupitant/palonosetron FDC alone (median Tmax of about 96 vs.12 h for M1 and 24 vs. 12 h for M3). Concomitant ketoconazole did not appear to have an impact on the time of the appearance of M2 in plasma. Mean maximum plasma concentration (Cmax) and overall plasma exposure (AUC0-tz) for all three metabolites M1, M2, and M3 were 32    Reference ID: 3537650   lower under co-administration with ketoconazole. Mean metabolite to parent ratios were 24.9%, 6.4%, and 15.1% for T1 compared to 30.3%, 12.1%, and 28.1% for R1.   Assessment of rifampicin effect Administration of the CYP3A4 inducer rifampicin with netupitant/palonosetron FDC alone decreased the exposure of netupitant and resulted in an AUC0-tz of 5.5 fold, AUC0-’ of 5.9 fold, and Cmax of 2.6 fold when the administration of the netupitant/palonosetron FDC alone was compared to the administration of netupitant/palonosetron FDC with the CYP3A4 inducer rifampicin. Coadministration of rifampicin resulted in a 15-20% decrease in palonosetron exposure. The primary PK parameters Cmax and AUC0-’ of netupitant showed a lower maximum plasma concentration and a lower overall plasma exposure after intake of netupitant/palonosetron FDC with rifampicin (T2) than after intake of netupitant and palonosetron FDC alone (R2) (Table 3).    Table 3 Mean±SD Netupitant Pharmacokinetic Parameters after Oral Administration of Netupitant/Palonosetron (300 mg/0.5 mg) with and without Rifampicin (600 mg q.d.) and Results of Analysis of Variance           Parameter Cmax [μg/L] AUC0- [h·μg/L]       T2 225.6±156.3 3463±2790 R2 498.1±225.6 16944±5915 PE%* 37.90 16.92         90%CI 28.81 - 49.86 12.70 - 22.55   3362±2766 15210±4977 18.05 13.56 - 24.01 AUC0-tz [h·μg/L] Values are arithmetic means±standard deviation (SD); *Point estimate (PE): ratio of geometric means (T2/R2) and 90% confidence interval (CI) T2: One capsule of netupitant/palonosetron (300 mg/0.5 mg) in combination with 600 mg q.d. (1 tablet of 600 mg) rifampicin (Test 2) R2: One capsule of netupitant/palonosetron (300 mg/0.5 mg) (Reference)   The primary pharmacokinetic parameters Cmax and AUC0-’ and of palonosetron showed a lower maximum plasma concentration and a lower ove rall plasma exposure after intake of netupitant/palonosetron FDC with rifampicin (T2) than after intake of netupitant and palonosetron FDC alone (R2) (Table 4).   Table 4 Mean±SD Palonosetron Pharmacokinetic Parameters after Oral Administration of Netupitant/Palonosetron (300 mg/0.5 mg) with and without Rifampicin (600 mg q.d.) and Results of Analysis of Variance           Parameter Cmax [ng/L] AUC0-’ [h·ng/L] AUC0-tz [h·ng/L]         T2 654.5±138.4 28354±7851 25557±7679 R2 772.2±206.0 35714±13467 32371±13055 33    Reference ID: 3537650 PE%* 85.44 81.03 80.64         90%CI 81.11 - 90.01 76.96 - 85.32 76.43 - 85.09   Values are arithmetic means ±standard deviation (SD); *Point estimate (PE): ratio of geometric means (T2/R2) and 90% confidence interval (CI) T2: One capsule of netupitant/palonosetron (300 mg/0.5 mg) in combination with 600 mg q.d. (1 tablet of 600 mg) rifampicin (Test 2) R2: One capsule of netupitant/palonosetron (300 mg/0.5 mg) (Reference)   After intake of netupitant/palonosetron FDC with rifampicin, the formation of metabolites was accelerated compared to intake of netupitant/palonosetron FDC alone (median Tmax of about 6 vs. 12 h for M1, 4 vs. 5 h for M2, 8 vs. 10 h for M3). For metabolites M1 and M3, mean maximum plasma concentration (Cmax) and overall plasma exposure (AUC0-∞) was slightly lower after co-administration with rifampicin. Mean metabolite to parent ratios for M1 and M3 were 34.9% and 45.5% for T2 compared to 29.3% and 26.8% for R2. For M2, mean maximum plasma concentration and overall plasma exposure were higher after co-administration with rifampicin. Mean metabolite to parent ratios for M2 were 176.9% for T2 compared to 11.0% for R2.   Reviewer’s comments: In vitro netupitant was metabolized mainly by CYP3A4 and to a lesser degree by CYP2C9 and CYP2D6. The significant effects of rifampin on the netupitant systemic exposure can reduce the efficacy of netupitant and the combination therapy. Although the dose-response relationship among the combinations with netupitant 100 mg, 200 mg and 300 mg was not evident for the delayed and overall phase. In the acute phase, the proportion of patients with CR was numerically higher with 300 mg netupitant (98%) than with lower doses of netupitant (92%). Nevertheless the overall CR rate in the acute phase was > 90% with combinations, the potential reduction of efficacy due to a decrease in the systemic exposure to netupitant would make the combination therapy without additional benefit over the monotherapy.   Study NETU-10-09: Netupitant/Palonosetron FDC with Chemotherapy   Title of Study A Single Dose, Open-Label, Randomized, Two Period, Cross-Over, Drug-Drug Interaction Study of Oral Palonosetron and Netupitant Fixed Dose Combination on the PK of Three Chemotherapeutics (Docetaxel, Etoposide, Cyclophosphamide) Metabolized by CYP3A4 in Cancer Patients   Methodology This multicenter, single dose, randomized, open label, 2 period, cross-over pharmacokinetic (PK) study was designed to evaluate the effects of oral netupitant administered as fixed dose combination with palonosetron on the PK profile of 3 different chemotherapeutic agents (docetaxel, etoposide, and cyclophosphamide) given to cancer patients.   Forty-eight (48) male and female patients • 18 years with histologically and / or cytologically confirmed malignant diseases and scheduled to receive at least 2 courses of 1 of the 3 chemotherapy agents and considered eligible to participate in this study, were planned to be enrolled into 1 of the 3 groups of 16 patients according to the chemotherapy regimen they were scheduled to receive. 34    Reference ID: 3537650     Within each group, all patients were to receive 1 of the 3 chemotherapeutic agents (docetaxel, etoposide or cyclophosphamide) for 2 consecutive cycles (hereafter referred to as treatment periods 1 and 2); Day 1 of the 2 treatment periods was separated by at least 3 weeks. The patients received a single oral dose of netupitant/palonosetron fixed dose combination (FDC; test Investigational Medicinal Product [IMP]) during either the first or the second treatment period. The antiemetic treatment for the alternate period was standardized, and all patients were given oral palonosetron 0.5 mg (Aloxi®, reference IMP). The treatment order was randomized within each group of patients receiving the same chemotherapeutic agent.   The patients received the test IMP on Day 1 of the test period (1 of the 2 treatment periods), 1 h before the start of their intravenous chemotherapy. During the alternate period, oral palonosetron (Aloxi®) was given 1 h before the start of chemotherapy for prevention of nausea and vomiting. Dexamethasone as antiemetic premedication was allowed provided it was given at both study periods. Rescue medication was allowed during the study if needed, according to the Investigator’s opinion, (e.g. prochlorperazine, thiethylperazine, metoclopramide, etc.). However, drugs that undergo CYP3A4-mediated metabolism, or are CYP3A4 inhibitors or inducers were to be avoided.   The PK profile of the 3 chemotherapeutic agents given with netupitant/palonosetron FDC (test IMP) was compared to the profile following a regimen of oral Aloxi® (0.5 mg palonosetron, reference IMP) alone. In addition, the safety profile and tolerability of the oral netupitant/palonosetron FDC, given together with an intravenous course of any of the 3 chemotherapy agents, was evaluated in this population of cancer patients. Furthermore, the PK of netupitant, netupitant metabolites and palonosetron in this population was explored.   PK Results   Docetaxel (N=8, PK set N=7). The mean plasma concentration curve obtained for docetaxel coadministered with netupitant/palonosetron FDC was overall similar in shape to that obtained for docetaxel with palonosetron alone. However, the mean docetaxel concentration curve in the netupitant/palonosetron FDC period was slightly higher than in the palonosetron alone period, primarily for the first few samples taken during and shortly after completion of the intravenous (IV) infusion. In particular, the concentration curves for 6 of the 7 patients who completed both treatment periods were slightly higher in the test than in the reference period, and the 7th patient had virtually the same concentrations in both periods. Exposure in the test period was approximately 37% higher for AUC0-t and 50% for Cmax than the exposure in the reference period.   The estimated mean ratios for the test to reference PK parameter values (expressed as % of the reference) were 135% and 149% for AUC0-t and Cmax, respectively; this suggests that there may be a drug-drug interaction between IV docetaxel and netupitant in the form of FDC with palonosetron. Also, the upper limits of the 90% CIs were >125.0% for AUC0-t and Cmax. 35    Reference ID: 3537650         Etoposide (N=12, PK set N=11). The mean concentration curve obtained for etoposide coadministered with netupitant/palonosetron FDC was overall similar in shape to that obtained for etoposide with palonosetron alone. However, the mean etoposide curve in the netupitant/palonosetron FDC co-administration period was slightly higher than in the palonosetron alone period, which was visible from the first post-dose sample onward. The exposure in terms of AUC0-t in the FDC period was approximately 21% higher than that in the reference period, while Cmax and AUC0-’ values were similar for both treatment periods.   36    Reference ID: 3537650     Cyclophosphamide (N=10, PK set N=10). The mean concentration curve obtained for cyclophosphamide co-administered with netupitant/palonosetron FDC was overall similar in shape to that obtained for cyclophosphamide with palonosetron alone. There was high variability between the patients, even during the infusion phase. In 7 of 10 patients, the plots of individual data revealed a lower cyclophosphamide Cmax in the netupitant/palonosetron FDC period than in the palonosetron alone period. In 3 patients, Cmax values were much higher in the netupitant/palonosetron FDC period than in the palonosetron alone period. For cyclophosphamide AUC0-t, 4 patients had higher values after palonosetron co-administration alone and 5 had higher values after FDC while 1 patient had the same values in both periods. The resulting very high individual test to reference ratios influenced the estimated mean ratio to such an extent that the data suggest a slight increase in PK parameters: cyclophosphamide exposure, in terms of mean Cmax, AUC0-t, and AUC0-∞, was 8%, 14%, and 14% higher, respectively, in the netupitant/palonosetron FDC co-administration period than after 37    Reference ID: 3537650   palonosetron. The estimated mean ratios for the test to reference parameter values (expressed as % of the reference) were between 119% and 127%; this suggests that there may be a minimal drugdrug interaction between IV cyclophosphamide and netupitant administered in the form of FDC with palonosetron. However, the large differences between results for the 2 treatments in individual patients did not result in consistent differences between the curves for the test and reference treatments.       Netupitant. Overall, geometric mean PK parameters of netupitant exposure when the netupitant/palonosetron FDC was co-administered with any chemotherapy were approximately 440 ng/mL for Cmax, approximately 14 h*μg/mL for AUC0-t and approximately 16 h*μg/mL for AUC0-∞. These values are in agreement with previous studies in healthy subjects.   38    Reference ID: 3537650   The mean netupitant concentrations obtained in each of the 3 chemotherapy groups after netupitant/palonosetron FDC co-administration were not essentially different, and elimination occurred at approximately the same rate. Netupitant was quantifiable in the last sample at 9 days post-dose in >50% of the patients.   Netupitant Cmax was observed approximately 4h (median tmax) after the netupitant/palonosetron FDC intake, irrespective of the chemotherapeutic agent. Netupitant AUC0-’ values could often not be derived with sufficient accuracy as the very long half-life of netupitant resulted in large extrapolations, despite sampling to 9 days after dose intake. The variability (geometric CV%) for Cmax was overall high (50% to 70%) and moderate for the other parameters. The mean t1/2z values, ranging between approximately 70 h and 90 h, were not essentially different across the 3 treatment groups. GeoMean CL/F values were 18 to 19 L/h across the 3 chemotherapy groups.     Netupitant metabolite M1. Overall exposure to netupitant metabolite M1 relative to netupitant was approximately 8% for Cmax and between approximately 30% (docetaxel and etoposide group) and 35% (cyclophosphamide group) for AUC0-t. These values are in agreement with previous studies in healthy subjects.   Netupitant M1 Cmax was observed approximately 7 h (median tmax) after netupitant/palonosetron FDC intake in the cyclophosphamide group, and at 12 h and 18 h postdose in the docetaxel and etoposide groups, respectively. Exposure to netupitant metabolite M1 in terms of GeoMean Cmax and AUC0-t was similar across the chemotherapy groups. AUC0-’ values for netupitant M1 for the docetaxel group could not be calculated as derived t1/2z values were not sufficiently accurate.   The variability for Cmax and AUC0-t (geometric CV%) was overall moderate. The mean t1/2z values were similar for the etoposide group (82 h) and cyclophosphamide group (91 h).   39    Reference ID: 3537650   Netupitant metabolite M2. Overall, exposure to netupitant metabolite M2 relative to netupitant in terms of Cmax ranged from approximately 45% in the etoposide (N=12) and cyclophosphamide (N=10) groups to 70% in the docetaxel group (N=8). The relative exposure to M2 in terms of AUC0-t ranged from 20% in the etoposide and cyclophosphamide groups to 30% in the docetaxel group. These values are in agreement with previous studies in healthy subjects.   Netupitant M2 Cmax was observed approximately 4 h (median tmax) after netupitant/palonosetron FDC intake irrespective of the chemotherapeutic agent. Exposure to netupitant metabolite M2 in terms of GeoMean Cmax and AUC0-t values was approximately 50% to 60% higher in the docetaxel group than in the etoposide and cyclophosphamide groups. AUC0-’ values were calculated for only half of the patients or less, as the derived elimination rates were often not reliable. The terminal elimination took place at mean concentrations of 10 to 20 ng/mL and the contribution of this phase to the overall exposure was limited despite the slow elimination with individual t1/2z values >80 h. The variability for Cmax and AUC0-t (geometric CV%) was overall moderate to high. The t1/2z values across the etoposide and cyclophosphamide groups were not essentially different (mean and median values of approximately 60 h).   Netupitant metabolite M3. Exposure to netupitant metabolite M3 relative to netupitant was approximately 14% for Cmax and approximately 34% for AUC0-t. These values are in agreement with previous studies in healthy subjects.   Netupitant M3 Cmax was observed 12 h (median tmax) after netupitant/palonosetron FDC capsule intake, irrespective of the chemotherapeutic agent. Exposure to netupitant metabolite M3 in terms of GeoMean Cmax and AUC0-t, and AUC0-’ as available, was similar across the 3 chemotherapy groups. The variability for Cmax, AUC0-t and AUC0-’ (geometric CV%) was overall moderate. The t1/2z values for netupitant M3 were calculated for most patients in each chemotherapy group. Although the terminal elimination took place at low mean concentrations, the contribution of this phase to the overall exposure exceeded 20% in several patients due to the slow elimination, with individual t1/2z values >100 h. The t1/2z values across the chemotherapy groups were not essentially different (mean and median values of approximately 65 h to 80 h).   40    Reference ID: 3537650   Palonosetron. Overall, geometric mean PK parameters of palonosetron exposure when netupitant/palonosetron FDC was co-administered with any chemotherapy were approximately 900 pg/mL for Cmax, approximately 50000 h*pg/mL for AUC0-t and approximately 57000 h*pg/mL for AUC0-’. These values are in agreement with previous studies in healthy subjects. Palonosetron Cmax was observed approximately 5 h (median tmax) after the netupitant/palonosetron FDC capsule intake, irrespective of the chemotherapeutic agent. Exposure to palonosetron in terms of GeoMean Cmax and AUC0-∞ values was approximately 30% (Cmax) to 65% (AUC0-∞) higher in the docetaxel group than in the etoposide and cyclophosphamide groups. The variability for these parameters (geometric CV%) was moderate overall. The mean t1/2z values were similar in the etoposide and cyclophosphamide groups, but appeared to be approximately 20 h longer in the docetaxel group. The GeoMean CL/F value was lower in the docetaxel group than in the etoposide and cyclophosphamide groups. Results shown in the docetaxel group, however, need to be interpreted with caution because of the small sample size (N=8). Reviewer’s comments According to the population PK analyses in phase 3 trial, the PK of netupitant and palonosetron in cancer patients was similar with in healthy subjects. Please see the Pharmacometrics review by Dr. Jingyu Yu for more details. Underlying reason for apparent high systemic exposure to palonosetron in the docetaxel is unclear. 41    Reference ID: 3537650       Study NP16602: Apomorphine Challenge in Healthy Volunteers   Title of Study Randomized, Double-Blind, Placebo-Controlled Evaluation of the Anti-Emetic Effect of RO0673189 (Netupitant) Following Apomorphine Challenge in Healthy Volunteers   Methodology Study NP16602 was a randomised, double-blind, placebo-controlled study in which 32 healthy subjects were assigned to 4 dosing groups of subjects each. Within each group, 6 subjects received a single dose of netupitant and two subjects received placebo. Eligible subjects were fasted overnight and received a standardized breakfast prior to dosing with netupitant or placebo. All subjects then received a subcutaneous injection of 50μg/kg apomorphine (an emetogen) at between 8 and 24 hours post-dose. Doses and timing of the apomorphine challenge for each group are shown in Table 1.   Table 1 Dose Groups and Timing of Apomorphine Challenge               Netupitant Dose Group Interval Between Netupitant Dose and Apomorphine Injection 100 mg (I) 24 h 100 mg (II) 8h 300 mg 12 h 450 mg 12 h   Emesis occurring during the 90-minute period following apomorphine injection was evaluated by the degree of nausea, measured at 10 minute intervals using a visual analogue scale, the occurrence of vomiting and the total number of vomits and retches.   Blood samples for pharmacokinetic analysis were collected pre-dose and at 15 and 45 min and 1, 1.5, 2, 3, 5, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 h post-dose.     42    Reference ID: 3537650   PD Results   Analysis of the results by plasma netupitant concentration at the time of apomorphine challenge showed a decrease in the incidence of vomiting with increasing netupitant levels. No subject in the highest concentration group (> 300 ng/mL, corresponding to 1 subject taking 300 mg and 5 subjects taking 450 mg) experienced vomiting, a statistically significant result compared to placebo (p = 0.010).   Subjects with lower netupitant concentrations also experienced less vomiting than the placebo group. In the three groups with lower netupitant concentrations, 50% of subjects experienced no vomiting (N=18), compared with 25% of subjects vomiting-free in the placebo group (N=8). In total, 15 out of the 24 subjects who received netupitant experienced no vomiting, with six subjects having fewer than 6 vomiting episodes. Only 2 of the 8 subjects receiving placebo experienced less than 6 vomiting episodes.   Retching was reduced in subjects treated with active drug, but no trend was observed between concentration groups. The results were skewed by one subject in the highest concentration group, who experienced a very high number of retches. Nausea tended to increase with netupitant concentration, with the exception of the lowest concentration < 50 ng/mL, which recorded the lowest levels.   Table 2   PK Results   Netupitant was absorbed in a first order fashion, reaching maximum plasma concentrations at approximately 5 h post-dose. The t1/2 was estimated to be approximately 50 h. These results suggest that netupitant provides better control for emesis compared with placebo following apomorphine challenge. A concentration-effect relationship was demonstrated with complete control of vomiting at plasma concentrations of > 300 ng/mL. However, there was a trend towards an increase in nausea with increasing plasma concentrations. The study medication was well tolerated by all subjects in this study. Reviewer’s comments: This study is exploratory only to guide the dose selection for a phase 2 trial.   43    Reference ID: 3537650   Study NETU-06-08: PET Study in Healthy Volunteers   Title of the Study A Positron Emission Tomography (PET) Study to Assess the Degree of Neurokinin-1 (NK1) Receptor Occupancy in the Human Brain After Single Doses of Netupitant in Healthy Male Subjects Using 11CGR205171 As Tracer     Methodology This was a single dose, randomized, open-label, PET study investigating the degree of occupancy of NK1 receptors in the human brain after single oral doses of netupitant in healthy male subjects. The study consisted of a screening visit, a baseline PET visit, a treatment period with up to 5 post dose PET scans and a follow-up visit. The screening assessments were performed within 28 days before dose administration, the baseline PET visit was performed within 7 days before dose administration and the follow-up visit was performed 14 ±2 days after dose administration.   At the baseline PET visit, eligibility criteria were re-checked and subjects still considered eligible were randomized and subjected to a baseline PET scan. On Day 1, subjects were admitted to the investigational site and a single dose of netupitant (100, 300 or 450 mg) was administered. Blood samples for determination of netupitant plasma concentrations were collected regularly for up to 97 hours after dose administration. PET scans were performed 6, 24, 48, 72 and 96 hours after dose administration. The subjects were discharged from the investigational site after the 24 hour post-dose PET scan and then returned for additional PET scans and PK blood sampling.   Results Median Tmax ranged from 5.56 to 5.74 hours indicating that the PET scans between 6 and 7 hours post dose were performed close to Cmax.   This study showed that netupitant showed that netupitant binds to NK1 receptor antagonist in the human brain with an ability to block NK1 receptors. The anticipated high NK1-RO (90% or higher) close to expected Cmax (6 hours post dose) was achieved for occipital cortex and frontal cortex for all investigated doses as well as for striatum (for 300 and 450 mg netupitant) and anterior cingulate (for 100 and 450 mg netupitant).   All doses showed a blockade of the NK1 receptors and for most regions the NK1-RO declined slowly until 96 hours post dose in a dose-dependent fashion. In the 100 mg dose group, 4 of 6 regions still had a mean NK1-RO over 70% at 96 hours post dose. In the highest dose group (450 mg), 5 of 6 regions had a mean NK1- RO near to 80% or higher at 96 hours post dose. A comparison of the results for the dose groups (100 mg, 300 mg and 450 mg) showed a general but low increase in NK1-ROs with increasing dose.        44    Reference ID: 3537650 Table l. receptor occupancy in striatum and frontal cortex hours after administration of netupitant by dose Table 11 NK, receptor occupancy in strlatum hours a?er administration of netupitant. by dose - PD set Net-phat Set-pliant 100 300 490 .V-Z SK. rmptor occupancy ?n 2 2 2 Mn. 84 92[cw- 8 3 receptor oeenpann- 2 2 2 1? .\lenn 70 80.VKI Raptor occupant) ,n 2 2 2 ?43% Mean 05.5 95.0 94SK. receptor occupant) 2 2 2 73 Mean 04.0 78.0 900 so 0.0 2.8 8.5 [cvu 4 9 NR. receptor occnpanry 2 2 2 .\ ean at 70In?. l6 2 9 of sobpets the speci?c n-thlbel of ubpeets dun available Table 13 NKI receptor occupancy in frontal cortex hours after administration of netupitant. by close - PD analysis set Netupltant Xempltant 100 mg 300 In: 450 mg receptor occupancy .n 2 2 2 6 Mean 90.0 93.5 95.0 rso 4.2 6.4 7.1 level. 5 7 7 receptor occupancy 2 2 2 2? =Mean 90.5 86.0 93.0 0.7 7.1 7.1 lcw. 1 8 8 receptor occupancy ,n 2 2 2 it 43 how Mean 83.0 87.5 95.5 SD 0.0 6.4 6.4 Icy-0 7 7 NK1 receptor occupancy in 2 2 2 72 50"" :Mean 82.5 90.0 94receptor occupancy 45 2 2 2 9? Mean 79.5 89.5 85.5 SD 2.1 4.9 2.1 chv. 3 6 2 N=Number of subjects in the speci?c group Reference ID. 3537650 naNumber of subiects with data available   Figure 1 NK1-ROs- Netupitant concentrations in (A) Striatum and (B) Frontal cortex   (A) Striatum   (B) Frontal cortex Summary  The anticipated high RO (90% or higher) close to expected Cmax (6 hours after dosing) was achieved after one single dose of netupitant for occipital cortex (100–450 mg), frontal cortex 46    Reference ID: 3537650   (100–450 mg), striatum (300 and 450 mg) and anterior cingulate (100 and 450 mg). Therefore it is expected that a minimum single oral dose between 100 and 300 mg netupitant would be necessary to provide an NK1-RO of 90% close to Cmax in the majority of the outlined brain regions. This was supported by the analysis of the PK/PD relationship in striatum.   All doses showed a relatively long duration of blockade of the NK1 receptors with moderate to high NK1-RO for all investigated brain regions at 96 hours post dose. There was a dose dependent decline in NK1-ROs over time with a slightly faster decline in the lowest dose group. For the highest dose of netupitant a mean NK1-RO higher than 90% was achieved in striatum, frontal cortex and anterior cingulate until 72 hours and in occipital cortex even up to 96 hours. Reviewer’s comments: NK1 receptors are widely distributed throughout the brain. Per the study report, the delineated regions of interest (ROIs) were selected to reflect changes in NK1-RO in the whole brain rather than the target region for the emesis control because of the ill-defined mechanism of emesis. Based on this study and the apomorphine-challenge study, netupitant doses 100 mg, 200 mg, and 300 mg were selected for the phase 2 trial.   47    Reference ID: 3537650   Study NETU-11-01: Single Ascending Dose PK Study for intravenous netupitant in Healthy Volunteers Title of the Study A Single Ascending Dose Study To Assess the Safety and Pharmacokinetics of Intravenously Administered Netupitant in Healthy Volunteers. A double-blind, placebo-controlled, unbalanced, phase I study. PK Results The PK of netupitant and its metabolites M1, M2 and M3 were investigated after IV administration of 4 single ascending doses of netupitant (25 mg, 50 mg, 75 mg, and 100 mg). Figure 1 Arithmetic mean concentrations of netupitant over (A)24 hours and (B)120 hours after start of infusion of netupitant 25, 50, 75, and 100 mg (A) (B) 48    Reference ID: 3537650   The netupitant plasma concentrations declined rapidly after end of infusion of the netupitant 25 mg dose and less than 10% of the initial netupitant concentration measured at the end of infusion was detected at 0.75 hours after start of infusion. The administration of the subsequent dose levels (i.e., netupitant 50 mg, 75 mg, and 100 mg) was performed with an infusion duration of 30 minutes to avoid episodes of very high netupitant plasma concentrations. The prolonged infusion duration resulted in reduced netupitant peak concentrations at the end of infusion with lower variability of data (CVs of about 37%, 23%, and 32% for the netupitant 50 mg, 75 mg and 100 mg doses, respectively). An increase of peak netupitant plasma concentrations with ascending netupitant doses infused over 30 minutes was observed. An increase of mean systemic netupitant exposure (AUC0-last) was also seen with ascending netupitant doses. The variability of AUC0-last was low with slightly higher variability for the 25 mg dose cohort (CV of about 19%) than for the other dose cohorts (CV of about 12% and 13%). The extent of exposure of all tested IV netupitant doses was lower than the exposure obtained after oral administration of netupitant 300 mg (i.e., the mean values of AUC0-tlast were <12,000 h*μg/L). After IV infusion of netupitant, the mean volume of distribution was high (about 493 L to 1524 L) and increased with ascending netupitant doses. The elimination of netupitant was slow with a long terminal elimination half-life (mean of 27 hours to 78 hours). The mean total clearance ranged between 12.7 L/h and 18.9 L/h. Netupitant metabolites M1, M2, and M3 were detected in plasma after IV administration of all tested dose levels. Netupitant was rapidly metabolized to metabolite M2 with the first quantifiable concentrations measured already at the end of infusion for all tested dose levels and a median Tmax of about 3 hours. For metabolite M3, the first quantifiable concentrations for the highest dose level of netupitant 100 mg were also observed at the end of infusion. For the lower dose levels, quantifiable concentrations were observed within the first 1.5 hours after start of infusion (a.s.i.). Median M3 Tmax was about 24 hours for all dose levels except for the highest dose level of 100 mg where the median Tmax was about 18 hours. For metabolite M1, first quantifiable concentrations were measured 1 hour a.s.i for the highest dose level of netupitant 100 mg. For all other dose levels, quantifiable concentrations were observed within the first 12 hours a.s.i. Median Tmax was about 24 hours for the 25 mg and 50 mg dose level and about 18 hours for the 75 mg and 100 mg dose levels. With the only exception of Cmax of netupitant after the faster infusion rate applied with the 25 mg dose, all mean exposure parameters 49    Reference ID: 3537650   (AUC0-tlast, AUC0-inf, and Cmax) of netupitant and metabolites M1, M2, and M3 increased with the IV netupitant doses of 25 mg, 50 mg, 75 mg, and 100 mg. Figure 2. Dose-normalized AUC by dose Table 1 Pharmacokinetic parameters of netupitant after infusion of netupitant 25, 50, 75, and 100 mg 50    Reference ID: 3537650 Reference ID: 3537650 Characteristic of . Negrpitant Nefupitanl Nesnpitant Nempimnt ne mpimn Statistics mg 5111112 .5612 109 m: 13:61? 0=61 (3:61 [h?pgl] 5 6 6 6 Mean 11184.2 24911.2 41110.0 4575.9 SD 365.67 299.32 4711.83 6111.41 19.41 11.93 11.46 13.14 Geo. mean 11157.9 2483.11 4153.5 4542.6 Gen.$1) 1.20 1.12 1.12 1.14 Median 1664.6 2371.9 395 7.7 4647.0 Minimum 161.1 1 2191 3751 3759 Maximum 2443 2933 5112 5435 [h?ugr'L] 5 6 6 6 Mean 2111 5 .6 2343.3 5291.4 5492.4 SD 374.33 370.72 1469.85 1267.18 CV 18.61] 13.04 27.711 23.07 Geo. mean 19119.11 2923.3 5143.3 5381.5 Geo. SD 1.20 1.14 1.23 1.24 Median 1848.1 2909.5 4637.5 541 1.7 Min imnm 1658 2400 4279 4277 Maximum 255 6 3241 81190 Mean 6.6 1.6 17.4 15.2 SD 3.117 11.64 16.56 3.311 CV 46.219 74.25 94.97 54.51 Geo.Mean 6.0 9.7 13.6 13 .3 (3:30.31) 1.59 1.85 1.99 1.31 Median 5.9 8.9 12.4 14.1 Min imnm 3 4 7 5 Maximum Mean 27 .116 43.66 77.77 61.01 SD 5.3.4 32.19 1113 .92 39.47 CV 19.72 73.74 133.62 64.69 Geo. mean 26.511 37.45 49.41 52.91 Gen. SD 1.25 1.73 2.44 1.75 Median 23.88 33.117 38.96 48.50 Minimum 111.411 24.32 26.77 27.16 Maximlun 32 .69 1113.5 9 289 .3 7 136.29 Yd 5 6 6 6 Mean 492.9 1076.7 1339.4 1523.6 SD 1 16.56 671.211 1262.37 635 .28 CV [?61 23.65 62.34 94.25 4 1.711 Geo. mean 4512.0 956.3 1038.3 14111.5 Geo. SD 1.27 1.64 2.03 1.51 Median 463.7 1177.2 933.11 1465.11 Minimum 373 55 5 507 916 Maximum 631.1 2417 3870 2528 51   Safety The netupitant infusion was locally not well tolerated except for the lowest dose of netupitant 25 mg. Overall 4 of the 24 subjects administered with IV netupitant developed an infusion site thrombosis (2 subjects at the highest dose level of 100 mg and 1 subject each at the 50 mg and 75 mg dose levels). These events were considered to have a possible (50 mg and 75 mg) or probable (100 mg) relation to the IMP. The dose escalation process was stopped after administration of 100 mg netupitant due to safety reasons. This IV netupitant formulation will not be further studied in humans due to the poor local tolerability profile observed in this study. Reviewer’s comments: The absolute oral bioavailability was not studied. In a cross-study comparison of PK parameters after single oral administration of 100 mg netupitant, the total clearance was lower after IV administration. The CL and CL/F were comparable. Table 2. Mean (%CV) PK Parameters for netupitant after single dose administration of oral or intravenous Netupitant at 100 mg in healthy subjects   1 Study RO16603: PK sampling up to 168 h post‐dose  2 NETU‐11‐01:  intravenous  infusion  over  15  min;  infusion  site  thrombosis  occurred  in  2  patients;  PK  sampling up to 120 h after start of infusion  52    Reference ID: 3537650   Table 3 Mean (%CV) PK Parameters for metabolites of netupitant after single dose administration of oral or intravenous Netupitant at 100 mg in healthy subjects   1 Study RO16603: PK sampling up to 168 h post‐dose  2 NETU‐11‐01: PK sampling up to 120 h after start of infusion  53    Reference ID: 3537650   In –Vitro Studies: Title: NK1 Receptor Antagonist Ro 67-3189: In Vitro Plasma Protein Binding and Blood/Plasma Partitioning in Man and Various Animal Species. Report No: 1006047 Specific Aims: To determine the in vitro protein binding of Ro 67-3189 in plasma of different species and to assess the blood/plasma partitioning. Study Date: 03/1999-05/1999 Test site: F. Hoffmann-La Roche Ltd., Basle, Switzerland, Dept. PRNS Sponsor: F. Hoffmann-La Roche, Ltd. Study Design: Test Item: Ro 67-3189 (NK1 receptor antagonist) Tested Concentration: 9-1300 ng/mL in human plasma Plasma reference: Table 1 Biochemistry values in plasma pools from the various species Species n total protein g/L albumin g/L AGP man 18 68 46 Dog 4 53 29 not measured Ra >20 61 32 not measured gerbil rats >6 46 29 not measured g/L 0.69 For in vitro blood/plasma partitioning, blood was obtained from one male healthy volunteer, two dogs and four rats. Study Method: Binding Study: The protein binding was evaluated by equilibrium dialysis at 37oC and pH 7.4 after addition of 14 C- or 3H-labeled drug to plasma. Binding to isolated human serum albumin (HSA), human α1acid glycoprotein (AGP), and to diluted bovine fetal serum (BFS) was assessed in addition. The time required to reach equilibrium was investigated for 14C-Ro 67-3189/003 at drug concentration of 2.6 µg/mL and pH of 7.4, and it was determined to be approximately 5 hrs. For all the subsequent binding studies with 3H-labeled Ro 67-3189/004 to determine the plasma protein binding in the various species, the dialysis time was set to 5.0 hours. 600 μL of blank plasma was added when sample were removed from the buffer solution from the dialysis cells to minimize the loss of substance due to non-specific adsorption to the material. The resulting dilution of the buffer samples was determined by weigh. 54    Reference ID: 3537650   The pH dependency of the protein binding was also determined between final pH values 7.0 and 7.8 by using 0.133 M Søerensen phosphate buffers. Blood/Plasma Partitioning: Freshly drawn blood was centrifuged to generate a small erythrocyte free plasma layer, and equilibrated at 37°C for 30 min. The erythrocyte-free plasma layer were then spiked with 14C-Ro 67-3189/003 where the drug concentrations ranged from 10 to 14000 ng/mL, and were immediately mixed at 37°C. After 30 min of incubation at 37oC, aliquots were removed, centrifuged and the drug concentrations were measured in plasma and whole blood by liquid scintillation counting. Selected rests of spiked blood samples were let to stay at 21° (RT) for 30 min to measure the influence of the temperature on the partitioning. The hematocrit (H) was determined in the freshly drawn blood using a hematocrit centrifuge and hematocrit reader. The reversibility of the partitioning was measured at the end of the incubation by re-suspending the erythrocytes in fresh blank plasma for 30 min at 37°C. Bioanalytical Method: Concentrations of 14C-Ro 67-3189/003 and 3H-Ro 67-3189/004 were determined in duplicate in buffer, plasma, protein solutions and whole blood (triplicate) by liquid scintillation counting. The limit of quantification in the buffer samples was 0.5 ng/mL for 14C-Ro 67-3189, and 0.0015 ng/mL for 3H-Ro 673189, Data Analysis: Protein Binding: The approximate time for protein binding to achieve equilibrium was calculated by fitting the % free drug versus time (t) data to the equation: %free = %freess·(1-e-kt) where %freess is the percent free at equilibrium, and k is a first order rate constant for the equilibration. The time to achieve equilibrium is taken as 5 times the equilibrium half-life (t1/2 = 0.693/k). The free fraction fu, was calculated as the ratio between the concentration found in the buffer dialysate (CB) and the concentration in the corresponding plasma or protein solution (CPe) dialysate at the end of the dialysis: fu = CB/CPe The free fraction was corrected for osmotic fluid volume shifts (which is considered to be relevant when plasma is dialyzed for time periods longer than 4 h) according to the equation given by Boudinot and modified by Lohmann: fu = CB·VPi / [CPe·VPe – CB·(VPe-VPi] where VPi and VPe are the initial and final plasma volume, respectively. Blood/Plasma Partitioning The blood/plasma concentration ratio (λ) was calculated from: λ = CW/CP = (H·CE/CP) + (1-H) where CW, CP and CE are the drug concentration in whole blood, plasma, and erythrocytes, respectively, and H the hematocrit value. The fraction fE of drug in erythrocytes was calculated from: fE = QE/QW = (λ+H-1)/λ 55    Reference ID: 3537650   where QE is the amount of drug in the erythrocyte compartment and QW the amount of drug in whole blood. Note that if no drug is bound the red blood cells, CE/CP tends to zero, and λ will simplify to: λ=1–H Results: Protein Binding in Human Plasma Equilibrium Kinetic: The time required to reach equilibrium in human plasma was determined with 14C-Ro 67-3189/003 at a concentration of 2600 ng/mL and found to be about 5 hours. All the values were corrected for fluid volume shift Determined in human plasma with 14C Ro 67-3189/003 at pH 7 4 and 2 6 µg/mL Influence of pH: Determined in human plasma at a concentration of 82 ng/mL; The time of dialysis was 5h; all the values were corrected for fluid volume shift Influence of Concentration: The protein binding in human plasma was constant (99.7% binding) over the whole concentration range tested (9-1300 ng/mL). Table 2: 3H-Ro 67-3189: In vitro binding to human plasma (pH 7.41) 56    Reference ID: 3537650   concentration of Ro 67-3189 (ng/mL) plasma buffer % free 2) % bound 2) 9.13 0.0376 0.36 99.64 24.7 24.7 39.9 74.9 249 251 425 786 797 867 865 1’300 0.0936 0.0958 0.121 0.298 0.927 0 987 1.69 4.03 4.16 3.27 2.67 4.41 0.33 0.35 0.27 0.35 0.34 0 35 0.36 0.45 0.46 0.34 0.28 0.30 0.33 99.67 99.65 99.73 99.65 99.66 99 65 99.64 99.55 99.54 99.66 99.72 99.70 99.67 MEAN5) SD correction factor 3) EtOH (%) 1.14 0.5 1.15 1.11 1.11 1.14 1.09 1 11 1.11 1.13 1.13 1.12 1.11 1.13 0.5 0.5 0.5 0.5 0.5 05 0.5 1.5 1.5 0.5 0.5 0.5 0.032 Binding to Isolated Human Plasma Proteins: The relative contribution of albumin (HSA) and α1-acid glycoprotein (AGP) to the overall plasma binding in man was determined at drug concentrations ranging from 20 to 2600 ng/mL, both proteins separately and/or in combination at physiological concentrations of 41 g/L (HSA) and 0.6 and 1.8 g/L (AGP). Binding of Ro 67-3189 to HAS was constant (fu=2.9%) within the concentration range tested (20 to 2’290 ng/mL) while binding to AGP was concentration dependent. Table 3: 3H-Ro 67-3189: In vitro binding to isolated human plasma Proteins matrix 1) HSA : 41 g/L concentration of 3H-Ro 67-3189 protein side ng/mL 20.3 66.1 214 732 2’290 buffer ng/mL 0.591 1.81 6.45 21.5 69.6 protein side µM 0.035 0.11 0.37 1.3 4.0 57    Reference ID: 3537650 % free % bound 2.9 2.7 3.0 2.9 3.0 97.1 97.3 97.0 97.1 97.0   AGP : 0.62 g/L HSA : 41 g/L + AGP : 0.62 g/L MEAN SD 17.6 52.6 200 636 1’810 MEAN SD 22.8 73.2 247 808 2’420 2.9 0.676 2.88 10.4 46.3 190 0.030 0.091 0.35 1.1 3.1 3.8 5.5 5.2 7.3 11 n.c. 96.2 94.5 94.8 92.7 89 n.c. 0.312 1.07 3.33 12.8 39.3 0.039 0.13 0.43 1.4 4.2 1.4 1.5 1.4 1.6 1.6 98.6 98.5 98.6 98.4 98.4 1.5 MEAN SD HSA : 41 g/L + AGP : 1.8 g/L FBS 2) : 10% 1) measured concentration 97.1 0.1 98.5 0.12 25.3 76.8 255 856 2’580 MEAN SD 0.150 0.446 1.66 4.91 17.6 59.6 197 621 1’790 4’930 MEAN SD 6.13 21.7 76.6 314 1’110 0.59 0.58 0.65 0.57 0.68 0.61 0.044 0.13 0.44 1.5 4.5 99.41 99.42 99.35 99.43 99.32 99.39 0.05 0.10 0.34 1.1 3.1 8.5 10 11 12 18 23 90 89 88 82 77 n.c. 2) foetal bovine serum n.c. n.c. not calculated Blood/Plasma Partition The mean blood/plasma concentration ratio (λ) in human was 0.69 at 37°C and 21°C. Partitioning was independent of the drug concentration (concentration range tested: 52-994 ng/mL). The fraction of drug in erythrocytes (fE) was 13%. The partitioning was reversible. Table 4: 14C-Ro 67-3189: In vitro blood/plasma concentration ratio (λ) at 37°C MAN Distribution concentration of Ro 673189 ng/mL blood plasma 52.0 100 308 610 994 75.1 147 448 895 1’420 MEN SD Reversibility 1) 0.201 0.320 at 21°C λ H fE 0.69 0.68 0.69 0.68 0.70 0.40 0.40 0.40 0.40 0.40 0.13 0.11 0.13 0.12 0.14 0.69 0.01 0.40 0.13 0.01 0.63 0.45 concentration of Ro 673189 ng/mL blood plasma 51.8 300 985 74.5 446 1’430 0.12 1) the hematocrite value was increased to 0.45 for the reversibility study because of the lack of blank plasma 58    Reference ID: 3537650 λ H fE 0.70 0.67 0.69 0.40 0.40 0.40 0.14 0.11 0.13 0.69 0.01 0.40 0.12 0.01 not measured   Reviewer’s Comment: 1. This review only focused on human data although animal data were also included in the study report. 2. Ro 67-3189 was found to be highly protein bound (>99%) in human plasma with 0.33% of mean percentage of free drug. The protein binding in human plasma was concentration independent up to 1300 ng/mL. 3. The blood/plasma concentration ratio (λ) in human was 0.69 and it was independent of the drug concentration up to 1 μg/mL. The fraction of drug in erythrocytes (fE) was about 13% in man. 4. The tested concentration of 9-1300 ng/mL of Ro67-3189 is acceptable as they approximately cover the expected Cmax values in human subjects at the clinical dose (300 mg) where the observed Cmax = 550-880 ng/mL. 59    Reference ID: 3537650   Title: RO0681133, RO0713001 and RO0731519, Metabolites of NK1 Receptor Antagonist RO0673189: In Vitro Plasma Protein Binding and Blood/Plasma Partitioning in Man, Dog and Rat. Report No: 1010388 Specific Aims: To determine the in vitro binding of the major metabolites of RO0673189, namely RO0681133, RO0713001 and RO0731519, to plasma proteins in man, dog and rat, and to assess the partitioning between blood and plasma. Study Date: 10/2001-06/2003 Test site: F. Hoffmann-La Roche Ltd., Basle, Switzerland, Dept. PRNS Sponsor: F. Hoffmann-La Roche, Ltd. Study Design: Test Item: 3 Metabolites of RO067-3189 : 14C-RO0681133 (desmethyl derivative, M1), RO0713001 (N-oxide derivative, M2) and 14C-RO0731519 (OH-methyl derivative, M3) 14 C- Plasma reference: The plasma pools for protein binding studies were obtained from healthy adult volunteers (n=11, male and female). For in vitro blood/plasma partitioning, blood was obtained from a single healthy male Volunteer. Study Method: Binding Study: The protein binding of RO0681133, RO0713001 and RO0731519, three major metabolites of the NK1 receptor antagonist RO0673189 was evaluated by equilibrium dialysis at 37oC and pH 7.4 after addition of 14C-labeled compounds to human plasma. The time required to reach equilibrium was investigated by conducting dialysis for different time period (0.5 h - 5.5 h). pH was measured at the end of dialysis. 600 μL of blank plasma was added when sample were removed from the buffer solution from the dialysis cells to minimize the loss of substance due to non-specific adsorption to the material. The resulting dilution of the buffer samples was determined by weigh. Blood/Plasma Partitioning: Freshly drawn blood was centrifuged to generate a small erythrocyte free plasma layer, and equilibrated at room temperature (21oC) and 37°C. The erythrocyte-free plasma layer were then spiked with aliquots of metabolites where the drug concentrations in blood ranged from 70 to 5900 ng/mL depending on the metabolites and species, and were immediately mixed at desired constant temperature. After 30 min of incubation, aliquots were removed, centrifuged and the drug concentrations were measured in plasma and whole blood by liquid scintillation counting. The hematocrit (H) was determined in the freshly drawn blood using a hematocrit centrifuge and hematocrit reader. The reversibility of the partitioning was measured at the end of the incubation by re-suspending the erythrocytes in fresh blank plasma for 30 min at the same temperature. Bioanalytical Method: Concentrations of 14C-labelled metabolites in buffer (single or duplicate) plasma (duplicate) and whole blood (triplicate) were determined by liquid scintillation counting. The limit of quantification (LOQ) was 10 ng/mL in blood and 0.3 ng/mL in plasma and buffer. 60    Reference ID: 3537650   Data Analysis: Protein Binding: The approximate time for protein binding to achieve equilibrium was calculated by fitting the % free drug versus time (t) data to the equation: %free = %freess·(1-e-kt) where %freess is the percent free at equilibrium, and k is a first order rate constant for the equilibration. The time to achieve equilibrium is taken as 5 times the equilibrium half-life (t1/2 = 0.693/k). The free fraction fu, was calculated as the ratio between the concentration found in the buffer dialysate (CB) and the concentration in the corresponding plasma or protein solution (CPe) dialysate at the end of the dialysis: fu = CB/CPe The free fraction was corrected for osmotic fluid volume shift (which is considered to be relevant when plasma is dialyzed for time periods longer than 4 h) according to the equation given by Boudinot and modified by Lohmann: fu = CB·VPi / [CPe·VPe – CB·(VPe-VPi] where VPi and VPe are the initial and final plasma volume, respectively. Blood/Plasma Partitioning The blood/plasma concentration ratio (λ) was calculated from: λ = CW/CP = (H·CE/CP) + (1-H) where CW, CP and CE are the drug concentration in whole blood, plasma, and erythrocytes, respectively, and H the hematocrit value. The fraction fE of drug in erythrocytes was calculated from: fE = QE/QW = (λ+H-1)/λ where QE is the amount of drug in the erythrocyte compartment and QW the amount of drug in whole blood. Results: Table 1: Comparison of in vitro Plasma Protein Binding in Man of Parent Drug RO0673189 and its Metabolites RO0681133, RO0713001 and RO0731519 free fraction (expressed as percentage) RO0673189 mean MAN 2) 1) 0.33 up to 1'300 ng/mL no RO0681133 0.91 up to 2'500 ng/mL no RO0713001 2.3 up to 2'500 ng/mL no RO0731519 0.88 up to 2'000 ng/mL no 1) Research Report No 1006047 2) Mean value calculated within the linear range of binding 3) max: value obtained at the highest concentration tested no : no influence of the concentration over the whole concentration range tested (b) (4) Table 2: Comparison of in vitro Blood/Plasma Partitioning in Man of Parent Drug RO0673189 and its Metabolites RO0681133, RO0713001 and RO0731519 blood/plasma concentration ratio (λ) RO0673189 1) RO0681133 61    Reference ID: 3537650 RO0713001 RO0731519   mean 2) 1.1 0.69 up to 1'000 ng/mL up to 2'500 ng/mL 0.69 up to 2'500 ng/mL MAN no no (b) (4) 1) Research Report No 1006047 2) Mean value calculated within the linear range of partitioning 3) max: value obtained at the highest concentration tested no : no influence of the concentration over the whole concentration range tested no 0.61 up to 2'300 ng/mL no RO068113: The time required to reach equilibrium in human plasma was determined with 14C-RO0 681133 at a concentration of 2300 ng/mL and found to be about 11 hours. However, dialysis time for subsequent protein binding study was set to 5.5 hr All the values were corrected for fluid volume shift. Determined in human plasma with 14C RO0681133 at pH 7.37 and 2.3 µg/mL The protein binding of 14C-RO0 681133 in human plasma was nearly constant (99.1% binding) over the whole concentration range tested (121-2550 ng/mL). Table 3: 14C-RO0 681133: In vitro binding to human plasma concentration of RO0681133 (ng/mL) buffer plasma 121 375 1'130 2'550 2'540 1.28 4.07 10.2 25.8 26.7 MEAN SD 1) FVS % free 2) % bound 0.93 0.97 0.81 0.90 0.94 2) 99.07 99.03 99.19 99.10 99.06 0.91 99.09 0.06 correction factor pH 1.14 1.12 1.12 1.12 1.12 7.45 nm nm nm 7.44 1.12 0.01 7.45 0.01 4) nm : not measured The mean blood/plasma concentration ratio (λ) in human was 1.1 at 37°C and 21°C. Partitioning was independent of the tested drug concentration (124 - 2460ng/mL). The partitioning was reversible. Table 4: 14C-RO0681133: In vitro blood/plasma concentration ratio (λ) in Man 62    Reference ID: 3537650     at 37°C  at 21°C    λ  concentration of RO0681133 (ng/mL) blood plasma    124 338 1'140 2'460 Distribution  114 312 1'040 2'190  1.09 1.08 1.10 1.12  MEAN SD  Reversibility 4)  1'210 concentration of RO0681133 (ng/mL) blood plasma  λ    336 317   2'240  2'450 1.10 0.02  1'060   MEAN SD  not measured  1.14 1.06   1.09  1.08 0.02  1) The hematocrite value was 0.38 (man) 4) The erythrocytes were resuspended in fresh blank plasma for 30 min at 37 C RO0713001 The time required to reach equilibrium in human plasma with 14C-RO0 713001 at a concentration of 2300 ng/mL was approximately 5 hours. The dialysis time was set to 5.5 hours to determine the plasma protein binding in the various species. All the values were corrected for fluid volume shift. Determined in human plasma with 14C RO0713001 at pH 7.37 and 2.3 µg/mL The protein binding of 14C-RO0713001 in human plasma was nearly constant (97.7% binding) over the whole concentration range tested (115-2500 ng/mL). Table 5: 14C-RO0713001: In vitro binding to human plasma concentration of RO0713001 (ng/mL) plasma buffer 115 347 1'120 2'480 2'500 2.82 8.72 29.8 63.8 70.3 MEAN SD 1) % free 2) % bound 2.2 2.3 2.4 2.3 2.5 2) 97.8 97.7 97.6 97.7 97.5 2.3 97.7 0.1 FVS correction factor 1.12 1.11 1.11 1.12 1.11 1.11 0.01 pH 4) 7.43 nm nm nm 7 44 7.44 0.01 The mean blood/plasma concentration ratio (λ) in human was 0.69 at 37°C and 21°C. Partitioning was 63    Reference ID: 3537650   independent of the tested drug concentration (74-2500 ng/mL). The partitioning was reversible. Table 6: 14C-RO0713001: In vitro blood/plasma concentration ratio (λ) in Man   at 37°C  at 21°C    λ  concentration of RO0713001 (ng/mL) blood plasma    73.8 337 1'190 2'500 Distribution  107 489 1'740 3'630  0.69 0.69 0.68 0.69  561 0.69 < 0.01  0.69 MEAN SD  Reversibility 3)  388 1) The hematocrite value was 0.38 (man), concentration of RO0713001 (ng/mL) blood plasma    λ    338 489   0.69 3'650  2'490 MEAN SD  not measured    0.68  0.69 0.01  3) The erythrocytes were resuspended in fresh blank plasma for 30 min at 37 C RO0731519 The time to reach equilibrium in human plasma with 14C-RO0731519 at a concentration of 2200 ng/mL was approximately 4.5 hours. The dialysis time was set to 5.5 hours to determine the plasma protein binding in the various species. All the values were corrected for fluid volume shift. Determined in human plasma with 14C RO0731519 at pH 7.39 and 2.2 µg/mL The protein binding of 14C-RO0 731519 in human plasma was mostly constant (99.1% binding) over the whole concentration range tested (114-2070 ng/mL). Table 7: 14C-RO0 731519: In vitro binding to human plasma concentration of RO0731519 (ng/mL) buffer  plasma 114 317 309 1'000 2'070 1'980 1.10 3.04 3.28 9.97 21.2 18.0  MEAN SD  1)   FVS % free 2) % bound 0.85 0.87 0.94 0.88 0.95 0.81 99.15 99.13 99.06 99.12 99.05 99.19  0.88 99.12 0.05 64    Reference ID: 3537650 2)   correction factor  1.13 1.10 1.13 1.14 1.08 1.13  1.12 0.02 pH 4)   7.40 7.38 nm 7.39 7.39 nm 7.39 0.01    The mean blood/plasma concentration ratio (λ) in human was about 0.62 and was independent of temperature (37°C, 31oC and 21°C). Partitioning was independent of the tested drug concentration (118-2310 ng/mL) at31oC. The partitioning was reversible. Table 8: 14C-RO0731519: In vitro blood/plasma concentration ratio (λ) in Man at 37°C    at 31°C concentration of RO0731519   (ng/mL) blood plasma λ concentra ion of RO0731519 (ng/mL) blood plasma       Distribution    352 566     Reversibility 4)     118 335 1'120 2'310 0.62     not measured λ concentration of RO0731519   (ng/mL) blood plasma     0.62 MEAN SD at 21°C  2) 196 552 1'830 3'620 MEAN SD 0.60 0.61 0.61 0.64   0.61 0.02   393     624 λ     0.63 336 551   2'480 3'960 0.61       0.63 MEAN SD 0.62 0.01       not measured 1) The hematocrite value was 0 43 (man) 2) The study was conducted at 31°C due to a technical problem affecting the temperature regulation 4) The erythrocytes were resuspended in fresh blank plasma for 30 min at 37°C Reviewer’s Comment: 1. This review only focused on human data although animal (rat and dog) data were also included in the study report. 2. The protein binding was 99.1% for RO0681133, 97.7% for RO0713001 and 99.1% for RO0731519 in human plasma. The protein binding was concentration independent over a concentration range which exceeds the maximum plasma concentrations expected in man. 3. The blood/plasma concentration ratio (λ) in human was 1.1 for RO0681133, 0.69 for RO0713001 and 0.61 for RO0731519. The ratio was independent of the drug concentration range which exceeds the maximum plasma concentrations expected in man. 4. The tested concentration for RO0681133 (121-2540 ng/mL) is acceptable as it covers the expected Cmax in human subject at the clinical dose of 300 mg where the Cmax of RO0681133 was 40-50 ng/ml. 5. The tested concentration for RO0713001 (74-2500 ng/mL) is acceptable as it covers the expected Cmax in human subject at the clinical dose of 300 mg where the Cmax of RO0713001 was 100-350 ng/mL. 6. The tested concentration for RO0731519 (115-2000 ng/mL) is acceptable as it covers the expected Cmax in human subject at the clinical dose of 300 mg where the Cmax of RO0731519 was 50-90 ng/mL.   65    Reference ID: 3537650   Title: In vitro metabolism of the NK1 receptor antagonist RO0673189: I. Kinetic parameters and Metabolites formed in incubations of liver microsomes, recombinant cytochromes and hepatocytes of different species, including man. Report No: 1003832 Specific Aims: The aim of this study was to evaluate the major metabolites formed during the elimination of RO0673189 in rats, dog, marmoset and man Study Date: 08/1998-05/2001 Test Site: F. Hoffmann-La Roche Ltd., Basle, Switzerland, Sponsor: F. Hoffmann-La Roche, Ltd. Study Design: Test Item: [14C]- RO0673189 (MW: 578.6 g/mol) Study Method: The major metabolic steps of RO0673189 have been studied in several in vitro incubation systems including human hepatocytes, liver microsome and microsomes containing recombinant enzymes. Hepatocytes: Hepatocytes from human liver tissue were prepared from hepatic surgical resections. Freshly prepared hepatocytes were seeded in collagen coated six-well plates at the density of 1.5 x106 cells (for human). When cell culture was ready, they were incubated with10 µM of test compounds for 24 hours. Liver Microsomes: Human liver microsomes were prepared from frozen human liver tissue (pooled tissue of 10 human livers, obtained from hepatic surgical resections). 10 µM of test compound was incubated with human liver microsome (100 µg protein/assays) for 20 minutes at 37oC in presence of NADPH. The reaction was terminated by the addition of 500 µl acetonitrile, centrifuged for 10 min at 15’000 g and the supernatant was analyzed by HPLC. Table 1: Characteristics of the microsomal preparations used: Recombinant Enzymes: The enzymes were expressed in E. coli and isolated as a membrane fraction. The radiolabeled test compound RO0673189 at a concentration of 5 μM (1 µM for CYP2C9) was incubated with four of the major human CYP450 isoenzymes (CYP3A4, CYP2C9, 2C19 and 2D6) at 100 to 600 pmol CYP450/ml and the incubations were initiated by the addition of NADPH (1 mM). After incubated for 30 min to 1 hour at 37°C, the reaction was terminated by the addition of 500 µl acetonitrile. After centrifugation for 10 min at 15’000 g and the supernatant was analyzed by HPLC. Table 2: Characteristics of the recombinant human CYP450 enzyme preparations used: 66    Reference ID: 3537650   Code rhCYP3A4 rhCYP2C9 rhCYP2C19 rhCYP2D6 Enzyme system Protein (mg/ml) 5.04 19.16 15.10 15.58 Rec. human CYP450 3A4 Rec. human CYP450 2C9 Rec. human CYP450 2C19 Rec. human CYP450 2D6 P450 (nmol/mg protein) 0.703 0.519 0.520 0.655 Preparation Date* 10.03.2000 25.05.1999 30.10.1998 05.05.1999 * all preparations were performed at Roche Basel at the dates indicated and stored frozen in aliquots at –80°C. Characterization of metabolites: The metabolites of the radiolabeled RO0673189-003 (580.6 g/mol) formed by human liver microsomes were analyzed by LC-MS and compared with the three synthesised metabolites RO0681133, RO0713001, and RO0731519. Bioanalytical Method: Samples were analyzed with HPLC and LC-MS. Results: Hepatocytes: Incubation of 10 µM of RO0673189 in human hepatocytes for 24 hours had resulted two metabolites (M1 and M2). Figure 1: Metabolite profiles obtained by incubation (24 hours) of Ro RO0673189-003 (10 μM) with Human hepatocytes. Liver Microsomes: Incubation of 10 µM of RO0673189 in human liver microsome for 20 minutes had also resulted two metabolites (M1 and M2). Figure 2: Metabolite profiles obtained by incubation of RO0673189 (10 μM) with human (20 min) liver microsomes (100μg protein/ml). 67    Reference ID: 3537650   Recombinant Enzymes: The contribution of different microsomal CYP450 enzymes to the metabolism of RO0673189 was studied by utilizing recombinant human enzymes. Based on the Figure 3, it appears that CYP2C9, 2C19 and 2D6 do not catalyst the formation of any metabolite of RO0673189 while CYP3A4 appears to metabolize RO0673189 to the same metabolites (M1 and M2) that were observed when RO0673189 was incubated with human liver microsomes and hepatocytes. Figure 3: Incubation (30 min - 1 hour) of Ro RO0673189-003 (1 - 5 µM) with recombinant CYP450 from E. coli membranes (100 – 500 pmol CYP450/ml) Kinetic Studies: The overall metabolism of the radiolabeled RO0673189 was studied in human, liver microsomes and membranes containing rhCYP3A4, and the initial velocity of the disappearance of RO0673189 (0.1 to 100 µM) was determined under linear product formation. 68    Reference ID: 3537650   Figure 4: Determination of the enzyme kinetic parameters of the formation of the metabolites of RO0673189 in human liver microsomes (100 μg/ml) For rhCYP3A4 incubations kinetic parameters were determined for the formation of the metabolites separately Figure 5: Determination of the enzyme kinetic parameters of the formation of the two main metabolites of RO0673189 catalyzed by rec. human CYP3A4 expressed in E. coli membranes (20 nmol P450/assay). Best Available Copy 69    Reference ID: 3537650   Characterization of Metabolites: Based on the similarity in retention time in HPLC analysis and results of MS analysis, RO0681133 fitted with one of the major metabolites, the N-demethylation product marked as M1 while RO0713001 fitted with the second major metabolite, a N-oxidation product, marked as M2. The minor metabolite M3 was found to be identical with RO0731519, showing a hydroxylation of the toloyl-methyl group of the molecule. Figure 6: Analysis of minor and major metabolites obtained after incubation (30 min) of Ro RO0673189003 (10 μM) with Human Liver Microsomes (250 μg protein) and comparison with synthetic reference Compounds 70    Reference ID: 3537650   Figure 7: Proposed metabolic pathway of RO0673189 Reviewer’s Comment: 1. This review only focused on the metabolic stability of RO0673189 in human although data for several animal species were also included in this study report. 2. It is not clear if the test systems (hepatocytes, liver microsome) were not properly validated in terms of various CYP enzymes prior to the use. 3. The sponsor did not provide detailed information about the hepatocyte (e.g. how many subjects, pooled vs. individual). 4. The experiment conditions did not include proper controls (both positive and negative) during the incubation. 5. The sponsor did not evaluate the potential of CYP1A2, 2B6 and 2C8 to metabolize RO067318. 6. The results of kinetic studies are difficult to interpret as the experimental conditions (e.g., duration of incubation, kinetic measurement of formation of metabolite vs. disappearance of parent) were not clear in the study report. 7. The concentration of 10 μM in hepatocytes and liver microsome and 1-5 μM in recombinant enzymes are acceptable as they approximately represent the expected Cmax and 10 times Cmax in human subject where the Cmax at the clinical dose was 550-880 ng/mL (≈ 1-.5 μM). 71    Reference ID: 3537650   Title: Netupitant: Reaction Phenotyping with Human Liver Microsomes and Human CYP1A2, CYP2B6 and CYP2C8 cDNA expressed enzymes. Report No: NETU-13-21 Specific Aims: The purpose of this study was to identify in vitro the major drug metabolizing enzymes in human that were responsible for the metabolism of netupitant. Study Date: 07/08/2013 - 07/23/2013 Test Site: (b) (4) Sponsor: Helsinn Healthcare, Swittzerland. Study Design: Test Item: Netupitant (MW: 578.6 g/mol) Test System: Commercially available Human Liver Microsomes (pool 50 donors mix gender) and Human CYP1A2, CYP2B6 and CYP2C8 cDNA expressed enzymes were used. Study Method: Test compound netupitant at the concentration of 10 μM was incubated with Human Liver Microsomes (0.8 mg/ml) in the presence and in the absence of different selective CYP isoforrn inhibitors and with recombinant CYP I A2, 2B6 and 2C8 (20 pmol P450) in Dulbecco's buffer, pH 7.4, for 60 minutes at 37°C in duplicates. Metabolism was started by the addition of NADPH (final concentration 1 mM) after 5 min pre-incubation time in 96 well plates. Aliquots of the incubation mixture were taken at time 0, and after 5, 10, 30 and 60 minutes incubation, the metabolism was stopped by the addition of an equal volume of acetonitrile containing deuterated standards of netupitant and its metabolites Ml, M2 and M3; samples were centrifuged and the supernatant was analyzed by LC-MS/MS to investigate both the disappearance of parent compound Netupitant and the formation of the metabolites Ml, M2 and M3. The inhibitors used for each CYP isoform were: 100 μM Furafylline (for CYPI A2), 100 μM Ticlopidine (CYP2B6), 100 μM Trimetoprim (CYP2C8), 100 μM Sulfaphenazole (CYP2C9), 100 μM Nootkatone (CYP2C 19), 100 μM Quinidine (CYP2D6), l μM Ketoconazole (CYP3A4) and 1000 μM Aminobenzotriazole (generic CYPs inhibitor). Controls: Specific probe substrates for each enzyme were incubated as positive controls to check the metabolic activity of the test systems used (Human Liver Microsomes and cDNA expressed enzymes): Tacrine (CYP1A2), Bupropion (CYP2B6), Paclitaxel (CYP2C8), Diclofenac (CYP2C9), S-Mephenytoin (CYP2Cl 9), Dextromethorphan (CYP2D6) and Midazolam (CYP3A4). Bioanalytical Method: The incubation samples were analyzed by LC-MS/MS to assess the formation of metabolites M l, M2 and M3 from the parent compound Netupitant Calculation: Intrinsic clearance (CLint) was calculated using the half-life approach where the half-life and CLint were determined from the concentration remaining at the different sampling points. By plotting the natural logarithmic (LN) value of the concentration of the compound remaining against the time, the slope was calculated by linear regression analysis and converted into the half-life (T112) and Clint expressed as 72    Reference ID: 3537650   μL/min/mg protein for human liver microsomes or μL/min/pmol P450 for cDNA expressed enzymes: Results: Based on study results of inhibition study in human microsome, it appear the metabolism of netupitant to M1, M2 and M3 is mainly mediated by CYP3A4 and lesser extent by CYY2C9 and CYP2D6. CYP1A2, CYP2B6, CYP2C8 and CYP2C19 do not appear to contribute to the netupitant metabolism. Study in CYPIA2, 2B6 and 2C8 cDNA expressed enzymes further confirms that these enzymes do not contribute to netupitant metabolism. 73    Reference ID: 3537650 Figure Netupitant Metabolites Formation Rate in HLM system. Metabolite: Formation with HLM 0.014 BEE .3 3 0.006 0M3 i 0002 0.000 (3., #9 eddy; waif? go?dod?ey ?fe Table 2 Newpitant metabolite Ml. M2 and M3 formation rate in HLM and expressed systems in the presence and in the absence of CYP: inhibitors. in Formula as I1 I2 Fem-Ion Fonndio u: Forrnalon Rue ?nation Rae we Rate 3 Metabolite Faun-lion Rae Data are expand as 74 Reference ID: 3537650   Reviewer’s Comment: 1. The choices for reference inhibitors were appropriate. 2. The choices of model substrates as positive controls were appropriate. The reported activities of the CYS enzymes (Clint) of these microsomes were within the historical range that was observed. 3. The concentration of 10 μM in liver microsome and in recombinant enzymes are acceptable as they approximately represent the expected Cmax and 10 times Cmax in human subject where the Cmax at the clinical dose was 550-880 ng/mL (≈ 1-1.5 μM). 4. Based on this study result, it appear the metabolism of netupitant to M1, M2 and M3 is mainly mediated by CYP3A4 and lesser extent by CYY2C9 and CYP2D6. 75    Reference ID: 3537650 Title: In Vitro Metabolism Of The Receptor Antagonist R00673189: H. Drug-Drug Interaction Studies with R00673189 and Major Metabolites (R00681133 and ROO713001), Involving Major Human Cytochrome P450 Isoenzymes Report No: 1003907 Speci?c Aims: To evaluate the in vitro inhibition potential of R00673189 for the major human cytochrome P450 isoenzymes CYP1A2. 2C9. 2C19. 2D6 and 3A4 utilizing human liver microsomes and isoform selective substrates. Study Date: 08/1998-03/2002 Test Site: F. Hoffmann-La Roche Ltd. Basel Switzerland Sponsor: Hof?narm?La Roche. Study Design: Test Item: R00673189 (netupitant) MW 578.6 g/mol) ROO681133 (M1): 567 g/mol ROO713001 (M2): 597 g/mol Test Concentration: R00673189 (netupitant): 0. 0.5. l. 10 and 100 ROO681133 (M1) and ROO713001 (M2): 0-30 nM Liver microsome: Microsomes were prepared from frozen htunan liver tissue. pooled tissue of 10 htunan livers. obtained from hepatic surgical resections. Study Method: Human liver microsomal protein was incubated with R00673189 (0. 0.5. 1. 10. and 1003M) and the corresponding selective model substrates at in the presence of NADPH generating system for speci?ed duration of incubation time. No pre-incubation was carried out. The enzymatic reactions were terminated by addition of methanol or acetonitrile. The inhibition potential of metabolites of R00673189. namely R00681133 and ROO713001 were evaluated for CYP3A4 enzyme only. The human liver microsome (bought from pool of 10 livers) was incubated with model substrate 20 uM testosterone and test compormds ROO681133 (M1) and ROO713001 (M2) at 0-30 nM for 20 minutes in presence of NADPH at 370C. The enzymatic reaction was temiination of addition of methanol. Table 1 Experimental Conditions to evaluate the inhibitmy potential of IT isoforms CYP m?del Metabolite HLM Protein Incubation substrate . Isoforms . substrate Amount TLme oncentrations . . CYP1A2 23:11:: l-hydroxy tacrme 0.5 12 mm 76 Reference ID: 3537650   CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4 CYP3A4 CYP3A4 diclofenac 2-50 µM S-mephenytoin 32.8 µM bufuralol 40 µM midazolam 2-50 µM testosterone 5- 20 µM nifedipine 20 µM simvastatin 3 µM 4-hydroxy diclofenac 0.1 mg/ml 5 min 4-hydroxymephenytoin 1 mg/ml 30 min 1- hydroxy bufuralol 1 mg/ml 30 min 1 hydroxymidazolam 0.1 mg/ml 10 min 6-β-hydroxytestosterone 0.075 mg/ml 20 min Oxidized nifedipine 0.2 mg/ml 10 min 0.02 mg/ml 5 min Bioanalytical Method: HPLC method. Data analysis: Not provided. Results: RO0673189, at concentration 0-100 µM, did not inhibit enzymes CYP1A2, 2C19 and 2D6 (IC50 >100 µM as demonstrated in Figure 1, 4 and 5, respectively. RO0673189 had shown some inhibition toward CYP2C9 with approximate IC50 value of 22.6 µM (Figure 2). Further studies with different concentration of model substrate had shown that RO0673189 inhibition of CYP2C9 is through competitive inhibition with Ki value of 25 µM as shown in the Dixon Plot (Figure 3). The inhibitory potential of RO0673189 for the CYP3A4 enzyme was evaluated with four different model CYP3A4 substrates, testosterone, midazolam, nifedipine and simvastatin. All of the model CYP3A4 substrate had demonstrated that RO0673189 is an inhibitor of CYP3A4 with IC50 value of 1.7-12 µM (Figure 6, 7, 10 and 11). Further studies with different concentrations of testosterone and midazolam had demonstrated that the CYP3A4 inhibition is a competitive inhibition as shown in Dixon plots with Ki value of 1.1 µM with testosterone (Figure 8) and 2.2 µM with Midazolam (Figure 9). The inhibition potential of RO0673189 metabolites, namely RO0681133 (M1) and RO0713001 (M2) were evaluated for CYP3A4 enzyme only with testosterone as the model substrate. RO0681133 (M1) appears to be an inhibitor of CYP3A4 with IC50 value of 1.2 µM (Figure 12). Due to solubility issue, RO0713001 (M2) was tested up to 1 µM, and notable inhibition was observed even at 1 µM (Figure 13). Table 2: Inhibition of CYP Enzyme by RO0673189 and its metabolites CYP450 isoenzme CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4 CYP3A4 CYP3A4 Substrate used Tacrine Diclofenac Mephenytoin Bufuralol Midazolam Testosterone Nifedipine Simvastatin Substrate conc (uM) 25 5 32.8 40 5 20 20 3 HLM conc. (mg/ml) 0.5 0.1 1 1 0.1 0.075 0.2 0.02 >>100 22.6 ± 3 >100 >>100 5.9 ± 1.0 1.7 ± 0.2 12.0 ± 0.5 10.5 ± 0.8 IC50 (uM) RO0673189 18.0 ± 6 RO0681133 (M1) RO0713001 (M2) 1.2 ± 0.5 > 1 µM 77    Reference ID: 3537650   Table 3: Inhibition of CYP450 metabolism; apparent Ki (µM) of netupitant CYP450 isoenzme CYP2C9 CYP3A4 CYP3A4 Substrate used Diclofenac Testosterone Midazolam Substrate conc.(uM) 2, 5, 10, 50 5, 10, 20 2, 5, 10, 50 HLM protein conc.(mg/ml) 0.1 0.075 1 Inhibitor conc. (uM) 0, 0.1, 0.5, 1, 5, 10 0, 0.2, 0.5, 1 0, 0.1, 0.5, 1, 5, 10 apparent Ki (uM) 25.0 ± 7.4 1.1 ± 0.2 2.2 ± 0.6 Inhibition mechanism competitive competitive competitive Figure 1: Inhibition potential of RO0673189 on tacrine hydroxylation, a reaction specific for CYP1A2. Tacrine (25 μM) was incubated with human liver microsomes (500 μg protein /ml) for 20 minutes with the inhibitor (n = 2). Figure 2: Inhibition potential of RO0673189 on diclofenac 4’- hydroxylation, a reaction specific for CYP2C9 Diclofenac (5 μM) was incubated with human liver microsomes (100 μg/ml) for 5 minutes with the inhibitor (n = 2). Results of different experiments. Figure 3: Interaction of RO0673189 with cytochrome P450 2C9, measured by the isoenzyme-specific 78    Reference ID: 3537650   hydroxylation of diclofenac in human liver microsomes. Dixon plot and nonlinear fitting. Figure 4: Inhibition potential of RO0673189 on S-mephenytoin hydroxylation, a reaction specific for CYP2C19. S-mephenytoin (32.8 μM) was incubated with human liver microsomes (1 mg/ml) for 30 minutes with the inhibitors indicated (n = 2). Figure 5: Inhibition potential of RO0673189 on bufuralol 1’- hydroxylation, a reaction specific for CYP2D6. Bufuralol (40 μM) was incubated with human liver microsomes (1 mg/ml) for 30 minutes with the inhibitor (n = 2). Figure 6: Inhibition potential of RO0673189 on CYP3A4, measured by testosterone 6-beta hydroxylation. 79    Reference ID: 3537650   Testosterone (20 μM) was incubated with human liver microsomes (75 μg/ml) for 20 minutes with the inhibitor indicated (n = 2). Figure 7: Inhibition potential of RO0673189 on CYP3A4, measured by midazolam 1’- hydroxylation. Midazolam (5 μM) was incubated with human liver microsomes (100 μg/ml) for 10 minutes with the inhibitor (n = 2). Figure 8: Interaction of RO0673189 with cytochrome P450 3A4, measured by the isoenzyme-specific 6βhydroxylation of testosterone in human liver microsomes. Dixon plot and non-linear fitting Figure 9: Interaction of RO0673189 with cytochrome P450 3A4, measured by the isoenzyme-specific 1hydroxylation of midazolam in human liver microsomes. Dixon plot and nonlinear fitting. 80    Reference ID: 3537650   Figure 10: Inhibition potential of RO0673189 on nifedipine oxidation, a reaction specific for CYP3A4. Nifedipine (20 μM) was incubated with human liver microsomes (0.2 mg/ml) for 10 min. with the inhibitor. Figure 11: Inhibition potential of RO0673189 on CYP3A4, measured by metabolization of simvastatin. Simvastatin (3 μM) was incubated with human liver microsomes (20 μg/ml) for 5 min. with the inhibitor. Figure 12: Inhibition potential of RO0681133 on CYP3A4, measured by testosterone 6-beta hydroxylation. Testosterone (20 μM) was incubated with human liver microsomes (50 μg/ml) for 20 minutes with the inhibitor indicated (n = 2). Figure 13: Inhibition potential of RO0713001 on CYP3A4, measured by testosterone 6-beta hydroxylation. 81    Reference ID: 3537650   Testosterone (20 μM) was incubated with human liver microsomes (50 μg/ml) for 20 minutes with the inhibitor indicated (n = 2). Reviewer’s Comment: 1. The study did not include positive control (known inhibitors) to evaluate the validity of the test system (human liver microsome) regarding CYP enzymes (optional). Nonetheless, the activity of CYP enzymes toward model substrates in absence of netupitant as inhibitor was within the historical data observed. 2. The concentration of RO0673189 at 0- 100 µM are acceptable as they approximately cover the Cmax and 10 times Cmax values to be expected in human subjects or patients taking this drug at the clinical dose of 300 mg. a. Observed Cmax = 550-880 ng/mL (≈ 1-1.5 µM) 3. Concentration of metabolites (M1 and M2) at 0-30 µM are acceptable as they approximately cover the Cmax and 10 times Cmax values for the corresponding metabolites to be expected in human subjects or patients taking this drug at the clinical dose of 300 mg: a. Observed Cmax for M1 (RO0681133) is 40-50 ng/ml (0.07-0.09 µM) b. Observed Cmax for M2 (RO0713001) is 100-350 ng/mL (0.17-0.58 µM). 4. The choices of CYP-specific model substrates and their concentrations to evaluate the inhibitory potential on each CYP isoforms were acceptable as covering the range around each Km value. 5. RO0673189 is not considered to be an inhibitor of CYP1A2, CYP2C19, and CYP2D6, as it did not produce a significant inhibitory effect on these CYP enzymes. 6. RO0673189 is shown to be an inhibitor of CYP2C9 with Ki of 25 µM. Since [I]/Ki= 1.5 µM /25 µM = 0.06<0.1, clinical in - vivo interaction with CYP2C9 is less likely. 7. RO0673189 is shown to be an inhibitor of CYP3A4 with Ki of 1.1-2.2 µM, and a follow-up in-vivo evaluation is recommended for the following reasons: a. Systemic exposure: Cmax/Ki = 1.5 µM /1.1 µM = 1.4>1 b. Gut exposure: [I]gut/Ki= 2074 µM / 1.1 µM = 1885>>>10 where [I]gut = dose/250 ml = 300 mg/250ml= 1.2 g/L. 8. The sponsor did not evaluate time-dependent inhibition potential with pre-incubation. 9. The sponsor did not evaluate the inhibition potential of RO0673189 for CYP2B6 and CYP2C8. 10. The sponsor only evaluated the inhibition potential of metabolites (M1 and M2) for CYP3A4, not other enzymes. 82    Reference ID: 3537650   Title: Netupitant, M1, M2, and M3: Determination of the potential inhibition (IC50) of CYP1A2, CYP3A4, CYP286, CYP2C8, CYP2C9, CYP2C19, CYP2D6 Report No: NETU-13-20 Specific Aims: To determine the potential inhibitory effect of Netupitant towards CYP2C8 and CYP286 and of its three major metabolites Ml, M2, M3 towards the major human liver CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2Cl 9, CYP2D6, and CYP3A4), using human liver microsomes. Study Date: 07/2013 Test Site: (b) (4) Sponsor: Hoffmann-La Roche, Ltd Study Design: Test Item: Netupitant and its metabolites M1, M2 and M3 Test Concentration: 0.3, l, 3, 10, 30 and 100 μM Liver microsome: Pooled human liver microsomes (from 50 individuals) used in this study were purchased from (b) (4) In Vitro Technologies. The microsomes were characterized by the supplier in respect to its CYP enzyme activities. Study Method: Human liver microsomal protein was incubated with test compound and the corresponding selective model substrates at 37oC in the presence of NADPH generating system for specified duration of incubation time. No pre-incubation was carried out. The enzymatic reactions were terminated by addition of ice-cold acetonitrile. For M1, M2 and M3 inhibition was evaluated towards the following CYP450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2Cl9, CYP2D6, and CYP3A4 (two substrates) using six test concentrations (e.g. 0.3, 1, 3, 10, 30 and 100 μM). For Netupitant inhibition was evaluated towards CYP2B6 and CYP2C8 using six relevant test concentrations (e.g. 0.3, 1, 3, 10, 30 and 100 μM). The quantity of CYP specific substrate metabolites produced during the incubation with and without the compound was evaluated in triplicate. In addition positive controls were included into each experiment. All incubations were performed under linear conditions with respect to time, protein concentration and amount of product formed. 83    Reference ID: 3537650 Table 1: Experimental Conditions to evaluate the inhibitorv potential of YP isoforms CYP CYP model substrate HLM Incubation . . . Protein . Posrtive Control Isoforms Concentrations Time Amount Tacrine . a-naphtho?avone CYP1A2 61M 0.1 mg/ml 10 mm 0.01. 0.1. 0.3 uM Bupropion . Ticlopidine CYP2B6 43 11M 0.1 mg/mL 20 nun 0'1. 1? 3 uM Paclitaxel . Quercetine CYP2C8 14 uM 0.1 mg/mL 20 nmi 0.6. 6. 18 uM Diclofenac . Sulfapheuazole CYP2C9 71M 0.1mg/ml 10 min 0.1 .1. 3t1M S-mephenytoin . Ticlopidine CYP2C19 71 0.2 mg/ml 40 nun 0.1 .1. 31M Dextromethorphan . Quinidine CYP2D6 5 uM 0.1mg/ml 10 nmi 0.01. 0.1. 0.3 uM midazolam . Ketoconazole CYP3A4 2.1 0.1 mg/ml 10 min 0.1. 031M testosterone . Ketoconazole CYP3A4 43 pM 0.1 mg/ml 20 nun 0.01. 0?1. 03 PM Bioanal?ical Method: Data analysis: ICSO were calculated with a non-linear regression (sigmoidal dose-response curve): =Bottom 1+ 10V where is the Logaritlmt of concentration and the of activity remaining. The percentage of CYP mediated enzyme activity remaining in the presence of a certain compound is given by: metabolized in presence of compound I00 activity remaining metabolized in absence of compound (control) For the prediction of likelihood of an in vivo inhibition the ratio of estimated intrinsic clearance values in absence and presence of an inhibitor (R) was calculated as follow: 1+ [1]/Ki Where is the Cmax value measured in plasma after single administration in human at the therapeutic dose of 300 mg of netupitant Results: Nutpitant showed weak inhibition toward both CYP2B6 and CYP2C8 with IC 50 values of 33.39 tiM and 50.4 uM. respectively. However. since Cmax/Ki are in vivo relevance of this interaction is unlikely. M1 showed inhibition toward CY 2B6. 2C8, 2D6. 3A4. and weak inhibition toward CYP 1A2. 2C9. 2C19. However. since Cmax/Ki >0.1 for only CYP3A4. an in vivo study is recommended 84 Reference ID: 3537650   for CYP3A4. The study had already conducted in vivo DDI study with netupitant concomitantly administered with CYP3A4 substrate midazolam.  M2 and M3 showed weak inhibition toward all evaluated CYP enzymes. Since Cmax/Ki<0.1, no in vivo follow up study is needed. 85    Reference ID: 3537650 M2 batch "35" 95200.1. R1 Ki lfKi (14M) CY Pl A2 TncrincBupmpion hydroxylalion 23.72 16.03 to 35.10 0.3 11.36 0.03 1.03 CY Paclilaxcl 100 NA NA NA NA NA CY P2C9 Diciofcnac NA NA NA NA NA CYP2CI9 4'-hydroxylalion 57.45 41.00 to 00.34 0.0 23.73 0.01 1.01 CYPIM 53.12 41.10 to 32.13 0.3 29.00 0.01 1.01 Midnzulam 33.114 32.20 10 40.05 0.9 19.42 0.02 1.02 nudazolam CY P301914 testosteron Tcsloslcrom 30.04 25.49 to 59.73 0.9 19.52 0.02 1.02 2 M3 batch l4-NETU.i22f62fl 95%(31. R2 Ki ma 111M) CY Pl A2 Tacrinc- -h}'dr0xylali0n NA NA NA NA NA Bupmpion hydruxylmicm 23.02 15.94 to 35.01 0.3 11.31 0.01 1.01 Paclilnxet ?-hydroxylmion 20.1.15 21.0? to 34.43 0.9 13.43 0.01 1.01 CY P2139 Diclurcnac 41-hydroxylaliun 7?.03 53.66 to 1 10.5 0.3 30.52 0.004 1 .00 74.0? 51.03 to 103.3 0.9 37.49 0.004 1.00 (3y Midazolam 10.95 3.30 to 13.63 0.9 5.43 0.03 1.03 madamlam CY PJA4 testeste run b?-hydroxylalion ?-45 5-22 *0 14-35 0-5 0'03 1'03 86 Reference ID: 3537650   Reviewer’s Comment: 1. The concentration of 0.3- 100 μM are acceptable as they approximately cover the Cmax and 10 times Cmax values of netupitant and its metabolites to be expected in human subjects or patients taking this drug at the clinical dose of 300 mg. a. Observed Cmax for netupitant = 550-880 ng/mL (≈ 1-1.5 μM) b. Observed Cmax for M1 is 40-50 ng/ml (0.07-0.09 μM) c. Observed Cmax for M2 is 100-350 ng/mL (0.17-0.58 μM). d. Observed Cmax for M3 is 50-90 ng/mL (0.08-0.144 μM) 2. The choices of CYP-specific model and their concentrations to evaluate the inhibitory potential on each CYP isoforms were acceptable as covering the range around each Km value. 3. Choices of model inhibitors as positive controls were acceptable. 4. The sponsor did not evaluate time-dependent inhibition potential with pre-incubation. 5. Netupitant showed weak inhibition toward both CYP2B6 and CYP2C8 with IC50 values of 33.39 μM and 50.4 μM, respectively. However, since Cmax/Ki are <0.1, no follow-up in vivo study is recommended. 6. M1 showed inhibition toward CYP 2B6, 2C8, 2D6, 3A4, and weak inhibition toward CYP 1A2, 2C9, 2C19. However, since Cmax/Ki >0.1 for only CYP3A4, an in vivo study is recommended for CYP3A4. The sponsor had already conducted in vivo DDI study with netupitant concomitantly administered with CYP3A4 substrate midazolam. 7. M2 and M3 showed weak inhibition toward all evaluated CYP enzymes. Since Cmax/Ki<0.1, no in vivo follow up study is needed. 87    Reference ID: 3537650   IC50 (µM) Ki CYP2B6 CYP2C8 32.39 50.43 16.2 25.22 1 1 1.5 1.5 0.062 0.040 0.093 0.059 CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 19.7 2.44 2.37 13.21 16.63 4.27 0.25 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.004 0.029 0.030 0.005 0.004 0.016 0.280 0.005 0.037 0.038 0.007 0.005 0.021 0.360 CYP3A4 39.39 4.89 4.7 26.4 33.26 8.54 0.51 0.7 0.35 0.07 0.09 0.200 0.257 M2 CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4 >100 23.72 >100 >100 57.45 58.12 38.84 39.04 NA 11.86 NA NA 28.73 29.06 19.42 19.52 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 NA 0.014 NA NA 0.006 0.006 0.009 0.009 NA 0.049 NA NA 0.020 0.020 0.030 0.030 M3 CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A4 >100 23.62 26.95 >100 77.03 74.97 10.95 9.45 NA 11.81 13.48 NA 38.52 37.49 5.48 4.72 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.144 0.144 0.144 0.144 0.144 0.144 0.144 0.144 NA 0.007 0.006 NA 0.002 0.002 0.015 0.017 NA 0.012 0.011 NA 0.004 0.004 0.026 0.031 Netupitant M1 Cmax Range (µM) 88    Reference ID: 3537650 Cmax/Ki Range   Title: In Vitro Evaluation of the Possible Induction Of CYP1A2, CYP 2C9, CYP 2C19 and CYP 3A4 By Netupitant, M1, M2 And M3 In Long-Term Monolayer Cultures Of Freshly Isolated Human Hepatocytes Report No: NETU-10-27 Specific Aims: The objective of this study was to determine the possible in vitro induction of the cytochrome P450 (CYP450) enzymes 1A2, 2C9, 2C19 and 3A4 by Netupitant and its metabolites M1, M2 and M3 in human hepatocytes. Study Date: 05/2010-08-2010 Test Site (b) (4) Sponsor: Helsinn Healthcare SA, Switzerland Study Design: Test Item: Netupitant (MW = 578.6 g/mol) and its metabolites M1, M2 and M3 Tested Concentrations: Netupitant: 0.2, 2 and 20 μM, M1, M2 and M3: 0.02, 0.2 and 2 μM Hepatocytes Preparation: Long-term monolayer cultures of freshly isolated human hepatocytes from 3 different donors were used (b) (4) for each enzyme evaluation. The hepatocytes were plated by the supplier in 24 well plates coated with collagen I at a density of 0.38 million hepatocytes per well. Human hepatocytes from six individuals were used.  First donor (for 1A2 and 3A4): male, 61 years, HEP220460  Second donor (for 1A2 and 3A4): male, 68 years, HEP220470  Third donor (for 1A2 and 3A4): female, 75 years, HEP220474    First donor (for 2C9+2C19): male, 66 years, HEP220473 Second donor (for 2C9 and 2C19): male, 70 years, HEP220477 Third donor (for 2C9 and 2C19): male, 62 years, HEP220486 The hepatocytes were characterized by the supplier in respect to various phase I (CYP1A2, CYP3A4/5, CYP2B6, CYP2D6, CYP2C19) and phase II (glucuronidation and sulfation) enzyme activities. In all six hepatocytes donor preparations used in the study, evaluated enzyme activities were within the historical range. Validation of Test System: The in vitro CYP induction study with human hepatocytes was considered acceptable if the following criteria were met: 89    Reference ID: 3537650    Phenacetin (substrate for CYP1A2), tolbutamide (substrate for CYP2C9), S-mephenytoin (substrate for CYP2C19) and midazolam (substrate for CYP3A4) are metabolised in the vehicle control incubations for not more than 30%. The positive control inducers should result in a >2-fold increase in enzyme activity of standard substrates as compared to the vehicle control (based on metabolite peak area).  Controls:  Known inducers, omeprazole for CYP1A1 and rifampicin for CYP2C9, CYP2C19 and CYP 3A4/5, were included as positive controls:  The study also had a vehicle control that did not contain any substrate and another untreated control as the negative control. Study Method: Hepatocytes from three different donors were incubated with 0.2, 2 and 20 μM Netupitant or 0.02, 0.2 and 2 μM M1, M2 and M3 or positive control inducers omeprazole or rifampicin for 72 hours at 37oC. The exposure medium was refreshed every 24 hours. Incubations were carried out in duplicate. In addition, two wells were left untreated to determine the basal CYP1A2, CYP2AC9, CYP2C19 and CYP3A4 activity of the hepatocytes. At the end of the 72 hours of incubation period, the activity of target enzyme CYP1A2, CYP2C9, CYP2C19 and CYP3A4 was assessed by incubating the hepatocyte with model substrate for each target enzyme and measuring the appearance rate of their respective metabolites. Table 1: Overview of substrates and inducers CYP isoenzyme 1A2 Species Human Phenacetin (60 µM) acetaminophen Known Inducer (positive control) Omeprazole (50 µM) 2C9 Human Tolbutamide (30 µM) 4-hydroxytolbutamide Rifampicin (30 µM) 2C19 Human S-mephenytoin (50 µM) 4’-hydroxymephenytoin Rifampicin (10 µM) 3A4 Human Midazolam (4 µM) 1’-hydroxymidazolam Rifampicin (50 µM) Model Substrate Metabolite Bioanalytical Method: The disappearance of model substrate and appearance of metabolites were determined with LC-PDA-MS method. Data Analysis: Enzyme induction of the positive control was calculated using the following equation Mean.Peak.area( MPA) positve control Fold.Induction  Mean.Peak. Area( MPA)vehicle The induction of the test substance was calculated as percentage of positive control using the following equation: %Positive  Control  MPA(test  sample)  MPA(negative control)  100% MPA( positive  control)  MPA(negative  control) in which MPA represents the mean peak area of the metabolite in the corresponding incubation conditions. A test substance is considered an inducer, if:  It produces a change in enzyme activity that is equal to or greater than 40% of the positive control  The induction is reproducible in hepatocytes from different donors 90    Reference ID: 3537650   Results: Netupitant, M1, M2 and M3 did not induce CYP1A2, CY2C9, CYP2C19 and CY3A4/5 enzyme activities when hepatocytes from three different human donors were treated with Netupitant up to 20 uM and M1, M2 and M3 up to 2 uM concentration after 72 hours of incubation based on induction threshold of 40% of the positive control. The test conditions were appropriate for measuring the target enzyme CYP1A2, CYP2C9, CYP2C19 and CYP3A4 activities as there were more than 2-fold induction in presence of positive controls (known inducer) and model substrate for each target enzyme were not metabolized in the vehicle control incubation for more than 30%. In the hepatocytes treated with Netupitant at a concentration of 20 μM, the cells were detached after the exposure period in one patch or no substrate metabolite was formed in another batch indicating is that Netupitant was likely cytotoxic at 20 μM. Table 2: CYP induction for the 3 donors after substrate incubation 91    Reference ID: 3537650 CYP 2C19 induction $est Fold Induction? Percentage of control Inducer Substance Concentration (Compared to vehlcle control) (uM) Donor Donor Donor Induction Donor Donor Donor Induction 1 2 3 1 2 3 Vehicle controlRifampicin1 10 5.6 5.9 7.5 Yes 100 100 100 Yes NetupitantNetupitantZ 2 2.3 2.7 2.3 Yes 28.4 34.5 19.8 No NetupitantM11 0.293 18.2 20.3 No Vehicle control1 1.1 1.1 0.8 0 0 0 RifampicinI 10 4.7 5.7 6.9 Yes 100 100 100 Yes M22 0.2.3 2.8 1.9 Yes 34.0 37.7 16.0 N0 M32 0.CYP 3A4 induction Test Fold lnductlonU Percentage of control Inducer Substance Concentration (Compared to vehlcle control) (uM) Donor Donor Donor Induction Donor Donor Donor Induction 1 2 3 1 2 3 Vehicle controlRifampicin1 50 4.5 2.2 2.8 Yes 100 100 100 Yes Netupitant2 0Netupitant2 Netupitant2 M11 0.Vehicle control? 1.6 1.0 1.1 0 0 Rifampicin1 50 4.2 2.1 2.4 Yes 100 100 100 Yes M22 0.02 0.9 1.1 1.0 No 0 13M32 0.02 1.2 1.2 0.9 No 6.6 19?Vehicle and inducer controls are the average of duplicate incubations per donor. 2Test substance incubations are the average of triplicate incubations per donor. Fold induction is the average of the metabolite peak area of the test substance incubation divided by the metabolite peak area of the vehicle control incubation. except for the fold induction of vehicle control which is the average of the metabolite peak area of the test substance incubation divided by the metabolite peak area of the medium control incubation. Reviewer ?s Comment: 1. dose of 300 mg. a. Observed max 550-880 ng/mL (2 ,uM) the clinical dose of 300 mg: (1. Observed Cmax for M1 is 40-50 ng/ml (0.07-0.09 pM) b. Observed max for M2 is 100-35071g/mL (0.1 7-0. 58 yM). c. Observed max for M3 is 50-90 ng/mL (0. 08-0144 pM). 3. The choice for positive controls (model inducers) are acceptable Reference ID: 3537650 92 The concentration of Netupitant at 0.2- 20 ,uM are acceptable as they approximately cover the Cm, and 10 times max values to be expected in human subjects or patients taking this drug at the clinical Concentration of metabolites (M1, M2 and M3) at 0.02-2 yM are acceptable as they approximately cover the me values of metabolites to be expected in human subjects or patients taking this drug at   4. The choices of CYP-specific model substrate to evaluate the CYP enzyme activities were acceptable. 5. Netupitant up to 20 µM and M1, M2 and M3 up to 2 µM are not considered to be inducers of CYP1A2, CY2C9, CYP2C19 and CY3A4/5 enzyme as it did not produce a change that is equal to or greater than 40% of the positive control. 6. The sponsor did not evaluate the potential of Netupitant and its metabolites M1, M2 and M3 to be an inducer of CYP2B6. APPEARS THIS WAY ON ORIGINAL 93    Reference ID: 3537650   Title: Interaction Studies of Netupitant with Human Pgp / MDR1 (ABCB1) Report No: NETU-06-13 Specific Aims: To evaluate the interaction of Netupitant with the ABC efflux transporter: human MDR1 (Pgp/ABCB1). Study Date: 11/2006 Test Site: (b) (4) Sponsor: Helsinn Healthcare SA, Switzerland Test Item: Netupitant: (MW = 578.6 g/mol) The sponsor evaluated the interaction of netupitant with P-gp transporter in 3 different assay methods, ATPase assay, Calcein Assay and bidirectional transporter assay on monolayer. Since Calcein assay did not provide any additional new information compared to bidirectional transport assay, it was not reviewed in detail. ATPase Assays Activation and inhibition Assay: Assay system: Membrane vesicles isolated from Sf9 insect cells overexpressing human MDR1 transporter Effect of Netupitant on MDR1-ATPase activation was measured in the presence of increasing concentrations of Netupitant (0.14, 0.41, 1.23, 3.70, 11.11, 33.33, 100 and 300 µM). Each concentration was tested in duplicate. Inhibitory effect of the netupitant on verapamil (40 µM) or digoxin (100 µM)-induced MDR1-ATPase activity was measured in the presence of the activator (varapamil or digoxin) and increasing concentrations of the netupitant. In the ATPase assay the amount of phosphate generated from the cleavage of ATP by the transporter is measured. If a test compound is a substrate of the given transporter, it will dosedependently increase the amount of phosphate generated in the system. If the activation type assay shows stimulation of ATPase activity with increasing drug concentration, then the test drug is likely to be a transported substrate. Inhibition type ATPase assay can reveal the interaction with the transporter, without distinguishing substrate and inhibitor. Figure 1. Activation and inhibition of MDR1 transporter by Netupitant measured in the ATPase assay (verapamil induced) 94    Reference ID: 3537650   Table 1. Activation and inhibition of MDR1 transporter measured in the ATPase assay Activation assay EC50 [µM] 0.37 Inhibition assay Maximal Efficacy IC50 [µM] Maximal Efficacy 29 % 7.2 100 % In the activation assay of standard MDR1 ATPase assay, netupitant shows 29 % maximal activation compared to verapamil (100%). In the standard inhibition assay, netupitant shows 100% inhibition of verapamil induced ATPase activity. Figure 2. Activation and inhibition of MDR1 transporter by Netupitant measured in the nonstandard (DDI model) ATPase assay Table 2. Activation and inhibition of MDR1 transporter measured in the ATPase assay Activation assay Inhibition assay EC50 [μM] 0.18 Maximal Efficacy 61 % IC50 [μM] 6.8 Maximal Efficacy 100 % In this, non-standard (DDI) activation assay Netupitant shows 61 % maximal activation compared to digoxin (100%) in MDR1 ATPase assay. In the inhibition assay, netupitant shows 100% inhibition of 95    Reference ID: 3537650   digoxin induced ATPase activity. Figure 3. Activation and inhibition of def MRP(negative control) transporter by Netupitant measured in the ATPase assay In this assay, netupitant shows no transporter specific interaction on control membrane to validate the test system. Reviewer’s Comment: Based on the ATPase activation assay, netupitant may be a substrate of P-gp. However, further studies are needed for confirmation. Caco-2 Monolayer: Bidirectional (A-B and B-A) permeability of 3H-digixin was evaluated in the presence of increasing concentration of netupitant (0.2, 1 5 µM) on Caco-2 cell line (on 24-well plate) at 37oC after 2 hours of incubation in duplicate. The paracellular permeability of the monolayer was assessed using 14C-mannitol (Papp(A/B) = 2.13x10-6 cm/s). 60 µM Verapamil (known P-gp inhibitor) was included as the positive control. Figure 4. Apparent permeability (Papp) of 3H-digoxin in the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) direction in the presence of different concentrations of Netupitant Table -3: Apparent permeability (Papp) of 3 H-digoxin in the apical-to-basolateral (A-B) and 96    Reference ID: 3537650 basolateral-to-apical (B-A) direction in the presence of different concentrations of Netupitant Apical to Basolateral Basolateral to Apical Ef?ux Ratio Papp (104S cm/sec) Papp (1045 cm/sec) Control 0.87 25.32 29 60 uM Verapamil 2.98 4.07 1.4 Netupitant (0.2 11M) 1.25 29.73 23.8 Netupitant (1 11M) 1.07 26.23 24.5 Netupitant (5 11M) 2.8 13.24 4.7 Reviewer ?s Comment: 1. The sponsor only evaluated potential of netupitant being an inhibitor of P-gp in Caco-2 cell monolayer in this study. The sponsor did not evaluate the potential of netupitant being a substrate of P-gp transporter in aco-2 cell monolayer (bidirectional transport assay) with net ?ux ratio information or evaluate the permeability of netupitant in the presence of potent P-gp inhibitor to predict the in vivo relevance of this interaction. 2. The study system appears to be reasonable as the mannitol permeability was within the erpected range, and the study had appropriate model substrate (digoxin) and appropriate positive control (Verapamil). 3. The concentration of netupitant at 0.2, 1, 5 are acceptable as they approximately cover the max value expected in human subjects or patients taking this drug at the clinical dose of 300 mg. Observed Cmax 550-880 ng/mL (2 ,uM) 4. Based on the result of this study, netupitant seems to inhibit P-gp at concentration dependent manner. However, IC 50 value was not determined in this study. Netupitant?s potential inhibition interaction with P-gp in vivo at clinical dose cannot be ruled out. The sponsor did conduct a follow up in-vivo drug-drug interaction study with digoxin concomitantly administered with netupitant. 5. Netupitant?s potential to induce P-gp transporter does not need to be evaluated since it has already been shown that netupitant and its metabolites do not induce CYP3A4 in in-vitro study NE U-I 0-2 7. 97 Reference ID: 3537650 Title: In vitro Interaction Studies of Netupitant and its three metabolites (M1, M2 and M3) with human BCRP, BSEP, MRP2 and MDR1 Efflux Transporters and with human OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 Uptake Transporters Report No: NETU-12-81 Specific Aims The purpose of this study was to provide data on the interaction of Netupitant, M1, M2 and M3 with the human ABC (efflux) transporters: BCRP (ABCG2/MXR), BSEP (ABCB11/sPgp), MRP2 (ABCC2) and MDR1 (ABCB1/P-gp) and the human uptake transporters: OATP1B1 (OATP2, OATP-C), OATP1B3 (OATP8), OAT1, OAT3, OCT1 and OCT2. Study Date: 01/2013-06/2013 (b) (4) Test Site: Sponsor: Helsinn Healthcare SA, Switzerland Test Item: Netupitant and its metabolites (M1, M2, and M3) Study Method: 1. Vesicular Transport Inhibition Assays for Efflux Transporter The sponsor had used vesicular transport inhibition assay to evaluate the inhibition potential of efflux transporter by netupitant and its metabolites. Vesicular transport assays were performed with inside-out membrane vesicles prepared from cells overexpressing human ABC transporters on 96-well plates. The netupitant, M1, M2, and M3 (at 0.01, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30 μM) were incubated with membrane vesicle preparations (total protein: 50 μg/well or 25 μg/well in case of BCRP) and the probe substrate in triplicates. Incubations were carried out in the presence of ATP or AMP to distinguish between transporter-mediated uptake and passive diffusion into the vesicles. At the end of the incubation period, the amount of probe substrate trapped in the vesicles was quantified by liquid scintillation counting. Table 1. Vesicular transport assay parameters Transporter Probe substrate Reference inhibitor human BCRP (ABCG2) human BSEP (ABCB11, sP-gp) human MRP2 (ABCC2) human MDR1 (ABCB1/P-gp) Ko134 (1 µM) cyclosporin A (20 µM) Benzbromarone (100 μM) Verapamil (100 µM) E3S (1 µM) Taurocholate (2 µM) E217βG (50 μM) NMQ (2 µM) Controls:  Incubation with AMP was included for background activity values for all data points.  Incubation without testing compounded (solvent only) was included to provide 100% activity values.  A reference inhibitor was included to serve as positive control for inhibition.  Membrane vesicle preparations from parental cells or Sf9 cells expressing defective transporters or beta-gal provided negative controls for function. Calculation: For all wells, the amount of the translocated probe substrate was determined in cpm. Relative activities were calculated with the following equation: 98 Reference ID: 3537650 Legend: A: amount of translocated substrate in the presence of TA and ATP B: amount of translocated substrate in the presence of TA and AMP C: amount of translocated substrate in the presence of solvent and ATP D: amount of translocated substrate in the presence of solvent and AMP 2. Uptake Transporters Inhibition and Substrate Assays Uptake transporters were evaluated using CHO cells or FlpIn293 cells stably expressing the respective uptake transporters. Table 2. Parameters of uptake transporter assays Transporter Incubation Time (inhibition) Probe substrate Reference inhibitor Negative Control Cell Line human OATP1B1 10 E3S (0.1 μM) Cerivastatin (100 µM) Parental CHO human OATP1B3 10 Fluo-3 (10 μM) Fluvastatin (30 µM) Parental CHO human OAT1 3 PAH (1.33 μM) Benzbromarone (200 μM) Parental CHO human OAT3 3 E3S (1 µM) Probenecid (200 µM) Mock transfected HEK293 human OCT1 20 Metformin (3.63 μM) Verapamil (100 μM) Parental CHO human OCT2 10 Metformin (3.63 μM) Verapamil (100 μM) Parental CHO Inhibition Assessment: Inhibition potential of netupitant, M1, M2, and M3 were evaluated by incubating netupitant, M1, M2, and M3 at 0.01, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30 μM concentrations with cells stably expressing the uptake transporter and the probe substrates on 96-well plate at 37 ± 1 °C in pH 7.4 buffer in triplicates. After the incubation, the cells were washed twice with buffer and lysed with 0.1 M NaOH (1 mM CaCl2 in 5% SDS in case of OATP1B3). Fluo-3 transport (OATP1B3) was determined by measuring fluorescence using 485 nm and 520 nm as the excitation and emission wavelengths, respectively. Radiolabelled probe substrate transport was determined with liquid scintillation counting. Controls: 1. Uptake transport in parental cells (non-transfected) provided background activity values for all data points. 2. Incubation without test compound (solvent only) provided 100% activity values. 3. A reference inhibitor served as positive control for inhibition. Calculation of relative activities: The amount of translocated probe substrate was determined for each well in cpm or RFU or nM. Relative activities were calculated from the equation: Legend: A: amount of translocated substrate in the presence of TA in transfected cells B: amount of translocated substrate in the presence of TA in parental cells C: amount of translocated substrate in the presence of solvent in transfected cells 99 Reference ID: 3537650 D: amount of translocated substrate in the presence of solvent in parental cells Substrate Assessment: The cellular uptake of netupitant, M1, M2, and M3 into cells was determined by incubating netupitant, M1, M2, or M3 at 1 and 10 μM concentrations with cells overexpressing the uptake transporter and control cells on 24-well plates at 37 ± 1 °C in pH 7.3 buffer for 2 and 20 min. After the incubation, the reactions were quenched by removing uptake buffer and the washing the cells twice. Cells were lysed by adding MetOH:H2O (3:1) and incubated for 10 minutes at 37 ± 1 °C. The amount of TA in the cell lysates was determined by LC/MS. The amount of protein in each well was quantified using the BCA kit for protein determination. Calculation of fold activation value The fold activation value was defined as the ratio of uptake of TA or probe substrate into transfected and parental cells: Fold activation = UPTTRP / UPTParental Legend: UPTTRP: accumulated amount of TA or probe substrate in transfected cells normalized by protein content [pmol/mg protein] UPTParental: accumulated amount of TA or probe substrate in parental cells normalized by protein content [pmol/mg protein] 3. MDCKII Monolayer for MDR1 and BCRP Substrate and Inhibition Assays The monolayer assays were performed using parental and MDR1 or BCRP transfected MDCKII cell monolayers cultured on the 24-well Transwell inserts. Substrate Assessment (bidirectional transport): Bidirectional transport through monolayers was determined by incubating of netupitant, M1, M2, and M3 (3, 10 and 30 μM) with parental and MDR1/BCRP transfected MDCKII cell monolayers (seeded on 24-well Transwell inserts) at 37 ± 1 °C. After the incubation, aliquots (100 μl) were taken from the receptor chambers to determine the amount of translocated TA. Samples were taken from the donor chambers before and after incubation to determine the initial concentration (C0) and recovery (R) of the test compound. Amount of netupitant, M1, M2, and M3 was determined by LC/MS. The digoxin/prazosin efflux ratio was determined as a positive control for MDR1/BCRP function. As a follow-up, bidirectional transport of M2 in parental and MDR1 transfected MDCKII cells was determined in the presence and absence of the MDR1 inhibitor PSC833 to confirm the specificity of the transport in MDCKII-MDR1 cells. Table 3: Monolayer assay parameters; MDCKII-MDR1/MDCKII-BCRP parental cells bidirectional permeability measurements Monolayer assay type MDCKII, MDCKIIMDR1 Compound Direction Concentration Incubation Time (min) M1 M2 M3 Lucifer yellow antipyrine digoxin A-B/B-A A-B/B-A A-B/B-A A-B/B-A A-B A-B/B-A 3, 10 and 30 µM 3, 10 and 30 µM 3, 10 and 30 µM 40 µg/ml 50 µM 5 µM 0, 15, 30, 60 and 120 0, 15, 30, 60 and 120 0, 15, 30, 60 and 120 120 30 120 100 Reference ID: 3537650 and MDCKII MDCKII, MDCKIIBCRP MDCKII, MDCKIIBCRP Netupitant M1 M2 M3 Lucifer yellow antipyrine prazosin M2 M2 + PSC833 A-B/B-A A-B/B-A A-B/B-A A-B/B-A A-B/B-A A-B A-B/B-A A-B/B-A-B A-B/B-A 3, 10 and 30 µM 3, 10 and 30 µM 3, 10 and 30 µM 3, 10 and 30 µM 40 µg/ml 50 µM 1 µM 30 µM 30 + 10 μM 0, 15, 30, 60 and 120 0, 15, 30, 60 0, 15, 30, 60 and 120 0, 15, 30, 60 and 120 120 30 60 120 120 Digoxin A-B/B-A 5 µM 120 Digoxin+PSC833 A-B/B-A 5 + 10 µM 120 Lucifer yellow A-BB 40 µg/ml 120 antipyrine A-B 50 µM 30 Inhibition Assessment: Bidirectional transport of model substrates (digoxin/prazosin) for MDR1 and BCRP in parental and MDR1/BCRP transfected MDCKII cells was determined in the presence and absence of Netupitant, M1, M2 and M3 (10 and 30 μM) or the reference inhibitor PSC833 or Ko134. The reference inhibitor (10 μM PSC833 or 1 μM Ko134) or the test compound, 30 μM TA, was added to both apical and basolateral chambers of the wells. After incubation at 37 ± 1 °C, aliquots (100 μl) were taken from the receptor chambers to determine the amount of translocated digoxin/prazosin via liquid scintillation. The donor compartments were sampled before and after incubation to determine the initial concentration (C0) and recovery (R) of digoxin/prazosin. Table 4: Treatment groups; MDCKII-MDR1/BCRP and parental cell permeability measurements Monolayer assay type MDCKII, MDCKIIMDR1 MDCKII, MDCKIIBCRP Incubation Time (min) 120 Model Substrate Direction Inhibitor Digoxin (5 µM) A-B/B-A Digoxin (5 µM) A-B/B-A Digoxin (5 µM) Lucifer yellow (40 µg/ml) Antipyrine (50 µM) Prazosin (1 µM) A-B/B-A NA M1, M2 and M3: 10 and 30 μM PSC833 (10 μM) NA A-B A-B/B-A NA NA 30 60 Prazosin (1 µM) A-B/B-A Netupitant, M1, M2 and M3: 10 and 30 μM 60 Prazosin (1 µM) Lucifer yellow (40 µg/ml) A-B/B-A Ko134 (1 μM) 60 A-B NA 120 Antipyrine (50 µM) A-B NA 30 A-B 120 120 120 Controls: 1. The transepithelial electric resistance (TEER) was determined for each well prior to the experiment to confirm the confluency of the monolayers to the experiment. Values above 150 Ω/cm2 were accepted. 101 Reference ID: 3537650 2. Monolayer integrity markers included the measurement of low (Lucifer yellow) and high (antipyrine) permeability compounds in each experiment. Values were accepted below 2 × 10-6 cm/s for LY and above 50 × 10-6 cm/s for antipyrine. 3. The efflux ratio of digoxin (positive control for MDR1-mediated active efflux) was accepted when above 3 for the digoxin control. The efflux ratio of digoxin in the presence of the reference inhibitor PSC833 (10 μM) was reduced to 1 ± 0.5. 4. The efflux ratio of prazosin (positive control for BCRP-mediated active efflux) accepted when above 3 for the prazosin control. The efflux ratio of prazosin in the presence of the reference inhibitor Ko134 (1 μM) was reduced to 1 ± 0.5. Calculation: The following equation was used for apparent permeability coefficient (Papp): Legend: dQ: amount of transported test drug dT: incubation time A: surface of porous membrane in cm2 (standard: 0.7) C0: initial concentration of the compound in the donor compartment Efflux ratio (ER) = Papp B-A / Papp A-B For MDCKII-MDR1/BCRP cells, efflux ratios were calculated as ERT/ERP where (ERT) and (ERP) are the efflux ratios for the transfected and the parental cells (used for negative controls), respectively. Recovery (R) was calculated according to the following formula to allow for estimation of metabolism and/or non-specific binding: Legend: QApical: amount of test drug detected in the apical chamber in pmol QBasolateral: amount of test drug detected in basolateral chamber in pmol Q0: amount of test drug detected at t = 0 in pmol Bioanalysis:  Digoxin/prazosin samples were analyzed with liquid scintillation counting.  Lucifer yellow samples were analyzed by measuring fluorescence, with excitation at 430 nm and emission at 520 nm.  Antipyrine and TA (netupitant, M1, M2, and M3) were analyzed with LC-MS system and HPLC-MS system. Results: 1. Vesicular Transport Inhibition Assays for Efflux Transporter 102 Reference ID: 3537650 Netupitant 0.01 0.1 1 IO IOO 0.0l O.l I IO IOO Netupitant concentration tiM) Ml concentration (it M) Figure 1. Inhibition of BCRP?mediated E38 transport by Netupitant and MI in the vesicular transport inhibition assay M2 M3 2 ISO- 2 IOO1 .3 125? 3 75. 3 loo0.0l (H ID IOO 0.0l l0 IOO M2 concentration (uM) M3 concentration (uM) Figure 2. Inhibition of BCRP-mediated E3S transport by M2 and M3 in the vesicular transport inhibition assay Netupitant ?:25 25 '33 25 0"r 0 0.01 (H IO IOO i? 0.0I l0 IOO Netupitant concentration l1 M) Ml concentration M) Figure 3. Inhibition of BSEP-mediated taurocholate transport by Netupitant and M1 in the vesicular transport inhibition assay 103 Reference ID: 3537650 1250.0I 0.I I I0 [00 0.0I 0.I I I0 100 M2 concentration (IIM) M3 concentration It MI Figure 4. Inhibition of BSEP-mediated taurocholate transport by M2 and M3 in the vesicular transport inhibition assay Netupitant M1 :5 125- C. elm-*ifH-0.0I 0.I I I0 I00 0.0I 0.I I I0 100 Netupitant concentration II M) Ml concentration (IIM) Figure 5. Inhibition of MRPZ-mediated E217BG transport by Netupitant and in the vesicular transport inhibition assay M2 M3 2 I50- I50 [8'25? 7? I003 0.0I 0.I I I0 [00 0.0I 0.I I I0 I00 M2 concentration (11M) M3 concentration Figure 6. Inhibition of MRPZ-mediated E217BG transport by M2 and M3 in the vesicular transport inhibition assay 104 Reference ID: 3537650 Table 5. Calculated Reaction Parameters from vesicular transport inhibition assays Reviewer’s Comments:  Netupitant, M1, M2 and M3 do not inhibit MRP2 up to 30 µM concentration and thus IC50 values were not determined for MRP2 transporter.  Netupitant, M2 and M3 slightly inhibit BSEP while M1 does not show any inhibition toward BSEP up to 30 µM concentration. Therefore, IC50 values could not be determined for BSEP transporter.  Netupitnat, M1, M2 and M3 inhibit BCRP in concentration dependent manner. Since total Cmax/IC50 are less for 0.1 for M1, M2 and M3, further studies are not needed for the metabolites. However, since total Cmax/IC50 is greater than 0.1 for parent drug netupitant, a follow up in vivo study may be recommended. 105 Reference ID: 3537650    o Netupitnat: Cmax/IC50= (1-1.5µM)/6 µM = (0.167-0.25)>0.1 o M1: Cmax/IC50=(0.07-0.09 µM)/8.6 µM =(0.008-0.01)<0.1 o M2: Cmax/IC50= (0.17-0.58 µM)/22.6 µM =(0.0075-0.026) <0.1 o M3: Cmax /IC50 = (0.08-0.144 µM)/10.6 µM = (0.0075-0.013) <0.1 M1, M2 and M3 inhibit inhibits MDR1in concentration dependent manner. However, since Cmax/IC50 <0.1 for all metabolites, not further studies are needed. However, inhibition potential of netupitant for MDR1 transporter was not evaluated in this experiment. o M1: Cmax/IC50=(0.07-0.09 µM)/4.95 µM =(0.014-0.018)<0.1 o M2: Cmax/IC50= (0.17-0.58 µM)/8.0 µM =(0.02125-0.0725) <0.1 o M3: Cmax /IC50 =( 0.08-0.144 µM)/5.35 µM = (0.014-0.027) <0.1 The reference inhibitors (positive controls) for all evaluated efflux transporters had adequate level of inhibition to confirm the function of the transporters in the applied vesicles. Negative controls with membrane vesicle prepared from parental cell had very minimum transport of model substrate into the vesicle. 2. Uptake Transporters Inhibition and Substrate Assays Inhibition Assessment: 106 Reference ID: 3537650 Netupitant M1 125- I25 :1 i i .8100} if, .31000.0! 1 l0 IOU 0.0I 0.1 ID 100 Netupitant concentration (uM) Ml concentration (pM) Figure 22. Modulation of 0ATPIB3-mediated luo-3 transport by Netupitant and in the uptake transporter inhibition assay 0.01 0.1 I l0 l0() 0.01 0.1 [0 IOO M2 concentration (HM) M3 concentration (pk/1) Figure 23. Modulation of OATPI B3?mediated Fluo-3 transport by MZ and M3 in the uptake transporter inhibition assay Netupitant 0.I00 Netupitant concentration Ml concentration (?Mi Figure 24. Modulation of OATl-mediated PAH transport by Netupitant and MI in the uptake transporter inhibition assay 107 Reference ID: 3537650 IQ H4 t?I?1 H?l H?t 25 0 0 0.01 0.l IOO 0.01 0.1 IOO M2 concentration (ttM) M3 concentration (pM) PAH uptake ofcontroluptake of control) FIG Figure 25. Modulation of OATl-mediated PAH transport by M2 and M3 in the uptake transporter inhibition assay Netupitant M1 2 '25? r; l250.0l 0.1 1 l0 100 0.01 0.1 10 l00 Netupitant concentration (uM) Ml concentration Figure 26. Modulation of OAT3-mediated E3S transport by Netupitant and in the uptake transporter inhibition assay I00 0.01 0.1 I I0 IOO M2 concentration (uM) M3 concentration (11M) Figure 27. Modulation of OAT3?mediated E38 transport by M2 and M3 in the uptake transporter inhibition assay 108 Reference ID: 3537650 Netupitant Ml .2 IZS- .2 125- ,53 .c WW a? 75? 1'0.01 0.1 I IO :00 5 0.01 0.l to 100 Netupitant concentration (pM) Ml concenttatiun Figure 28. Modulation of OCTl-mediated metformin transport by Netupitant and M1 in the uptake transporter inhibition assay 0.0] 10 mo 4 0.01 DJ I 10 l00 M2 concentmtion (0M) M3 concentration (pM) Figure 29. Modulation of OCTl-mediated metformin transport by MZ and M3 in the uptake transporter inhibition assay Netupitant Ml '00 Ig?? I Mctfonnin uptake ol'control) Mctfon?nin uptake nl?control) 0 0 0.01 I 10 100 0.01 OJ 1 10 100 Netupitant concentration (pM) Ml concentration (ttM) Figure 30. Modulation of OCTZ-mediated metformin transport by Netupitant and M1 in the uptake transporter inhibition assay 109 Reference ID: 3537650 M2 M3 150 5 Mctforrmn uptake of control) 0 l-H Metfonmn uptake of control33? 031?"631' 'i nb'""iBo olin? '61! {0 "Too M2 concentration (pM) M3 concentration (pM) Figure 31. Modulation of OCTZ-mediated metformin transport by M2 and M3 in the uptake transporter inhibition assay 110 Reference ID: 3537650 Reviewer’s Comments:  OATP1B1: Netupitant, M1 and M3 showed weak inhibition toward OATP1B1, and thus, IC50 values could not be estimated. However, M2 did show some inhibition toward OATP1B1 with IC50 of >30 µM. Since total Cmax/IC50 = 0.58 µM /30 µM =0.02 < 0.1, a follow-up in-vivo study is not needed.  OATP1B3: Netupitant and M1 showed weak inhibition toward OATP1B3 and IC50 value would not be estimated up to 30 µM. M2 and M3 inhibited OATP1B3 with IC50 values of 4.3 and 9.6 µM. Since Cmax/IC50 = 0.144 µM /9.6 µM = 0.015<0.1 for M3, an invivo study for to evaluate the inhibition potential of M3 toward OATP1B3 is not needed. Although total Cmax/IC50 = 0.58 μM /4.3 μM = 0.13 >0.1 for M2, R-value = 1+ (fu x I in,max/IC50) = 1.08 <1.25 and thus, in vivo study is not needed.  OAT1: Netupitant, M1, M2 and M3 do not appear to inhibit OAT1 significantly up to 30 µM concentration and thus, IC50 value could not be determined.  OAT3: Netupitant, M1, M2 and M3 do not inhibit OAT3. 111 Reference ID: 3537650  OCT1: Netupitant, M1, M2 and M3 all appear to inhibit OCT1 in concentration dependent manner. For netupitant, although Cmax/IC50 =0.19 for OCT1, it is not substantially larger than 0.1. Since Cmax/IC50 <0.1 for OCT2, and OCT1 and OCT2 have overlapping substrate specificities, we do not anticipate a significant in-vivo OCT1 interaction for netupitant. o Netupitant: Cmax/IC50 = (1-1.5 µM) /7.9 µM = (0.13-0..19) >0.1 o M1: Cmax/IC50 = (0.07-0.09 µM) /19 µM = (0.0037-0.0047)<0.1 o M2: Cmax/IC50 = (0.17-0.58 µM)/7.4 µM =(0.023-0.078)<0.1 o M3 Cmax/IC50 = (0.08-0.144 µM) /4.4 µM =(0.018-0.033) <0.1  OCT2: Netupitant appears to inhibit OCT2 in concentration dependent manner with IC50 value of 22.3 µM while M1, M2 and M3 did not show significant inhibition toward OCT2. Since Cmax/IC50 = (1-1.5 µM) /22.3 µM = (0.045-0.07) <0.1, in-vivo follow up study is not needed.  The reference inhibitors (positive controls) for all evaluated uptake transporters had adequate level of inhibition to validate the test system to confirm the function of the transporters. Substrate Assessment: 112 Reference ID: 3537650 ((HO-K - (?llO-K 12ml Mill (Hill!) 100 JOHN loo 2mm protein) l'mnspon (pmolmg protein) ll 3 minutes IHM) 20 minute? lpM) 2 minutes IDLIM) 30 minutes 1 IOPM) Figure 34. Accumulation of M2 in expressing and control cells in the uptake transporter substrate feasibility assay I?ll? - r: L: .x'minutes linll 20 minuics (lpM) 2 minutes (10? M) 20 minutes Figure 35. Accumulation of M3 in OATPIBI expressing and control cells in the uptake transporter substrate feasibility assay Netupitant (1 uM) Netupitant (10 uM) I I CHO-K :400- 16000~ :200- 14mm .5 :3 10mm .5 $00? 71 snow 5 6m" ouuo~ ?55 low E. a 4000 20m 33 wow i? 2 minutes l?uM) 20 minutes M) 2 minutes IOHM) 20 minutes M) Figure 36. Accumulation of Netupitant in OATPIB3 expressing and control cells in the uptake transporter substrate feasibility assay 113 Reference ID: 3537650 MI (I 11M) Ml (1011M) CI l?P 83 P13 - Clio-K - Hm ?(no so ?ll UN) 'l?mnspon (pmol?mg protein) 'l'mnspon (pmol mg protein) 2 minutes IHM) 20 minutes lpM) 3 lniuuleu IOHM) 30 milmles ?th Figure 37. Accumulation of in 0ATPIB3 expressing and control cells in the uptake transporter substrate feasibility assay M2 (l 11M) MZ (10 pM) - CIIU-K . Imm- l4000~ i 5 MW 5 moon 800')? 9 5- (muo? r' if? 300- g? 4000- 2000- t: I) 3 minutes 'llM? 2? minutes( lit?) 2 minutes IOHM) 20 minutes Figure 38. Accumulation of MZ in OATPIB3 expressing and control cells in the uptake transporter substrate feasibility assay ms (1 pM) (10 pM) - t'llU-K - l-H'Ull Ilmm 15mm 12000 T?tm '20? mm m? mum (moo mm anmlcul?ptw ZomimucsumM) 30o lranspurl (pmol mg pmlcim 'l tampon (pmol mg prolcim Figure 39. Accumulation of M3 in OATPIB3 expressing and control cells in the uptake transporter substrate feasibility assay 1 14 Reference ID: 3537650 thupitant (1 11M) thupitant (10 11M) I: - - 1000- 10001)- 80th 3 1101111? 2" 30 (11104 1101111? 2 201% 2001minutes IHM) 2111inutes(l()11M) 20 minutes( l0pM) Figure 40. Accumulation of Netupitant in expressing and control cells in the uptake transporter substrate feasibility assay M1 (1 11M) M1 (10 pM) - (?llO?K - 1111111 15 13111111 :3 ?3 H1100 121111 :7 - 1111111 E) 11111111 8. a 1.1111 11111111 3 41111 a 31111 3111111 ,minutes WM) 10 nunutcs 111M1 2 minutes (IUHM) 201ninutcs 1011M) Figure Accumulation of MI in OCTI expressing and control cells in the uptake transporter substrate feasibility assay M2 11 pM) M2 (10 pM) CHO-DCTI - I (THO-K 511 411 a 411.1 j. 311 5- 31111 fr:? 211 311.1 g} 111 11111 1? 11 s? 11 2 minutes( IHM) 20 minutes IHM) 2 minutes IUHM) 20 minutes IUHMI Figure 42. Accumulation of M2 in expressing and control cells in the uptake transporter substrate feasibility assay Reference ID: 3537650 Reviewer’s Comments:  As Netupitant and its metabolites are primarily eliminated through hepatobiliary route, the sponsor choice to evaluate the potential of Netupitant and its metabolites being substrate for OATP1B1 and OATP1B3 was appropriate.  As none of the test compound showed ≥2 fold increase in uptake in transfected cells compared to parental cell, netupitant, M1, M2 and M3 do not appear to be substrates for OATP1B1, OATP1B3 and OCT1.  Regarding the positive controls for these transporter, fold increase in uptake of model substrate (positive controls) in transfected cells compared to parental cell were not reported. However, the sponsor evaluated the uptake of model substrates in absence and presence of model inhibitor of for these specific transporters to validate the test system. Uptake of these model substrates were substantially inhibited in the presence of model inhibitor based on the raw data. 116 Reference ID: 3537650 3. MDCKII Monolayer for MDR1 and BCRP Substrate and Inhibition Assays Substrate Assessment (bidirectional transport): Calculated reaction parameters from MDCKII-MDR1 studies Calculated Reaction Parameters From MDCKII-BCRP Studies 117 Reference ID: 3537650 Reviewer’s Comments:  The study system was appropriately validated with positive controls. Based on the raw data, positive control digixon and prazosin as model substrate for MDR1 and BCRP had net flux ratio > 2 in all experiments, and net flux ratio of these model substrates were substantially reduced in the presence of model inhibitor for these transporter (PSC833 and Ko134 were used as model inhibitors for MDR1 and BCRP, respectively).  The sponsor did not evaluate the potential of netupitant being a substrate of MDR1 (Pgp).  As the net flux ratio for M1 and M3 were below 2 at all concentrations, M1 and M3 are not substrate of MDR1.  The net flux ratio of M2 for MDR1 was > 2 at all tested concentration. The sponsor further evaluated the potential for M2 being a substrate for MDR1 in presence of MDR1 inhibitor. Efflux of M2 in was further reduced in presence of MDR inhibitor suggesting that M2 is a substrate for MDR.  Netupitant, M1, M2 and M3, are not substrates of BCRP transporter o Although the net flux ratio when corrected for parental cell are > 2 for under certain conditions, it appears that it was due to very low flux ratio in parental cells. Based on efflux ratio in BCRP transfected cells alone, none of the tested compounds are substrates of BCRP transporter as efflux ratio for all of them were less than 2 in BCRP transfected cells. o Repeated experiments at 10 µM reconfirmed that Netupitant, M1, M2 and M3, are not substrates of BCRP transporter as both efflux ratio in transfected cells alone and net efflux ratio when corrected for parental cells are <2 for all tested compounds. Inhibition Assessment: Calculated reaction parameters from MDCKII-MDR1 studies APPEARS THIS WAY ON ORIGINAL 118 Reference ID: 3537650 digoxin:24. 3 4.46 1523:4415 d: 0.08 PSC83311.43 i020 1.47 i 0.22 bidirectional M130 M: M: 1.1166013 Digexin permeability of 1 0206] 1 12:: digoxin with Ml digc-xin: 1.86: 0.48 digoxin: 31.63de2.66 17.04i4.67 PSC833: 0.98 0.03 0.02 1.30 i 0.05 7.334066 1.25 i 0.06 9.21 0.67 digoxin: 1.47 :t 0.13 digoxin: 40.21 i: 5.46 22.45 i 5.46 09840.10 1304:0151 1.32i0.18 bidirectional M2130 M2 (30 PM): 23-88 i 2'54 Digoxin permeability of L2 i 0-12 23-5 i 2-53 M2 101309;:ng ??111 digoxin: 1.86 4 0.48 digoxin: 31.63 4 2.66 12.04 4 4.67 PSC833: 0.98 i 0.03 PSC833: 1.28 :t 0.02 1.30 i 0.05 +Mzimpm}; 19.84245 1.45i0.11 28.68i2.81 Digoxin bidirectional dig-021111: 1.54 i 0.15 digoxin:24. 13 i 4.46 15.? i 446 M3 permeability of PSC833: 0.92 3: 0.03 1.43 4 0.20 1.42 4 0.22 dieoxin with M3 (30 0M): M3 (30 2.12 0.32 M3 l.00i0.06 2.13:1: 0.32 diguxin: 1.86 d: 0.48 digoxin:31.63 2.66 12.04 :e 4.62 PSC833: 0.98 0.03 PSC833: 1.28 d: 0.02 1.30 d: 0.05 013(1061141: 7.234: 1.62 1.16i0.06 8.44:1: 1.82 Calculated reaction parameters from MDCKII-BCRP studies Test Assay ER Net ER MDCKII MDCKII- prazosin: 0.91 0.09 :t 0.83 18.97 i 0.84 i 0.20 0.91 i 0.08 1.04 i 0.22 . b?d?re?iji'l9?1al 1? Netupila111?30 Netupitant (30 12M): 6.86 0.30 pennea'] It): 0 0?9] 0.0i 6.25 (2.30 Netupitant prazosm W101 thupitant prazosin: 0.971 0.08 prazosin: 14.28 1.18 14.76 i 1.68 0904:0114 4:01?) 1.461016 Netupitant (10 Netupitant (10 0M): 20.35 d: 2.51 0.86 0.08 17.56 1.40 Prazos bidirectional prazosin: 0.91 i 0.09 prazosin: 17.35 i 0.83 18.97 i 0.84 I permeability of 0.37 4: 0.20 4 0.03 1.04 0.22 Reference ID: 3537650 119 Reviewer’s Comments:  The study system was appropriately validated with positive controls with model inhibitor and substrates. Based on the raw data, model substrates digixon and prazosin for MDR1 and BCRP had net flux ratio > 2 in all experiments, and net flux ratio of these model substrates were substantially reduced in the presence of model inhibitor for these transporter (PSC833 and Ko134 were used as model inhibitors for MDR1 and BCRP, respectively).  The sponsor did not evaluate the potential of netupitant being an inhibitor of MDR1 (Pgp) in this study.  While M2 did not inhibit MDR1 and BCRP at both 10 µM and 30 µM, M1 and M3 inhibited MDR1 in concentration dependent manner. However, IC50 values were not determined in this monolayer cell system. Based on rough estimate of IC50 around 10 µM or based on the IC50 values from the vesicular system, an in-vivo study is not needed for M1 and dM3.  Netupitant, M1 and M3 inhibited BCRP in concentration dependent manner where no inhibitions were observed at 10 µM and inhibition was observed at 30 µM. However, IC50 values were not determined in this monolayer cell system. Since no significant P-gp inhibitory effect of netupitant was observed with Digixin in in-vivo where 5 μM netupitant have inhibited P-gp transporter in vitro, we do not anticipate a significant BCRP inhibitory effect of netupitan in vivo since netupitnat at 10 μM did not inhibit BCRP transporter in vitro. 120 Reference ID: 3537650 Overall Reviewer’s Comment: 1. The tested concentration of Netupitant, M1, M2 and M3 up to 30 µM was acceptable as they approximately cover the Cmax and 10 times Cmax values to be expected in human subjects or patients taking this drug at the clinical dose of 300 mg.  Observed Cmax of Netupitant is 550-880 ng/mL (≈ 1-1.5 µM)  Observed Cmax for M1 is 40-50 ng/ml (0.07-0.09 µM)  Observed Cmax for M2 is 100-350 ng/mL (0.17-0.58 µM).  Observed Cmax for M3 is 50-90 ng/mL (0.08-0.0.144 µM). 2. Although the sponsor did not evaluate the potential of netupitant to inhibit MDR1 (P-gp) in both vesicular transport system and monolayer system in this study, the sponsor did conducted an in-vivo drug-drug interaction study with digoxin administered concomitantly with netupitant to evaluate the inhibition potential of P-gp by netupitant. 3. The sponsor did not evaluate the potential of netupitant being a substrate or inhibitor of P-gp transporter in this study. APPEARS THIS WAY ON ORIGINAL 121 Reference ID: 3537650   Title: Determination Of The Permeability Of [14C]-Netupitant Using the Parallel Artificial Membrane Permeability Assay (PAMPA) Report No: 494368-NETU-10-26 Specific Aims: The aim of this study was to determine the permeability of [14C]-Netupitant using the parallel artificial membrane permeability assay (PAMPA). Study Date: 07/07/2010 – 07/15/2010 Test Site: (b) (4) Sponsor: Helsinn Healthcare SA, Switzerland Study Design: Test Item: [14C] Netupitant Tested Concentrations: 0.1, 0.5, 2, 10 and 50 μM Test System: BD GentestTM Pre-Coated PAMPA plate system: A 96-well microtiter plate assembled with a 96 well filter plate containing an artificial lipid membrane barrier mimicking the intestinal epithelium was used. Controls: As controls, the reference compounds [3H]-propranolol (high permeability) and sulfasalazine (low permeability) were included in the assay at one concentration in triplicate. Study Method: The permeability of [14C]-netupitant was determined at five different concentrations (0.1, 0.5, 2, 10 and 50 μM) in triplicate in PAMPA. [14C]-Netupitant, [3H]-propranolol or sulfasalazine were applied at the donor site of the pre-coated filter plate wells. The filter plate was coupled with the receiver plate and the assembly was incubated at room temperature for 5 hours. At the end of the incubation, the plates were separated and 150 μL solution from each well of the filter plate and the receiver plate exposed to [14C]Netupitant or [3H]-propranolol was transferred to scintillation vials and analyzed using liquid scintillation counting. Solutions containing sulfasalazine were transferred to a UV-transparent 96-well plate and analyzed using a spectrophotometer at 360 nm. Data Analysis: The permeability of a compound (in cm/s) and the mass retention (in %) were calculated using the following formula: Mass retention: Where: Pe = estimated permeability C0 = initial compound concentration in donor well (mM) CD(t) = compound concentration in donor well at time t (mM) 122    Reference ID: 3537650   CA(t) = compound concentration in acceptor well at time t (mM) VD = donor well volume (= 0.3 ml) VA = acceptor well volume (= 0.2 ml) Cequilibrium = [CD(t)VD + CA(t)VA] / (VD + VA) A = filter area = 0.3 cm2 t = incubation time = 18000 s (= 5 hr) Acceptance Criteria: The PAMPA assay was considered acceptable if it meets the following criteria: (b) (4)  The permeability of sulfasalazine should be below (b) (4)  The permeability of propranolol should be above Evaluation  If Pe  If Pe permeability  If Pe a compound is considered to have low permeability nm/s) a compound is considered to have medium (b) (4) (b) (4) a compound is considered to have high permeability. Results: Sulfasalazine and Propranolol had acceptable level of low and high permeabilities validating the PAMPA system. Based on the mass retention, while sulfasalazine has low non-specific binding to the filter, propranolol has approximately 70% non-specific binding to the surface of the plate or is trapped inside the artificial membrane. Table-1: Permeability of sulfasalazine (exposure concentration: 100 μM) Table-2: Permeability of propranolol (exposure concentration: 213 nM) Based on the measured and theoretical concentration of netupitant on the donor side, it appears that netupitant did not dissolve well in the buffer used in the experiment. Therefore, the actual concentration of netupitant that is exposed at the donor side is much lower than what is stated theoretically. 123    Reference ID: 3537650   Table-3: [14C]-Netupitant concentrations in the initial spiked samples Spike Theoretical Concentration (Bq/mL) Measured Concentration (Bq/mL) Recovery (%) 0.1 µM Netupitant 46.4 25.3 54 0.5 µM Netupitant 232 30.6 13 2 µM Netupitant 928 85.4 9 10 µM Netupitant 4640 2104 45 50 µM Netupitant 23200 13134 57 Permeability at theoretical concentrations of 0.1, 0.5 and 2 μM were not determined since the concentrations of [14C]- Netupitant in the receptor compartment were below the limit of detection (which is 25 dpm). At theoretical concentrations of 10 and 50 μM, the permeability of [14C]-Netupitant was determined to be 1.1x10-6 cm/s (= 10.6 nm/s) and 2.5x10-6 cm/s (24.6 nm/s), respectively. According to the sponsor’s criteria, netupitnant’s permeability would correspond to medium to high permeability. At both 10 and 50 uM concentration, netupitant had about 65-70% non-specific binding or is trapped inside the artificial membrane. Table -4: Permeability of [14C]-Netupitant 124    Reference ID: 3537650   Title: Determination Of Passive Diffusion Of [14C]Netupitant Using Bi-Directional Assay In Caco2 Cells Report No: 494369-NETU-10-25 Specific Aims: The aim of this study was to determine the passive diffusion and the apparent permeability of [14C]Netupitant using Caco-2 cells. Study Date: 06/28/2010 – 08/16/2010 Test Site: (b) (4) Sponsor: Helsinn Healthcare SA, Switzerland Study Design: Test Item: [14C] Netupitant Tested Concentrations: 1, 10 and 100 μM Test System: Caco-2 cells plated in a 24-transwell plate Controls: As controls of monolayer integrity, the reference compounds [3H]-propranolol (high permeability) and [3H]-mannitol (low permeability) were included in the assay. Additionally, the integrity of the Caco-2 cell monolayer was checked by measuring the transepithelial electrical resistance (TEER) (>1000 Ohm.cyou m2). Study Method: Permeability of [14C]Netupitant at three concentrations (1, 10, and 100 μM) were evaluated from apical side to the basolateral side (A→B) and from the basolateral side to the apical side (B→A) on Caco-2 cells in triplicate wells and was repeated on two different days. Before the start of the experiments, the medium on the apical and basolateral side was refreshed and cells were incubated for 30 minutes. The experiment was started by adding transport buffer containing a test substance or a vehicle to the apical and/or the basolateral compartment. The apical compartments were filled with 300 μL of transport buffer and the basolateral compartments were filled with 900 μL of transport buffer. After 30, 60, and 120 minutes of incubation (at 37.0 ± 1.0°C and 5.0 ± 0.5% CO2), a 50 μL sample was drawn from the receiver compartment, which was immediately replaced with transport buffer. The samples were analyzed using liquid scintillation counting (LSC). At the end of the experiment, samples from both the apical and basolateral compartments were analyzed to determine the recovery. Data Analysis: The apparent permeability (Papp) of test items across the monolayer was calculated as follows: Papp = (Vr/C0)(1/S)(dC/dt) Where Papp is apparent permeability, Vr is the volume of medium in the receiver chamber, C0 is the concentration of the test drug in the donor chamber, S is the surface area of monolayer, 125    Reference ID: 3537650   dC/dt is the linear slope of the drug concentration in the receptor chamber with time after correcting for dilution. The sponsor states that as netupitant showed high non-specific binding (table-3), the apparent permeability for netupitant was calculated using the final donor concentration at the termination of the incubation instead of C0 to avoid an underestimation of the permeability. Acceptance Criteria: The bi-directional transport assay with Caco-2 cells was considered acceptable if it meets the following criteria:  The TEER value should be (b) (4) Ωcm2 above background value (transwell without cells). (b) (4)  Permeability values of mannitol were below cm/s (<50 nm/s).  The propranolol:mannitol permeability ratio was (b) (4) Evaluation    (b) (4) If P If Pe If Pe compound is considered to have low permeability compound is considered to have medium permeability (b) (4) a compound is considered to have high permeability. (b) (4) Results: Mannitol and Propranolol had acceptable level of permeability validating the Caco-2 monolayer cell system. Mannitol permeability was below 50 nm/s in both directions and propranolol/mannitol permeability ratio was > 5 for all experiment. Table-1: Permeability of Mannitol and Propranolol Experiment 1 2 Mannitol PA/B PB/A (nm/s) (nm/s) 0.7 3.0 7.3 10.7 Propranolol PA/B PB/A (nm/s) (nm/s) 263 388 262 471 (PB/A)prop/(PB/A)man 130 44.0 Based on the measured and theoretical concentration of netupitant on the donor side, the actual concentration of netupitant that is exposed at the donor side is much lower than what is stated theoretically at 1 and 10 µM concentrations. The sponsor states that the lower concentration measured in buffer can be explained by the poor aqueous solubility of netupitant. Table-2: [14C]-Netupitant concentrations in the initial spiked buffer samples Experiment Contents 14 1 µM [ C]Netupitant in buffer 1 Measured concentration (DPM) Recovery (%) 640 337 52.5 14 6405 3507 54.8 14 64050 66371 104 14 644 294 45.5 14 6675 3683 55.2 14 63600 71792 113 10 µM [ C]Netupitant in buffer 100 µM [ C]Netupitant in buffer 1 µM [ C]Netupitant in buffer 2 Theoretical 14 [ C]Netupitant concentration (DPM) 10 µM [ C]Netupitant in buffer 100 µM [ C]Netupitant in buffer The sponsor stated that because netupitant showed high non-specific binding (with low recovery % in table 3), the apparent permeability was calculated using the final donor concentration at the end of the 126    Reference ID: 3537650   incubation and the apparent permeability was determined with the actual measured concentrations for the calculations (table 4). The permeability of [14C]Netupitant from the A-B side was above 200 nm/s and the permeability from the B-A was above 20 nm/s. According to the sponsor’s criteria, [14C]Netupitant would be considered to have medium to high permeability under the conditions used in this study. Table-3: Measured [14C]Netupitant concentrations in the initial spiked buffer, donor and receptor samples Experiment Contents spike 1 µM [ 1 1) A→B 14 10 µM [ 1 µM [ 1 µM [ 1 µM [ C]Netupitant in buffer C]Netupitant in buffer 14 14 11 19 20 19 14 13 13 337 134 141 131 3507 2310 2426 2393 66371 26010 26340 27570 294 75.3 67.5 77.4 3683 662 695 701 71792 9323 10380 9073 294 164 179 170 3683 2361 2399 2220 71792 24074 24385 22747 8.3 15.3 11.3 179 160 188 704 843 1040 16.3 16.6 16.3 52.3 56.1 50.6 924 917 935 63.2 76.0 82.2 180 203 205 1779 2942 9313) 41 43 40 68 71 70 40 40 42 42 40 43 22 23 23 17 18 17 63 70 67 36 37 34 34 35 32 14 C]Netupitant in buffer C]Netupitant in buffer 14 C]Netupitant in buffer 14 C]Netupitant in buffer 14 10 µM [ 0.0 5.2 1.4 43.8 42.6 35.0 8073) 594 536 14 100 µM [ 2 2) B→A 47.4 31.9 32.7 533 576 569 6892 7016 7174 66371 C]Netupitant in buffer 14 10 µM [ 337 14 100 µM [ 2 1) A→B [ C]Netupitant concentration in receptor Compartment (DPM) 3507 C]Netupitant in buffer 14 10 µM [ [1 4 C]Netupitant concentration in donor compartment (DPM) 14 100 µM [ 1 2) B→A C]Netupitant in buffer Measured concentration in spike (DPM) C]Neutpitant in buffer 14 100 µM [ C]Netupitant in buffer 14 C]Netupitant in buffer 14 1) Apical to basolateral transport; donor compartment = apical side, receptor compartment = basolateral side 2) Basolateral to apical transport; donor compartment = basolateral side, receptor compartment = apical side 3) Outlier, due to an analytical error Table-4: Permeability of [14C]Netupitant at the initial concentrations Co of 1, 10, and 100 µM Experiment C0 (µM) PA/B (nm/s) PB/A (nm/s) 1 1 10 100 n.a. 336 342 126 102 50.9 127    Reference ID: 3537650 Recovery (%)   2 1 10 100 853 292 439 666 127 165 14 n a : not applicable Could not be determined since no detectable [ C]Netupitant was present in receiver compartment) Reviewer’s Comment:  The tested concentration for this permeability study was 1, 10 and 100 µM. The recommended concentration of drug for permeability studies are 0.01, 0.1, and 1 times the highest dose strength dissolved in 250 ml which would correspond to approximately 20, 200 and 200 µM (the proposed dose is (300 mg/250 mL) / (578.6g/mol)= 2074μM.  This study is not adequate to categorize the drug for BCS classification as the suitability of this method was not evaluated with sufficient number of model drugs. Generally, to demonstrate suitability of a permeability method intended for application of the BCS, a rank-order relationship between test permeability values and the extent of drug absorption data should be established using a sufficient number of model drugs (20 models drugs for in vitro cell culture methods) to allow precise differentiation between drug substances of low and high intestinal permeability attributes.  Expression of P-gp was not characterized in the Caco-2 cell monolayer system with a model substrate.  Passive permeability cannot be assumed for netupitant for following reasons: o Netupitant does not have linear PK as Netupitant systemic exposure increased more than dose-proportional manner with dose increase from 100 mg to 300 mg. o in-vitro permeability of netupitant changes with initial concentration of drug o The rate of transport from apical-to-basolateral is different than the rate of transport from basolateral-to-apical direction for netupitant  Due to solubility issue, the actual concentration in donor compartment is different that the theoretical concentration.  Netupitant showed high non-specific binding (30-90%).  The apparent permeability was calculated using the final donor concentration at the end of the incubation instead of initial donor concentration.  Overall, the result of this study is difficult to interpret for above reasons.   128    Reference ID: 3537650 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------INSOOK KIM 07/07/2014 DILARA JAPPAR 07/07/2014 SUE CHIH H LEE 07/07/2014 Reference ID: 3537650 ADDENDUM to CLINICAL PHARMACOLOGY REVIEW NDA 205-718 Submission Date(s) September 29, 2013 Brand Name Generic Name Akynzeo® Netupitant and palonosetron Reviewer Insook Kim, Ph.D., Dilara Jappar, Ph.D. Team Leader Sue-Chih Lee, Ph.D. PM Reviewer Jingyu “Jerry” Yu, Ph.D. PM Team Leader Nitin Mehrotra, Ph.D. OCP Division OND Division Division of Clinical Pharmacology 3 Division of Pharmacometrics Division of Gastroenterology and Inborn Errors Products Sponsor Helsinn Relevant IND(s) 73,493 Submission Type Original Formulation; Strengths; Fixed dose combination of 300 mg netupitant and 0.5 mg palonosetron in oral capsule Each capsule contains three 100 mg tablets of netupitant and 0.5 mg soft-gel capsule of palonosetron  Prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy1  Prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy1 One AKYNZEO capsule administered approximately one hour prior to the start of chemotherapy AKYNZEO can be taken with or without food Proposed indication Dosing Regimen 1 NME Executive Summary This is an addendum to the clinical pharmacology review of NDA 205-718 dated May 30, 2014 to discuss the Post-Marketing Study Recommendations. The application is submitted in support of an approval of AKYNZEO®, a fixed dose combination of 0.5 mg palonosetron, a 5-HT3 receptor antagonist and 300 mg netupitant, a NK1 receptor antagonist for prevention of chemotherapy induced nausea and vomiting (CINV). One AKYNZEO capsule contains one Reference ID: 3533088 capsule of 0.5 mg palonosetron and three tablets of 100 mg netupitant. Palonosetron, one of two active moieties of AKYNZEO®, has been approved for the prevention of CINV as an intravenous injection and oral capsule. On the other hand, netupitant a new molecular entity has not been approved for any indications. The sponsor intends to market netupitant only as a combination product but not as a single component product. 1.1 Post-Marketing Studies We recommend following post-marketing studies to improve the labeling of AKYNZEO pending its approval.  In vivo drug interaction study to evaluate the duration of inhibitory effects of AKYNZEO on CYP3A4 enzyme activity beyond 4 days after single dose administration of AKYNZEO. Rationale: Co-administration of a single dose of netupitant increased the exposure to dexamethasone, a substrate of CYP3A4 by 1.7-fold on Day 1 and up to 2.4-fold on Day 2 and Day 4. The potential inhibitory effect of netupitant on CYP3A4 was not studied beyond Day 4. Given AKYNZEO will be used in patients who require multiple medications for underlying disease treatment as well as supportive care, a study is necessary to provide adequate information for use of AKYNZEO with concomitant medications that are CYP3A4 substrates.  In-vitro study to evaluate the potential of netupitant being a substrate for P-gp transporter in bi-directional transport assay system Rationale: The potential of netupitant being a substrate for P-gp in ATPase activation assay suggested that netupitant is likely a substrate for P-gp. However, information is lacking whether netupitant is a substrate for P-gp on bi-directional transport assay system, which is considered a confirmatory study. 2 Reference ID: 3533088 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------INSOOK KIM 06/27/2014 DILARA JAPPAR 06/27/2014 JINGYU YU 06/27/2014 NITIN MEHROTRA 06/27/2014 SUE CHIH H LEE 06/27/2014 HAE YOUNG AHN 06/27/2014 Reference ID: 3533088 BIOPHARMACEUTICS REVIEW Office of New Drug Quality Assessment NDA Submission Date Brand Name Generic Name Formulation; Strength(s) 205-718 September 27, 2013 Indication Treatment of nausea and vomiting associated with cancer chemotherapy Helsinn Healthcare S.A. Limited Assadollah Noory, Ph.D. Tapash Ghosh, Ph.D. Richard Lostritto, Ph.D. ONDQA-Biopharmaceutics DGIEP Applicant Reviewer Team Leader Acting Supervisor OPQ Division OND Division Stamp Dates AkynzeoTM Netupitant / Palonosetron 300 mg/0.5 mg capsule September 27, 2013; January 17, 2014; March 27, 2014 EXECUTIVE SUMMARY Under the provisions of 505(b)(1), Helsinn Healthcare S.A. submitted for approval of their AkynzeoTM netupitant/palonosetron (300 mg/0.5 mg) capsule, for the prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of moderately and highly emetogenic cancer chemotherapy. This biopharmaceutics review involves review of two bioequivalence (BE) studies that provided a bridge between the late Phase 1 formulation, Phase 3 formulation, and the to-be-marketed formulation. In addition, dissolution method development and validation reports as well as proposed regulatory dissolution acceptance criteria were also reviewed. BE Studies: The application for netupitant/palonosetron (300 mg /0.5 mg) capsule includes two bioequivalence study reports. Study NETU-09-07 provides a bridge between a late Phase 1 formulation with a Phase 3 formulation and Study NETU-11-02 provides a bridge between two Phase 3 formulations from two manufacturing facilities (to-be-marketed formulation). The studies were randomized, open-label, balanced, two-treatment, two-sequence, four-period, single-dose, replicate crossover under fasting conditions. Study NETU-09-07 Forty-seven subjects completed this balanced, randomized, open-label, two-treatment, two-sequence, four-period, single-dose, replicate crossover bioequivalence study under fasting conditions study. Plasma PK parameter estimates, point estimates as ratio of test over reference expressed as percent, and the 90% confidence intervals are shown in the following table. Reference ID: 3523196 Table 1: Summary Statistics PK-Parameter Test Reference Point Estimate (%) 90% Confidence Interval 434.12 + 242.09 431.76 + 260.33 106.92 92.91 – 123.04 AUC0-t (ng/mL×h) 12321.44 + 5209.58 12058.30 + 5609.79 105.92 96.24 – 116.58 AUC0-∞ (ng/mL×h) 14401.61 + 7307.75 14375.41 + 7335.11 101.57 91.17 – 113.16 Cmax (ng/mL) 1.53 + 0.39 1.53 + 42 100.18 97.15 – 103.31 AUC0-t (ng/mL×h) 52.19 + 95 52.05 + 17.50 100.19 97.10 – 103.38 AUC0-∞ (ng/mL×h) 56.71 + 18.59 57.10 + 34 99.37 96.54 – 102.28 Netupitant Cmax (ng/mL) Palonosetron The 90% confidence limits for netupitant and palonosetron are within 80% to 125% for AUC and Cmax indicating that the late Phase 1 and Phase 3 formulations are bioequivalent under fasting conditions. Study NETU-11-02 Eighty-two subjects completed this randomized, open-label, two-treatment, two-sequence, fourperiod, single-dose, replicate crossover bioequivalence study under fasting conditions. Plasma PK parameter estimates, point estimates as ratio of test over reference expressed as percent, and the 90% confidence intervals are presented in the following table. Table 2: Summary Statistics PK-Parameter Test Reference Point Estimate (%) 90% Confidence Interval 453.96 + 238.01 486.83 + 268.02 92.72 86.41 – 99.50 AUC0-t (ng/mL×h) 12736.28 + 4892.81 13627.64 + 5745.25 93.93 89.35- 98.74 AUC0-∞ (ng/mL×h) 13862.50 + 5761.88 15031.75 + 6858.15 92.62 87.34 – 98.22 1.27 + 0.33 1.24 + 0.31 102.36 100.38 – 104.37 AUC0-t (ng/mL×h) 44.68 + 12.41 44.32 + 13.07 101.11 99.32 – 102.94 AUC0-∞ (ng/mL×h) 48.17 + 12.70 47.59 + 13.40 101.08 99.23 – 102.96 Netupitany Cmax (ng/mL) Palonosetron Cmax (ng/mL) The 90% confidence limits for netupitant and palonosetron are within 80% to 125% for AUC and Cmax indicating that the formulation manufactured in Ireland is bioequivalent to formulation (b) (4) manufactured under fasting conditions. 2 Reference ID: 3523196 In summary, Study NETU-O9-O7 demonstrated that late Phase 1 formulation manufactru'ed in atalent Philadelphia is bioequivalent to the Phase 3 formulation manufactm?ed in atalent Philadelphia, and study 1-02 demonstrated that the Phase 3/to-be-marketed formulation manufactured in Ireland is bioequivalent to the Phase 3 formulation manufactru'ed in (we) Dissolution: The following table shows the dissolution methods and acceptance criteria proposed by the Sponsor for both the intermediate and the fmished ?xed dose combination products. Drug Name Dosage Form USP Apparatus Speed Medium Volume (rpm) Criteria Acceptance Palonosetron Capsule/ Combination Capsule Netupitant Tablet/Combination Capsule (4) In the mid-cycle communication with the Sponsor, the Agency recommended the following dissolution acceptance criteria for both the intermediate and the ?nished products. Active component USP Apparatus Speed (rpm) Dissolution Medium Medirun Acceptance Volume Criteria Palonosetron (W4) Netupitant In response to the mid-cycle commrmication the Sponsor proposed the following acceptance criteria for the intermediate products. proposed speci?cation FDA request llelsinn llealtheare response for dissolution . (4) Intermediate Palonosctron Softgel Intermediate _\'ctupitant Tablet With respect to the ?nished product. the sponsor proposed the following acceptance criteria 1mtil the evaluation of additional dissolution data from ?ve new batches (shown below); the Sponsor will then revisit the netupitant acceptance criteria for their FDC capsule. Reference ID: 3523196 NDA proposed speci?cation for Palonosetron dissolution Netupitunt? Palonosetron combination capsule ctupitant- Palonosetron combination capsule I FDA request Ilelsinn Ilealthcare response (5) (4) The following table summarizes the dissolution acceptance criteria for palonosetron and netupitant intermediate products and the ?nished ?xed-dose combination product. Drug Name Dosage Form USP Speed Medium Volume Acceptance Apparatus (rpm) Criteria Intermediate Products Palonosetron Capsule/Combination USP Paddle 75 0.01 HCL 500 mL 09(4) in 30 Capsule minutes Netupitant Tablet/Combination USP Paddle 100 0.07M Phosphate 900 mL (4) in 45 Capsule buffer pH 6.8 minutes containing 1% sodium SDS Finished Product Palonosetron Capsule/Combination USP Paddle 75 0.Capsule minutes Netupitant Tablet/Combination USP Paddle 100 0.07M Phosphate 900 mL in 60 Capsule buffer pH 6.8 minutes containing 1% sodi1m1 SDS In essence, the sponsor did not propose any change for palonosetron either for intermediate or for the FDC ?nished product. However, for netupitant, they accepted the Agency?s proposal for the intermediate product but for the FDC ?nished product, they still wanted (W) in 60 minutes until they gather more information. The proposal is acceptable by the Agency. I Recommendation: Based on the ONDQA-Biophannaceutics review, NDA 205-718 is recommended for approval. The sponsor agreed to submit dissolution data as a post approval supplement (PAS) ??om ?rst ?ve batches following approval of the product and will revisit the netupitant dissolution acceptance criteria in the ?nal ?xed dose combination product. Signature 06/11/2014 Tapash Ghosh, Team Leader Of?ce of New Drug Quality Assessment Signature 06/11/2014 Assadollah N001y, Biophaimaceutics Reviewer Of?ce of New Drug Quality Assessment Reference ID: 3523196 BACKGROUND Netupitant-Palonosetron combination fixed-dose combination capsules are composed of the following: • Three (3) intermediate 100 mg netupitant tablets; (b) (4) • One (1) intermediate palonosetron softgel capsule containing 0.50 mg of palonosetron (0.56 mg of palonosetron hydrochloride); • One (1) size 0 hard gelatin capsule consisting of a white body with black imprint “HE1” and a caramel cap. Thus the dosage delivered by one capsule of the drug product is 300 mg netupitant and 0.5 mg palonosetron. The design intent was to develop an oral fixed dose combination to allow administration of two drug substances in a single dosage form prior to each chemotherapy cycle. The 100 mg netupitant tablet and the 0.50 mg palonosetron softgel are produced as intermediate drug products. They are referred to by the applicant as Intermediate Netupitant Tablet and Intermediate Palonosetron Softgel. It should be mentioned that 0.50 mg palonosetron softgel capsule was always used as the approved product (NDA 22233), manufactured jointly by both Catalent Pharma Solutions, USA and Helsinn Birex Pharmaceuticals, Ireland for Helsinn Healthcare SA, Switzerland. Palonosetron hydrochloride injectable (Aloxi®) was approved on July 25, 2003 for the prevention of chemotherapy-induced nausea and vomiting, but netupitant is a new molecular entity (NME). The empirical formula of netupitant is C35H32F6N4O with a molecular weight of 578.6. Netupitant is (b) (4) a white to off-white powder; it is very slightly soluble in water; ; soluble (b) (4) in isopropanol, ethanol, and Netupitant is classified as a BCS Class 2, poorly soluble and highly permeable based on the Biopharmaceutics Classification System. The empirical formula of palonosetron hydrochloride is C19H24N2O•HCl with a molecular weight of 332.9. Palonosetron hydrochloride is a white to off-white crystalline powder; it is freely soluble in (b) (4) water; soluble in propylene glycol; slightly soluble in (b) (4) ethanol, and Palonosetron hydrochloride is classified as a BCS Class 1, highly soluble and highly permeable based on the Biopharmaceutics Classification System. 5 Reference ID: 3523196 Composition of the Fixed-Dose Netupitant-Palonosetron Combination Capsule is given in the following Table: Ingredient I Reference I Function I %w/w I Quantity (mg) Intermediate Netupitant Tablet 4 Netupitant Internal I Active ingredient . . (19(4) Microelystalline cellulose Eur Sucrose acid esters Internal Povidone K-3O Eur roscannellose sodium Eur Plui?ed water Elu' Silicon dioxide/ Eur Sodium steai'yl fumarate 4? Eur Magnesium stearate (W I Eur Total I Intermediate Palonosetron Softgel Palonosetron Internal I Active ingredient (W4) (19(4) Ph. Eur. Glycerin Em? oleate (4) Internal Pmi?ed water Em? Butylated hydroxyanisole (BHA) Eur 09(4) Eur. Theoretical Fill Weight . I 5' (4) Gelatin (W4) Eur I Internal Intemal Elu? Elu? .T . (4) . - Eur (W4) Internal Internal Netupitant?Palonosetron Combination Capsule Size 0 hard gelatin capsule. white body caramel Internal Capsule shell 1 capsule cap. HIEI printed in black on the white body6 6 Reference ID: 3523196 INDIVIDUAL STUDY REVIEWS BIOANALYTICAL: The concentrations of netupitant and palonosetron in hrunan plasma were determined by using liquid chromatography mass spectrometry methods. Validation of the bioanalytical methods performance used for the determination of concentrations of netupitant and palonosetron in plasma are presented in the following table. Analytical Parameters Netupitant Palonosetron Analytical Range 2.00 to 500.00 ng/mL 45.00 to 1500.00 Pg/mL Between?batch Precision 2.92 to 3.62 2.2% to 5.6% Between-batch Accuracy 0.41 to 1.92 0.0% to 1.8% Within?batch Precision 1.45 to 4.88 1.1% to 7.2% Within?batch Accuracy ?1.58 to 3.56 0.1% to 3.3% Recovery 62.0 95.6 Freeze?thaw Stability LQC (three cycles) 6.28 -1.7 Freeze?thaw Stability HQC (three cycles) 0.74 2.1 Freezer Stability LQC 5.50' 41.48" Freezer Stability HQC -0.43* 44.90? 34 Months at 22 Months at The bioanalytical method is acceptable for the determination of concentrations of netupitant and palonosetron ??om the plasma samples. Bioequivalence study NE 7. This study was conducted to bridge Phase II and Phase studies, using an extemporaneous combination of Netupitant 300 mg (two Netupitant Capsules, 150 mg) plus Palonosetron 0.50 mg softgel versus Netupitant- Palonosetron Combination Capsules (300 mg/0.50 mg). Dissolution data for both netupitant and palonosetron was provided. The data indicated that the formulations were bioequivalent and that the Phase II and Phase formulations were bridged. 6.1. Study NETU-09-07 Title: Bioequivalence study of a new netupitant/palonosetron ?xed dose combination (300 mg/0.50 mg) versus extemporaneous combination of netupitant 300 mg and palonosetron 0.50 mg a?er single dose administration to healthy male and female volrmteers Principal Investigator: Milko Radicioni. MD Cross Research S.A.. Phase I Unit - Via F.A. Giorgioli 14 CH-6864 Arzo. Switzerland I Study Start Date: July 22. 2009 I Study End Date: January 27. 2010 I Reference ID: 3523196 Treatments: Test: Netupitant/palonosetron ?xed dose combination, 300 mg/0.50 mg hard gel capsules, atalent Philadelphia, USA. Batch release N. 29702-005 (bulk batch Expiration January 2010. References: Netupitant 2 150 mg capsules, batch release N. 29702-005 (bulk batch Expiration January 2010 plus Palonosetron 0.50 mg so?gel capsules. Catalent Pharma Solutions. USA. batch release N. 29702-005 (bulk batch Expiration January 2010. Objective: To assess the bioequivalence of netupitant and palonosetron of a ?xed dose combination (300 mg/0.50 mg hard gel capsules, atalent Philadelphia, USA) versus netupitant 2 150 mg capsules plus palonosetron 0.50 mg softgel capsules (C atalent Pharma Solutions, USA) in healthy male and female subjects Imder fasting conditions and to monitor the safety and tolerability of test and reference products following a single dose administration. Study study was a balanced, randomized, open-label, two-treatment, three-sequence, form-period, single-dose, replicate crossover bioequivalence study under fasting conditions. The study medications were administered with 180 mL of mineral water. The wash-out period was 28 days. Study Population: Forty-seven of ?fty subjects em'olled completed the study. Three subjects withdrew their consent. The following table contains subjects? demographics. Table 5: Study Subjects Subjects Demographics Gender 24 Male; 26 Female Age (yr) 32.3 i 6.1 (19-45) Weight (kg) 68.7 i 12.6 (51.0-91.6) Height (cm) 170.3 i 9.7 (154?195) BMI (kg/m2) 23.5 i 2.4 (19.2-28.8) Race 41 White, 7 Hispanic, 2 Mestizo Note: Data resented as mean i SD . n_e Sample Collection for Pharmacokinetic Measurements: Blood samples were collected at the following speci?ed times dru?ing each period for the determination of concentrations of netupitant and palonosetron in plasma: prior to dosing (zero horn4.5, 5, 5.5, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192 and 216 hours post dosing. Pharmacokinetic and Statistical Analysis: The pharmacokinetic parameters were determined using WinNonLin? version 5.2 for netupitant and palonosetron, shown in the following table. Reference ID: 3523196 Table 6: Summar PK?Parameter Netupitant Test of Pharmacokinetic Parameters, Mean SD. Reference Cmax (ng/mL) 434.12:i:242.09 431.76i260.33 Tum 5.00 (2.00-12.00) 5.00 (ZOO-12.00) (ng-h/mL) 12321.442t5209.58 12058.30zt5609.79 AUCM (ng-h/mL) 14401.612t7307.75 Tn: (111?) 95.62:i:58.84 Palonosetron Cm, (ng/mL) l.53i0.39 1.53i0.42 Tum 5.00 (LOO-12.00) 4.50 (LOO-12.00) (ng-h/mL) 52.19zt17.95 52.05il7.50 AUCM (ng-h/mL) 56.71:i:l8.59 57.10il8.34 Tn: (111') 44.15:t15.l6 43.28il4.31 - Mean Range) The plasma concentration time pro?les for netupitant are shown in the following ?gure. Reference ID: 3523196 BEST AVAILABLE 3 I. 1" a u;I.rI 1 rf'?t' :Ipuku ,il :Iuhn 3".1 HA I Him?? 3110:! TI COPY lw?The plasma concentration time pro?les for palonosetron are shown in the following ?gure. BEST AVAILABLE -D"Neuphn12 I. a; upah hunch. ?1 7-1 a. m?pl uphill COPY ?ne! no a. bid '2 zip-in murmualwuw?mt software for Windows release 9.2 was used for the statistical analysis of this bioequivalence study. The procedure was used with treatment as the random effect. The results are shown in the following table. Table7: Summary Statistics Netupitant Geometric mean ratio Treatment comparison Parameter 90% Test vs. Reference Cum 106.92% 92.91 123.04% 105.92% 96.24 116.58% 101.57% 91.17 113.16% Palonosetron Geometric mean ratio Treatment comparison Parameter 90% CI Test VS. Reference max 100.18%; 97.15 - 103.31% 100.19% 97.10 - 103.38% 99.37% 96.54 - 102.28% A statistical reanalysis performed by this reviewer using SAS version 9.3 con?rmed that netupitant/palonosetron ?xed combination, 300 mgr/0.50 mg hard gel capsules, atalent Philadelphia, USA has similar bioavailability to netupitant 2 150 mg capsules plus one 0.50 mg softgel palonosetron capsule, 00(4) under fasting conditions. Conclusion: The 90% con?dence limits for netupitant and palonosetron are within 80% - 125% for AUC and Cmax indicating that netupitant/palonosetron ?xed combination, 300 mg/O.50 mg hard gel capsules, atalent Philadelphia, USA is bioequivalent to netupitant 2 150 mg capsules plus one 0.50 mg softgel palonosetron capsule, one under fasting conditions. 10 Reference ID: 3523196 6.2. Study 1-02 Bioequivalence Study NETU-11-02. This study was conducted to b1idge manufacturing sites of the Netupitant-Palonosetron Combination Capsule used dining development (4) to the commercial site, HBP (Damastown, Mulhudda1t-Dublin 15, Ireland). Title: Bioequivalence study of the netupitant/palonosetron ?xed dose combination product by Helsinn Birex Phannaceuticals (Ireland) versus the netupitant/palonosetron ?xed dose combination product by M0 after a single dose administration to healthy male and female vollmteers. Principal InvestigatorzMilko Radicioni, MD Cross Research S.A., Phase I Unit - Via FA. Giorgioli 14 H-6864 A120, Switzerland I Study Start Date: May 5, 2011 I Study End Date: October 30, 2011 I Treatments: Test: Netupitant/palonosetron ?xed dose combination, 300 mg/0.50 mg hard gel capsule, Batch 31000861. Expiration November 2012, Helsirm Birex Pharmaceuticals Ltd., Ireland. References Netupitant/palonosetr on ?xed dose combination, 300 mg/0. 50 mg hard gel capsule, Batch 0901640. Expiration Decembe1 2011, ObjectivezTo assess the bioequivalence between two different production batches at two different manufacturing sites of netupitant/palonosetr on 300 mg/0. 50 mg ?xed dose combination capsules, [(test) manufacttu ed by Helsinn Birex Phannaceutical Ltd., I1e1and, batch 31000861 and (1efe1ence) manufactmed by M4) batch and to assess the safety and tolerability of test and reference after single dose administration 1mde1 fasting conditions. Study Design: The study was a randomized, open-label, two-treatment, mic-sequence, fom-period, single-dose, replicate crossover bioequivalence study Imder fasting conditions. The study medications were administered with 180 mL of mineral water. The wash-out peliod was 28 days. Study Populatioanighty-two (82) of eighty-eight (88) subjects em'olled completed the study. Four subjects withdrew their consent, one subject showed positive test for barbitm?ate, and one subject showed positive test for pregnancy. The following table contains subjects? demographics. Sub ects Dem hics Gender 69 Male; 19 Female 33.6 8.0 19 50 Wei 73.6+11.8 51?103 Hei 173.6+8.9 150?201 BMI 24.3 2.4 19.3 29.0 Race 80 white; 3 black, 2 3 others Note: Data as mean i SD 1 1 Reference ID: 3523196 Sample Collection for Pharmacokinetic Measurements: Blood samples were collected at the following speci?ed times during each peiiod for the detelmination of concentrations of netupitant and palonosetron in plasma: prior to dosing (zero hour4.5, 5, 5.5, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192 and 216 hom's post dosing. Pharmacokinetic and Statistical AnalysiszThe pharmacokinetic parameter determined using WinNonLin? version 5.2 for netupitant and palonosetron are shown in the following table. Table 9: Summary of Plasma Pharmacokinetic Parameters, Mean SD Test rameter Reference Netupitant Cm, (ng/mL) 453.96i238.01 486.83i268.02 Tmax (111?) 5.00 (ZOO-24.00) 5.25 (2.00-8.00) (ng-h/mL) 12736.28i489281 13627.64i57452 AUCinf (ngOh/mL) 13862.50i5761.88 15031.75i6858.15 Tm (hr) 7662:2883 77.78:t29.99 Palonosetron Cunx (Pg/mL) 1270.50i327.47 1240.62i309.75 Tum (111?) 300 (200?1200) 3.00 (2.00-8.00) AUCM (pg-h/mL) 44679.60i12408.91 44323.39il306724 AUCinf (pg-h/mL) 48165.20i12696.09 47592.96zt13403.20 Tn: (111?) 37.22i10.82 38.16:t11.76 me: median (range) N=l47; N=l46 The plasma concentration time pro?les for netupitant are shown in the following ?glu'e. Reference ID: 3523196 BEST AVAILABLE COPY 12 The plasma concentration time pro?les for palonosetron are shown in the following ?gm?e. Edna. am "le; E- BEST AVAILABLE COPY software for Windows release 9.2 was used for the statistical analysis of this bioequivalence study. The procedlu?e was used with treatment as the random effect. The results are shown in the following table. Table 10: Summary Statistics Netnpitant Geometric mean ratio Treatment comparison Parameter PE Test vs. Reference 92.72 86.41 - 99.50 AUCH 93.93 89.35 - 98.74 92.62 87.34 98.22 Palonosetron Geometric mean ratio Treatment comparison Parameter PE Test vs. Reference 102.36 100.38 104.37% AUCH 101.11 99.32 102.94% 101.08 99.23 102.96% A con?rmatory statistical reanalysis was performed by this reviewer using SAS version 9.3. The procediu?e was used with subject within the sequence as the random effect. A comparative result of 90% con?dence limits reported by the sponsor and analyzed by this reviewer is shown in the following table. I 90 con?dence Limits Netnpitant Parameter Sponsor Reported Reviewer Analysis 86.41 99.50 85.21 100.90 AUCM 89.35 98.74 88.51 99.60 AUCMB 87.34 98.22 87.11 100.00 Palonosetron Parameter Sponsor Reported Reviewer Analysis 100.38 104.37% 100.00 104.76 AUCM 99.32 - 102.94% 99.01 103.25 99.23 102.96% 98.81 103.25 Reference ID: 3523196 13 The 90% con?dence limits for netupitant and palonosetron are within 80% - 125% for AUC and max indicating that the formulation manufactured at Helsinn Birex Pharmaceuticals Ltd., Ireland is bioequivalent to the fonnulation manufactlu?ed at (low under fasting conditions. Conclusion: The 90% con?dence limits for netupitant and palonosetron are within 80% - 125% for AUC and Cmax indicating that the formulation manufactured at Helsinn Birex Phannaceuticals Ltd., Ireland is bioequivalent to the formulation manufactured at (mo imder fasting conditions. Bridging Support with Dissolution data: Dissolution data were provided for: Combination Capsule batch 30005284 (encapsulated by HBP using Intermediate Netupitant Tablet batch 30004380 (manufactured by HBP, Ireland) and Intermediate Palonosetron Softgel batch 10JM- 164 (manufactlu?ed by atalent)). Netupitant- -Palonoset10n Combination Capsule batch N0901409 (encapsulated by? M4) using Intermediate Netupitant Tablet batch N0901098 (manufactured by (W) and Intelmediate Palonosetlon Softgel batch 09JM- 270 (manufactm ed by atalent). Calculations of f2 were perfonned for these dissolution pro?les. The f2 value for the netupitant dissolution pro?les was 524, indicating that they are similar. The f2 value for the palonosetron dissolution pro?les was 71.3, indicating that they are similar. The same variability for the combination capsule (?nal drug product) was obsewed. The data for netupitant is reproduced below. Data for the Mean of 12 tablets was used to calculate f2. The data indicated that the fonnulations were bioequivalent and that the fonnulations manufactured at the different facilities were bridged. I 081 inspection The memorandum from 081 dated May 29, 2014 indicates that the inspection repo?s of clinical and analytical sites of study 1-02 is satisfact01y by the Agency. DISSOLUTION Sponsor developed an in vitro dissolution method for the netupitant in the netupitant-palonosetron combination cansules (IN) (hm (hm The Sponsor adapted the dissolution method for palonosetron from the approved method for palonosetron capsule (NDA 22-233). The approved dissolution methodology for palonosetron is USP Apparatus 2 (paddle0.01N dissolution medium at 37.0i0.5? C. 14 Reference ID: 3523196 Netu itant Anal 'cal Method A UV specirophotometric assay at was used to detect netupitant in dissolution medium. The analytical parameters are shown in owing table. Analytical Parameters Netupitant Analytical Range Evaluated 0.0827831 -0.40432 mg/mL Linearity (10, 25, 50, 75, 150 and 200%) Rz=0.9996 Between Analyst Precision Analyst l-2 4.3% Within Run Precision 0.5% Accuracy 99.0 to 100.4% Recovery 99.4 to 102% Solution Stability in medium at RT for 9 days 100.52% Method Speci?city: A placebo hard gelatin capsule containing one palonosetron HCI 0.5 mg so?gel was analyzed to evaluate and con?rm method speci?city and possible interference. The placebo interference was -An overlay scan of netupitant in dissolution medium, and the netupitant working reference standard are shown in the following ?gure. Figure 1. Overlaid Spectra of Dissolution Medium (Blank), Netupitant Working Standard Solution, Placebo Sample Solution and Dissolution Accuracy Solutions for Over Encapsulated Palonosetron HCI Softgel, 0.5 mg] Netupitant Tablets, 300 mg Combination Product The ?gure con?rms that at- there was no signi?cant interference from palonosetron and/or any other material on netupitant absorbance. Dissolution Method Robustness for Intermediate Netupitant Tablet: 1 5 Reference ID: 35231 96 The Sponsor demonstrated that their dissolution method is robust and it can be used as product release methodology. The selection of 0. 07M Phosphate Bu?'er pH 6.8 1% SDS as the dissolution medium for netupitant is acceptable. Palonosetron Analy?cal Method A validated reverse phase HPLC procedure with a gradient mobile base and UV detection a- nm was used for determination of alonosetron concentrations. The analytical parameters are shown in the following table. Analytical Parameters Palonosetron Analytical Range Evaluated 0.25245 to 1.5147 mcg/mL Linearity (10, 25, 50, 75, 150 and 200% Between Analyst and HPLC Systems Precision 0.2 to 1.4% Within Run Precision 0.2 to 0.4% Accuracy 99.5 to 100.6% Recovery 99.5 to 100.1% Solution Stability in dissolution medium at 5? for 98.9 to 99.9% 5 days 16 Reference ID: 3523196 The analytical methods for determintion of concentration of netupitant and palonosetron are acceptable. DISSOLUTION METHOD DEVELOPMENT (b) (4) 3 Pages Have Been Withheld In Full As b4 (CCI/TS) Immediately Following This Page 17 Reference ID: 3523196 In response to the mid-cycle communication the Sponsor proposed the following acceptance criteria for the intermediate products. 7 NBA proposed speci?cation 7 FDA request 7 Helsinn Healthcare response for dissolution Intermediate Palonosetron Softgel Intermediate Nempitant Tablet With respect to the ?nished product, the sponsor proposed the following acceptance criteria for the ?nished product until the evaluation of additional dissolution data from ?ve new batches and then they will revisit the netupitant acceptance criteria. A NDA proposed speci?cation - FDA request . Helsinn Healthcare response for Palonosetron dissolution Netupitant- Palonosetron combination capsule NDA proposed specification 7 FDA request 7 Helsinn Healthcare response for Netupitant dissolution thupitant- Palonosetron combination capsule In essence, the sponsor did not propose any change for palonosetron either for intermediate or for the FDC ?nished product. However, for netupitant, they accepted the Agenc ?s roposal for the intermediate product but for the FDC ?nished product, they still wanted in 60 minutes until they gather more information. The proposal is acceptable by the Agency. 2 1 Reference ID: 35231 96 The following Table summarizes the dissolution acceptance criteria for palonosetron and netupitant intermediate products and the finished fixed-dose combination product. Drug Name Dosage Form Palonosetron Capsule/Combination Capsule Tablet/Combination Capsule Netupitant USP Speed Apparatus (rpm) Intermediate Products Medium Volume USP Paddle 75 rpm 0.01 N HCL 500 mL USP Paddle 100 rpm 0.07M Phosphate buffer pH 6.8 containing 1% sodium SDS 900 mL Acceptance Criteria (b) (4) in 30 minutes (b) (4) in 45 minutes Finished Product Palonosetron Netupitant Capsule/Combination Capsule Tablet/Combination Capsule USP Paddle 75 rpm 0.01 N HCL 500 mL USP Paddle 100 rpm 0.07M Phosphate buffer pH 6.8 containing 1% sodium SDS 900 mL (b) (4) in 30 minutes (b) (4) in 60 minutes 22 Reference ID: 3523196 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------ASSADOLLAH NOORY 06/11/2014 TAPASH K GHOSH 06/11/2014 Reference ID: 3523196 OFFICE OF CLINICAL PHARMACOLOGY REVIEW NDA 205-718 Submission Date(s) Brand Name 9/27/13; 11/8/13; 12/16/14; 12/20/14; 1/9/14; 4/4/14; 4/30/14; 5/12/14; 5/14/14; 5/21/14; 5/22/14 Akynzeo® Generic Name Netupitant and palonosetron Reviewer Insook Kim, Ph.D., Dilara Jappar, Ph.D. Team Leader Sue-Chih Lee, Ph.D. PM Reviewer Jingyu “Jerry” Yu, Ph.D. PM Team Leader Nitin Mehrotra, Ph.D. OCP Division OND Division Division of Clinical Pharmacology 3 Division of Pharmacometrics Division of Gastroenterology and Inborn Errors Products Sponsor Helsinn Relevant IND(s) 73,493 Submission Type Original Formulation; Strengths; Fixed dose combination of 300 mg netupitant and 0.5 mg palonosetron in oral capsule Each capsule contains three 100 mg tablets of netupitant and 0.5 mg soft-gel capsule of palonosetron • Prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy 1 • Prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy1 One AKYNZEO capsule administered approximately one hour prior to the start of chemotherapy AKYNZEO can be taken with or without food Proposed indication Dosing Regimen NME Table of Contents 1 Executive Summary .....................................................................................................2 1.1 Recommendations .................................................................................................2 1.2 Post-Marketing Studies .........................................................................................2 1.3 Summary of Clinical Pharmacology and Biopharmaceutics Findings..................2 Reference ID: 3516218 Question-Based Review ...............................................................................................8 2.1 General Attributes of the drug...............................................................................8 2.2 General Clinical Pharmacology ..........................................................................11 2.5 General Biopharmaceutics ..................................................................................72 3 Major Labeling Recommendations ............................................................................81 4 Appendices .................................................................................................................82 4.1 Table of Clinical Pharmacology Studies .............................................................82 4.2 Pharmacometric Review .....................................................................................87 4.3 Summary review of the tQT review by the IRT-QT …………………………..88 4.4 OCP Filing Form .................................................................................................91 2 1 Executive Summary The application is submitted in support of an approval of AKYNZEO®, a fixed dose combination of 0.5 mg palonosetron, a 5-HT3 receptor antagonist and 300 mg netupitant, a NK1 receptor antagonist for prevention of chemotherapy induced nausea and vomiting (CINV). One AKYNZEO capsule contains one capsule of 0.5 mg palonosetron and three tablets of 100 mg netupitant. Palonosetron, one of two active moieties of AKYNZEO®, has been approved for the prevention of CINV as an intravenous injection and oral capsule. On the other hand, netupitant a new molecular entity has not been approved for any indications. The sponsor intends to market netupitant only as a combination product but not as a single component product. 1.1 Recommendations The Office of Clinical Pharmacology found the submission acceptable from a clinical pharmacology standpoint provided a mutual agreement on labeling languages is reached. 1.2 Post-Marketing Studies Post-marketing are currently under discussion. . 1.3 Summary of Clinical Pharmacology and Biopharmaceutics Findings In support of AKYNZEO, 23 studies were conducted for PK or PD. In addition 15 in vitro studies were conducted. Pharmacokinetics of palonosetron and netupitant was studied in healthy subjects, cancer patients and patients with hepatic impairment after administration of AKZYNZEO. PK and PD of netupitant alone were also studied during the early development phase prior to combination with palonosetron. General clinical pharmacology of palonosetron mainly relies on the previous findings from the approval of Aloxi products. AKYNZEO is proposed to be available only as one strength i.e. 300 mg netupitant and 0.5 mg palonosetron and to be administered as a single dose at one hour prior to the initiation of chemotherapy. As such 2 Reference ID: 3516218 except on study for multiple dose PK of netupitant, PK of netupitant and palonosetron was characterized after single dose administration. Exposure (Dose)-Response Relationship Efficacy In a dose-finding study (NETU-07-07), the proportion of patients with complete response (CR) 2 was compared between palonosetron monotherapy at 0.5 mg and the combinations of 0.5 mg palonosetron with netupitant at three different doses i.e.100 mg, 200 mg, and 300 mg. The CR rate was evaluated during the 0-24 h (acute phase), 24-120 h (delayed phase) and 0-120 h (overall phase) after the administration of chemotherapeutics. The study was designed to show the difference between the combination therapy and palonosetron alone but not between doses. The concentration-response relationship was not studied because PK samples were not collected. No evident dose-response relationship was observed among doses for the CR rate in the delayed and overall phases. Compared to palonosetron monotherapy, all three combinations of palonosetron and netupitant showed statistically significant difference in the proportion of patients with CR during the delayed and overall phases. On the other hand, only the combination with 300 mg netupitant showed statistically significant difference for the CR rate in the acute phase in comparison to palonosetron monotherapy. The combination with netupitant 300 mg showed a numerically higher CR rate for the acute phase CINV than lower doses. Study NETU-07-07 is proposed to establish the contribution of netupitant component to the prevention of CINV in addition to palonosetron component and the clinical efficacy of the combination product for the prevention of CINV associated with HEC, cisplatin-based chemotherapy 3. Based on the statistically significant difference in the prevention of CINV in the acute, delayed and overall phases compared to palonosetron monotherapy, the combination of 0.5 mg palonosetron and 300 mg netupitant was selected for phase 3 clinical trials. The combination of 0.5 mg palonosetron and 300 mg netupitant resulted in the significantly higher proportion of patients with CR over palonosetron alone with respect to the CR in the delayed phase, the acute and overall phases after the administration of an anthracycline and cyclophosphamide regimen for the treatment of a solid malignant tumor (NETU-08-18). Please see the statistics review for more details. Safety: Overall the most common adverse reactions (an incidence ≥2 %), assessed by investigators as treatment related, were headache and constipation. In the dose-finding study, the incidence of TEAEs was higher with combinations of 200 mg or 300 mg netupitant (NETU) (50% and 54%, respectively) than with 100 mg NETU (40%). The overall rate of TEAE was higher after the combination treatment with NETU 300 mg/PALO 0.5 mg compared to oral PALO 0.5 mg alone 2 3 Defined as no emesis and no rescue medication http://www.cancer.gov/cancertopics/pdq/supportivecare/nausea/HealthProfessional/page5#Reference5.2 3 Reference ID: 3516218 (70% vs. 61%, respectively) in the integrated safety analysis. For the detailed review of safety profile, please see the clinical review by Dr. Nancy Snow, Medical Officer of DGIEP. Effects on QTc interval To assess the potential effect of the combination therapy, a thorough QT study was conducted at doses up to 600 mg NETU in combination with 1.5 mg PALO in healthy subjects. No significant QTc interval prolongation was observed when single dose 600 mg NETU and 1.5 mg PALO was co-administered 4 . Consistently, the exposure-response relationship was not evident between ddQTcF and concentrations of NETU and its metabolites as well as concentrations of PALO and its metabolites. No significant effect of PALO on the QTc interval was consistent with the previous report of no effect of PALO on the QTc doses up to 2.25 mg after intravenous administration 5. The supratherapeutic dose in this study provides the safety margin of 2 fold for netupitant and 3 fold for palonosetron. The supratherapeutic dose provided higher Cmax and similar AUC for NETU in patients with moderate hepatic impairment. Pharmacokinetic/ Biopharmaceutics Properties This review is mainly focused on netupitant while general PK characteristics of palonosetron were previously reviewed during the approval of single ingredient products6. AKYNZEO After single dose administration of AKYNZEO in healthy subjects, the peak plasma concentrations for netupitant and palonosetron were reached in about 5 hours. Concomitant food did not significantly affect the systemic exposure to netupitant and palonosetron. In cancer patients, the rate and extent of absorption of netupitant and palonosetron were similar to those in healthy subjects. No significant PK interactions between netupitant and palonosetron were observed. Netupitant Distribution Population PK analysis indicates that the apparent central and peripheral volume of distribution (Vz/F) was estimated to be 486 L and 1170 L, respectively. Human plasma protein binding of netupitant is greater than 99.5% at drug concentration ranging from 10-1300 ng/ml and protein binding of its major metabolites (M1, M2 and M3) are greater than 97% at drug concentrations ranging from 100 to 2000 ng/mL. Metabolism In in vitro studies netupitant is metabolized mainly by CYP3A4 and by CYP2C9 and CYP2D6 to a lesser degree. Three major metabolites were identified desmethyl derivative, M1; N-oxide 4 Study NETU-07-20. For more details, see the IRT-QT team reviews of the thorough QT study dated 1/19/2010 (IND 73,493 SDN 024) and 3/3/14 (NDA 205-718) 5 Aloxi Package Insert 6 Clinical pharmacology review of original NDA 21-371 4 Reference ID: 3516218 derivative, M2; OH-methyl derivative, M3 in vivo and were all shown to bind to human substance P/neurokinin 1 (NK1) receptor in vitro. Mean AUC for metabolites M1, M2, and M3 was 29%, 14% and 33% of netupitant, respectively. Elimination In cancer patients, the apparent median elimination half-life of netupitant was 88 hours and the estimated median systemic clearance was 20.5 L/h based on population PK analysis. Upon oral administration of labeled netupitant, about 50% and 75% of the administered radioactive dose was recovered from the excreta (urine and feces) collected over 120 h and 336 h (2 weeks), respectively. Over 2 weeks the total of 3.95 % and 70.7 % of the radioactive dose was recovered in urine and feces, respectively. The mean fraction of oral dose of netupitant excreted unchanged in urine was less than 1 % suggesting renal clearance is not a significant elimination route for netupitant. Specific populations Currently no dosage adjustment for palonosetron is recommended by renal or hepatic impairment. Age In cancer patients population PK analysis indicated that age (within the range of 29 to 75 years old) did not influence the pharmacokinetics of netupitant or palonosetron. Gender The Cmax for netupitant was 35 % higher in females than in males but the AUC was similar between males and females. For palonosetron 25-35% higher AUC and Cmax were observed in female subjects than in male subjects consistently with the previous observation. Hepatic Impairment In patients with mild or moderate hepatic impairment, the mean Cmax for netupitant was about 30% higher and the mean AUC0-∞ was 56% and 107% higher, respectively than in healthy subjects. The Cmax for netupitant in two patients with severe hepatic impairment was 63% and 463% higher compared to the mean Cmax in healthy subjects. . In patients with mild or moderate hepatic impairment, the mean Cmax for palonosetron was about 35-40% higher and the mean AUC0-∞ was 35% and 55% higher, respectively than in healthy subjects. Renal Impairment There was no dedicated PK study to evaluate the effect of renal impairment on PK of netupitant. On the other hand, no significant effect of CLCR on PK of netupitant was noted in the population PK analysis while the effect of CLCR was noted for palonosetron consistently with the current labeling for palonosetron. The pharmacokinetics has not been studied in subjects with end-stage renal disease for either palonosetron or netupitant. In vitro studies for evaluation of drug interaction potential assessment 5 Reference ID: 3516218 Based on the in-vitro studies, netupitant and M1 are inhibitors of CYP3A4. netupitant is an inhibitor of P-gp and BCRP transporters. In addition, CYP inhibition: In in vitro studies, netupitant and its metabolite M1 are inhibitors of CYP3A4. A follow-up in vivo study with CYP3A4 substrate midazolam was conducted. Netupitant did not inhibit CYP1A2, CYP2C19, and CYP2D6 in vitro. In vivo drug interactions via inhibition of CYP2B6, 2C8 and 2C9 at the clinical dose of 300 mg are unlikely based on weak inhibition of toward these enzymes in in vitro studies. M1 showed inhibition toward CYP2B6, 2C8, 2D6, and 3A4, and weak inhibition toward CYP 1A2, 2C9, 2C19. However, since Cmax/Ki >0.1 for only CYP3A4, in vivo drug interaction via M1 inhibition toward CYP enzyme is unlikely except for CYP3A4. M2 and M3 showed weak inhibition toward all major CYP enzymes. Since Cmax/Ki<0.1 for all enzymes, in vivo drug interaction via M2 or M3 inhibition individually toward CYP enzyme is unlikely. CYP induction: Netupitant up to 20 μM and its metabolites (M1, M2 and M3) up to 2 μM are not inducers of CYP1A2, CYP2C9, CYP2C19 and CYP3A4. The sponsor did not evaluate the potential of netupitant and its metabolites to induce CYP2B6. Transporters: Netupitant is an inhibitor of P-gp and BCRP transporters based on in vitro studies. Potential of netupitant being a substrate for P-gp was not evaluated adequately. In addition, M2 is shown to be a substrate for P-gp. Based on in vitro data, in vivo interaction of netupitant as a substrate for BCRP, OATP1B1, OATP1B3, and OCT1, or as an inhibitor of BSEP, MRP2, OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 is unlikely. In addition, based on the in vitro data, in vivo interaction of three major metabolites, M1, M2 and M3 as substrates of BCRP, OATP1B1, OATP1B3, and OCT1, or as inhibitors of MDR1, BCRP, BSEP, MRP2, OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 are unlikely. In vivo drug interactions (A) Effect of other drugs on the PK of netupitant and palonosetron CYP3A4 inhibitor: Co-administration of AKYNZEO with ketoconazole increased the mean Cmax and AUC of netupitant by 25% and 140%, respectively compared to those after administration of AKYNZEO without ketoconazole. Co-administration of ketoconazole increased the mean AUC and Cmax for palonosetron was about 10-15%. The labeling should include a cautionary statement about co-administration of AKYNZEO with strong CYP3A4 inhibitors when necessary. 6 Reference ID: 3516218 CYP3A4 inducer: Co-administration of AKYNZEO with rifampicin decreased the mean Cmax and AUC of netupitant by 62%, and 82%, respectively compared to those after AKYNZEO alone. Co-administration of rifampicin decreased the mean Cmax and AUC of palonosetron by 15% and 20%, respectively. Use of AKYNZEO in patients who have been on CYP3A4 inducers at the time of AKYNZEO administration is not recommended to ensure the efficacy of combination therapy. (B) Effect of netupitant or Akynzeo on PK of other drugs Drugs that are CYP3A4 substrates Netupitant component of AKYNZEO is a moderate CYP3A4 inhibitor and the increase in the systemic exposure to concomitant drugs that are CYP3A4 substrates was observed to a various degree when AKYNZEO or netupitant alone was co-administered. The significant inhibitory effect was shown for 4 days. While there is no study done beyond 4 days, the inhibitory effect on CYP3A4 is estimated to last at least for 6 days after single dose administration of AKYNZEO. Close monitoring for sign of adverse events for concomitant CYP3A4 substrates especially with narrow therapeutic window is recommended. Midazolam: When netupitant 300 mg was co-administered with oral midazolam, the mean Cmax and AUC of midazolam was increased by 36% and 226%, respectively. Based on this result, netupitant is considered a moderate CYP3A4 inhibitor. The effect of netupitant on midazolam was studied only on the day of co-administration. Dexamethasone: The potential effect of netupitant on PK of dexamethasone, a CYP3A4 substrate was studied. Palonosetron is not an inhibitor of CYP3A4; therefore, its effect was not studied. The coadministration of a single dose of netupitant (300 mg) with oral dexamethasone regimen (20 mg on Day 1, followed by 8 mg b.i.d. from Day 2 to Day 4) significantly increased the exposure to dexamethasone in a dose-dependent manner. When netupitant was co-administered on Day 1, the mean AUC of dexamethasone was increased by 1.7-fold on Day 1 and up to 2.4fold on Day 2 and Day 4. The potential inhibitory effect of netupitant on CYP3A4 was not studied beyond Day 4. Therefore the sum of individual [I]/Ki 7 for netupitant and its metabolites was calculated. The mean [I]/Ki ranged 0.0134-0.167 (ranged 0.089-0.285) on Day 4 when the AUC of dexamethasone was still 2-fold higher than the control. The mean total [I]/Ki decreased to below 0.1 on Day 6 and was 0.093 (0.049- 0.210) at 140 h post-dose. This estimation suggests that drug interaction via CYP3A4 inhibition by netupitant is less likely on Day 6 but cannot be ruled out. Of note in this study the half-life of netupitant and the metabolite M1 of which the Ki for CYP3A4 was lower than that of netupitant, was estimated to be shorter than those observed in other studies suggesting a possibility of underestimation of the duration of inhibitory effects on CYP3A4. Chemotherapeutics 7 The sum of [I]/Ki values for netupitant, M1, M2, and M3 using observed plasma concentrations up to 120 h post-dose and extrapolated plasma concentrations beyond 120 h. Ki values were obtained from in-vitro inhibition studies. 7 Reference ID: 3516218 The systemic exposure to intravenously given chemotherapeutic agents (docetaxel, etoposide,) that are metabolized by CYP3A4 was increased to a different degree (10-49%) when AKYNZEO was co-administered than when chemotherapeutic agents were coadministered with palonosetron alone in cancer patients. When co-administered with AKYNZEO the mean Cmax and AUC of intravenously administered docetaxel were 49% and 35% higher, respectively. The systemic exposure to intravenously administered etoposide and cyclophosphamide was also increased when AKYNZEO was coadministered by 10-28% Oral contraceptive AKYNZEO, when given with a single oral dose of 60 μg ethinyl estradiol and 300 μg levonorgestrel increased the mean AUC of levonorgestrel by 46% while AKYNZEO had no significant effect on the mean AUC of ethinyl estradiol. P-glycoprotein substrate Digoxin When netupitant 450 mg was concurrently administered with digoxin, the systemic exposure and urinary excretion of digoxin at steady-state was not significantly affected. 2 Question-Based Review 2.1 General Attributes of the drug 2.1.1 What pertinent regulatory background or history contributes to the current assessment of the clinical pharmacology and biopharmaceutics of this drug? Netupitant is a NK1 receptor antagonist and a new molecular entity. It is proposed as an oral fixed dosage form in combination with palonosetron, a 5-HT3 receptor antagonist, for the prevention of chemotherapy-induced nausea and vomiting after moderately or highly emetogenic chemotherapy. Netupitant is not approved for any indications. Currently aprepitant (EMEND®), NK1 receptor antagonist is commercially available for oral and intravenous administration for the CINV and PONV. Palonosetron hydrochloride is available as an IV formulation (ALOXI®) marketed in the US since September 2003 for prevention of chemotherapy-induced nausea and vomiting (CINV) and post-operative nausea and vomiting (PONV). The oral formulation, soft gelatin capsule (0.50 mg) of palonosetron was approved in the US for CINV (2008) but has not been marketed in the US. Aloxi Capsule is indicated for: • Moderately emetogenic cancer chemotherapy -- prevention of acute nausea and vomiting associated with initial and repeat courses Aloxi for injection is indicated for: 8 Reference ID: 3516218 Moderately emetogenic cancer chemotherapy - prevention of acute and delayed nausea and vomiting associated with initial and repeat courses 0 Highly emetogenic cancer chemotherapy -- prevention of acute nausea and vomiting associated with initial and repeat courses The indication for PONV is not proposed in this application. To conform to the combination rule. the sponsor conducted the ef?cacy trials in comparison to oral Aloxi. In addition. to establish the contribution of oral palonosetron to the prevention of nausea and vomiting induced by HEC. a non-inferiority trial in comparison to intravenous Aloxi was conducted. The sponsor does not propose HEC-CINV indication for oral Aloxi based on the non-inferiority trial. With recent re-classi?cation of emetogenicity of anthracycline-cyclophosphamide regimen used in a clinical trial for MEC from MEC to HEC. the exact indication for regarding the prevention of nausea and vomiting by the degree of emetogenicity of chemotherapy is lmder discussion. The discussion on the indication is deferred to the clinical review. 2.1.2 What are the highlights of the chemistry and physical-chemical properties of the drug substance, and the formulation of the drug product as they relate to clinical pharmacology and biopharmaceutics review? Netupitant Netupitant is white to off-white powder very soluble in water. Netupitant is not hygroscopic. Molecular formula: Molecular weight 578.61 g-mol'l Log at pH (5M4) 00(4) Log at pH Log pKal pKaZ Figure 1. Structure of Netupitant Reference ID: 3516218 Palonosetron Palonosetron hydrochloride is a white to off-white crystalline powder. It is freely soluble in water. Palonosetron hydrochloride is essentially non-hygroscopic. Palonosetron hydrochloride drug substance is synthesized solely as the (b) (4) isomer. Figure 2. Structure of palonosetron Akynzeo®, a netupitant-palonosetron fixed-dose combination (FDC) capsule contains three 100 mg netupitant tablets and one palonosetron 0.5 mg softgel capsule. The palonosetron 0.5 mg softgel capsule in combination capsule is similar to the currently approved Aloxi capsule with the (b) (4) exceptions of and capsule size. In the dose-finding study and the study establishes the contribution of netupitant and the efficacy of combination therapy for HEC induced nausea and vomiting, netupitant and palonosetron were also given as an extemporaneous combination. In this trial netupitant was formulated in a standard hard gelatin capsule, while palonosetron was the soft gelatin capsule currently approved. A bioequivalence study was conducted between the FDC and the extemporaneous combination. 2.1.3 What are the proposed mechanism(s) of action and therapeutic indication(s)? Netupitant is a substance P/neurokinin 1 (NK1) receptor antagonist. Palonosetron is a 5-HT3 receptor antagonist. Chemotherapeutic agents exert their emetic stimulus via processes that involve the release of serotonin and substance P and subsequent activation of the 5-HT3 and NK1 receptors. The proposed indications are as below. • • prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy 10 Reference ID: 3516218 The proposed indications have been previously granted. With updated classification of emetogenicity of certain chemotherapeutic regimens, the indications are currently being reconsidered. 2.1.4 What are the proposed dosage(s) and route(s) of administration? Akynzeo® is a solid oral Netupitant/Palonosetron 300mg/0.50 mg fixed dose combination capsule (FDC), composed of one size 0 hard gelatin capsule. Each capsule contains three intermediate netupitant tablets (3×100 mg netupitant tablets) and one intermediate palonosetron 0.5 mg softgel capsule. The proposed dosage regimen is 1 hour before chemotherapy by oral administration without regard of food. 2.2 General Clinical Pharmacology 2.2.1 What are the design features of the clinical pharmacology and clinical studies used to support dosing or claims? The clinical efficacy and safety of Akynzeo is supported by three clinical trials in cancer patients (NETU-07-07, NETU-08-18, NETU-10-29). In addition, an efficacy trial PALO-10-01 was conducted to establish the contribution of oral palonosetron to the efficacy in prevention of CINV associated with cisplatin-based highly emetogenic chemotherapy. In PALO-10-01, the efficacy of oral palonosetron was evaluated in comparison to intravenous palonosetron, which is approved for the prevention of acute CINV associated with HEC. In clinical pharmacology program, pharmacokinetics of netupitant and its metabolites was extensively studied and 14 clinical pharmacology related in-vitro studies were conducted. Some clinical pharmacology studies were conducted for netupitant alone and some studies were conducted for the combination product. Because the proposed dosage regimen is a single dose per chemotherapy cycle, the clinical pharmacology studies for netupitant and for FDC were mostly conducted after a single dose administration except multiple dose PK for netupitant. On the other hand the clinical pharmacology of palonosetron was mostly referenced to the previously conducted studies in support of oral and intravenous Aloxi. For the list of studies, please see Appendix 1. 2.2.2 What is the basis for selecting the response endpoints and how are they measured in clinical pharmacology and clinical studies? The proposed indication is to prevent chemotherapy induced nausea and vomiting (CINV). 11 Reference ID: 3516218 Accordingly clinical efficacy was evaluated based on the proportion of patients with complete response (CR) (defined as no emesis, no rescue medication) in the 0-24 h (acute phase), the 25120 h (delayed phase) and the 0-120 h (overall phase) post chemotherapy. Other efficacy endpoints such as time to first emetic episode, time to first rescue medication, time to treatment failure (based on time to the first emetic episode or time to the first rescue medication, whichever occurs first) were also evaluated. In two phase 1 pharmacodynamics studies, NK1-receptor occupancy in brain and the prevention of apomorphine-induced nausea and vomiting were explored for netupitant. 2.2.3 Are the active moieties in the plasma and urine appropriately identified and measured to assess pharmacokinetic parameters and exposure-response relationships? Yes. Both netupitant and palonosetron were quantified in the plasma. Netupitant was also measured in urine. See Section 2.6 for more details. 2.2.4 Exposure-Response Evaluation 2.2.4.1 What are the characteristics of the exposure-response relationships for efficacy? Clinical efficacy The dose-response relationship to evaluate the contribution of netupitant at different dose levels to the CR rate in addition to palonosetron was explored in patients receiving cisplatin-based highly emetogenic chemotherapy (HEC) (NETU-07-07). In Study NETU-08-18 in which the combination of 300 mg netupitant/0.5 mg palonosetron was studied in patients receiving anthracycline-cyclophosphamide regimen, PK samples were collected. However, a formal assessment of exposure-response relationship could not be made due the limited PK data collected in the clinical studies as only 117 patients out of 726 (~16%) in AKYNZEO arm in the study NETU-08-18. In NETU-07-07, there was no significant dose-response relationship in the proportion of patients with CR in overall and delayed phase among three netupitant doses of 100 mg, 200 mg and 300 mg. PK samples were not collected so the concentration-response relationship was not studied. In this study netupitant was co-administered with oral palonosetron at the approved dose of 0.5 mg as well as with oral dexamethasone (Dexa) as a standard of care. The dosage regimen for oral dexamethasone was reduced in combination treatment arms for the increase in systemic exposure to dexamethasone due to inhibition of metabolizing enzyme by netupitant. As such for Palo only treatment, oral Dexa was given at 20 mg on Day 1 and 8 mg twice daily on Days 2-4. For treatment arm with netupitant, oral Dexa dose was reduced to 12 mg on Day 1 and 8 mg once daily on Days 2-4. This study was designed to compare the complete response rate with the Palo 12 Reference ID: 3516218 alone treatment so was not powered to show differences among the combinations with different netupitant doses. (Table 1and Figure 3) The complete response rate over 120 hours after treatment with PALO+NETU ranged 87.4% and 89.6% and was similar among netupitant doses. All treatment with PALO+NETU showed statistically higher CR rate in overall phase and delayed phase while only combination with NETU 300 mg was statistically better than PALO alone for prevention of CINV in acute phase. Based on this study the combination of PALO 0.5 mg with NETU 300 mg was selected for subsequent efficacy trials. Study NETU-07-07 also established the contribution of netupitant component to the prevention of CINV in addition to Palo component and the clinical efficacy of the combination therapy for the prevention of CINV associated with HEC, cisplatin-based chemotherapy. The review of statistical analysis of NETU-07-07 is deferred to the biostatistics reviewer. Table 1 Complete Response Rate (NETU-07-07) Source: NETU-07-07, Table 16 and 18 1 p-value from logistic regression analysis including gender as covariate 13 Reference ID: 3516218 Figure 3 Complete Response Rate in the Overall, Acute and Delayed Phase (NETU-07-07) The time to first emetic episode was significantly longer for patients treated with netupitant compared to palonosetron alone. The Kaplan-Meier plot showed that the 300 mg dose appeared to have the largest effect, with the curves starting to diverge approximately 6-8 hours after chemotherapy (Figure 4). Curves show the higher efficacy of netupitant 300 mg from 24 through 44 hours compared to lower netupitant doses. Figure 4 Time to first emetic episode BEST AVAILABLE COPY In a phase 3 trial, AKYNZEO demonstrated superiority to PALO monotherapy in prevention of delayed (primary efficacy endpoint), acute, and overall time periods (secondary efficacy endpoints) (Table 2). 14 Reference ID: 3516218 Table 2 Complete Response delayed, acute and overall cycle 1 (NETU-08-18) Pharmacodynamics Study Doses of netupitant used in study NETU-07-07 (100 mg, 200 mg and 300 mg) were selected based on exploratory pharmacodynamics studies such as apomorphine challenge study and NK1 receptor occupancy study. An initial study NP16602 using an apomorphine challenge model showed that administration of netupitant reduced the incidence of vomiting induced by apomorphine compared to placebo. Netupitant appeared to reduce the incidence of emetic episodes in a concentration dependent manner. No vomiting occurred when plasma concentrations were >300 ng/mL at the time of the challenge compared with 75% of the subjects receiving placebo (Table 3). 15 Reference ID: 3516218 Table 3 Apomorphine challenge study: Summary of Vomiting episodes and Area under the Nausea VAS 8 The extent of receptor occupancy (RO) by netupitant was measured by Positron Emission Tomography (PET) technology (NETU-06-08) after single dose administration of netupitant at 100 mg, 300 mg and 450 mg. In this study, the RO (90% or higher) close to the expected Cmax was achieved after a single dose of netupitant in the occipital cortex (100-450 mg), frontal cortex (100–450 mg), striatum (300 and 450 mg) and anterior cingulate (100 and 450 mg). The timeand concentration-dependent NK1-RO was apparent in Striatum region but it was not as apparent in other brain regions. In an exploratory PK/PD analysis using the Sigmoid Emax model, 225 ng/mL was estimated to correspond to an NK1-RO of 90% in stratum region. A comparison of the results for the dose groups (100 mg, 300 mg and 450 mg) showed a general but low increase in NK1-ROs with increasing dose. (Table 4, Figure 5) Together the individual Cmax values in the receptor occupancy study and the mean Cmax observed in other single oral dose studies (from 92 to 168 ng/mL after 100 mg and from 335 to 747 ng/mL after 300 mg), a single oral dose between 100 and 300 mg of netupitant was suggested to be needed to reach an NK1-RO level in striatum of at least 90% close to the expected Cmax in the majority of the brain regions evaluated in the PET study. Initially 200 mg netupitant was proposed to be the clinical dose so the tQT study was conducted for a combination with netupitant 200 mg and the 3-fold higher supratherapeutic dose. Later the the combination with 300 mg netupitant was selected for clinical trials and for marketing (NETU07-07). 8 The degree of nausea was recorded using a 100 mm visual analogue scale (VAS) ranging from 0 (no nausea) to 100 (severe nausea) 16 Reference ID: 3516218 Table 4 Average Neurokinin-1 Receptor Occupancy (NK1-RO) in Striatum at 6, 24, 48, 72, and 96 Hours after Administration of a Single Dose of 100, 300, and 450 mg Netupitant Figure 5 NK1-RO-netupitant concentration in (A) Striatum and (B) Frontal cortex (A) Striatum 17 Reference ID: 3516218 (B) Frontal cortex 2.2.4.2 What are the characteristics of the exposure-response relationships for safety? In Study NETU-07-07, the netupitant dose-dependent increase in TEAE was not evident. The incidence of TEAEs was generally similar among treatment groups (Table 5). The overall rate of TEAE was higher after the combination treatment with NETU 300 mg/PALO 0.5 mg compared to oral PALO 0.5 mg alone i.e. 70% vs. 61% (Table 6). For detailed review of safety profile, please see the clinical review. Table 5 Summary of subjects with treatment emergent adverse events 18 Reference ID: 3516218 Table 6 Overview of Treatment-emergent Adverse Events combined – Cycle 1 (Phase 2/3 Cancer Patients) 2.2.4.3 Does this drug prolong the QT or QTc interval? No significant QTc interval prolongation was observed when a combination of 600 mg NETU and 1.5 mg PALO was administered to healthy subjects (NETU-07-20) 9 (Table 7, Figure 6). No significant effect of PALO on QTc interval was previously shown at doses up to 2.25 mg after intravenous administration of PALO alone. Consistently there was no evident exposure-response relationship between ddQTcF and concentrations of netupitant and its metabolites, M1, M2, and M3 as well as concentrations of palonosetron and its metabolite M4 and M9 in the tQT study with doses up to netupitant 600 mg/palonosetron 1.5 mg. Therefore this study demonstrates the lack of effects on QTc interval by addition of NETU up to 600 mg. At the time of the tQT study, 200 mg NETU/0.5 mg PALO was predicted to be a therapeutic dose so the 3-fold higher dose i.e. NETU 600 mg/PALO 1.5 mg was chosen as a supratherapeutic dose. The dose-proportional increase in the systemic exposure was observed for both netupitant and palonosetron. The supratherapeutic dose in this study provides the safety margin of 2 fold for the proposed dose for Akynzeo® i.e. NETU 300 mg/PALO 0.5 mg. The supratherapeutic dose cover the Cmax observed in patients with moderate hepatic impairment and in healthy subjects when ketoconazole, a strong CYP3A4 inhibitor was co-administered. 9 Please see the IRT-QT team reviews of the thorough QT study dated 1/19/2010 (IND 73,493 SDN 024) and 3/3/14 (NDA 205-718) for more details. 19 Reference ID: 3516218 Table 7 The Point Estimates and the 90% CIs Corresponding to the Largest Upper Bounds for netupitant/palonosetron (200 mg/0.50 mg and 600 mg/1.50 mg) and the Largest Lower Bound for Moxifloxacin (Analysis by FDA IRT-QT team) Figure 6 Mean (90% CI) ddQTcF-time profile From IRT-QT review of tQT study (1/20/2010), Page 24) 2.2.4.4 Is the dose and dosing regimen selected by the sponsor consistent with the known relationship between dose-concentration-response, and are there any unresolved dosing or administration issues? No. The dose-response relationship was not evident among the combination with netupitant 100 mg-300 mg and the CR rate greater than 85% for primary and key secondary endpoints at all doses. However, the proposed dose is supported by efficacy trials and NETU 300 mg/PALO 0.5 mg combination was the only combination that showed significantly higher CR rate than PALO alone for all primary and key secondary endpoints to support the efficacy of the combination and the contribution of netupitant to the prevention of CINV associated with cisplatin-based highlyemetogenic chemotherapy. 20 Reference ID: 3516218 2.2.5 Pharmacokinetic Characteristics 2.2.5.1 What are the PK characteristics of netupitant and its major metabolite? This review will mainly discuss the PK characteristics of netupitant which is a new molecular entity. The general PK characteristics for palonosetron were previously studied for the approval of oral and intravenous palonosetron. Netupitant After oral administration, netupitant is absorbed slowly reaching peak in approximately 4-5 hours. Plasma concentrations were measurable between 0.25 and 3 hours and plasma concentrations followed a first order absorption process. Netupitant was eliminated from the body in a multi- exponential fashion, with an apparent mean terminal half-life ranging, on average, from 30 to 100 hours (for doses of 30 mg to 450 mg). Substantial distribution of netupitant into tissues was indicated by a large volume of distribution (Vz/F) ranged from approximately 850 to over 2000 L. The apparent oral plasma clearance (CL/F) ranged from approximately 10 to 35 L/h, indicating that the compound is slowly cleared from blood. The renal clearance of netupitant ranged 4.2-39 mL/h (Figure 7). Netupitant undergoes extensive metabolism to form three major active metabolites in plasma. Metabolites M1 and M3 reached the maximum concentration much later (17-32 h on average) than netupitant and metabolite M2 (approximately 5 h). Metabolites M1, M2 and M3 accounted on average for 29%, 14%, and 33% of parent exposure, respectively, in terms of AUC0-t in the ADME study. A minor metabolite identified later during the development, M4 accounted for about 3% of parent drug exposure. All four metabolites were shown to bind to human NK1 receptor in vitro. Figure 7 Mean (±SD) plasma netupitant concentration (ng/mL) vs. time profiles after single oral administration of AKYNZEO (T) and Netupitant and Palonosetron extemporaneous combination (R) used in the dose-finding study (NETU 09-07) (A) 21 Reference ID: 3516218 Best Available Copy (B) Palonosetron 10 After single dose administration of AKYNZEO in healthy subjects, the peak plasma concentrations for palonosetron were reached in about 5 hours (Figure 8). Distribution Palonosetron has a volume of distribution of approximately 8.3 ± 2.5 L/kg. Approximately 62 % of palonosetron is bound to plasma proteins. Metabolism Palonosetron is eliminated by multiple routes with approximately 50 % metabolized to form two primary metabolites: N-oxide-palonosetron and 6-S­hydroxy-palonosetron. These metabolites each have less than 1 % of the 5-HT3 receptor antagonist activity of palonosetron. In vitro metabolism studies have suggested that CYP2D6 and to a lesser extent, CYP3A4 and CYP1A2 are involved in the metabolism of palonosetron. However, clinical pharmacokinetic parameters are not significantly different between poor and extensive metabolizers of CYP2D6 substrates. Elimination (b) (4) 10 Package Insert for Aloxi I.V. 22 Reference ID: 3516218 Figure 8 Mean (±SD) plasma palonosetron concentration (ng/mL) vs. time profiles after single oral administration of AKYNZEO(T) extemporaneous combination (R) (NETU 07-07) and Netupitant and Palonosetron Best Available Copy (A) (B) 2.2.5.2 What are the single dose and multiple dose PK parameters? Multiple dose PK was studied for netupitant only in this development program. After once daily dosing for 7 days, the systemic exposure gets about 3 fold higher than that after single dose as expected from a long half-life of netupitant. There was a greater than doseproportional increase in the systemic exposure as the dose increases from 100 mg to 300 mg after single and multiple doses while the dose-proportional increase in the systemic exposure was observed as the dose increases from 300 mg to 450 mg (Table 8,Table 9, Table 10). Table 8 Mean ± SD (%CV) Pharmacokinetic Parameters of netupitant and palonosetron after single dose administration of Akynzeo® in healthy subjects (NETU-09-07) Tmax (h) 434.1 ± 242.1 5 (55.7) (2-12) 1.5 ± 0.4 5 Cmax (ng/ml) Netupitant (n=47) Palonosetron AUCt (ng*h/ml) 12321 ± 5209.6 (42) 51.2 ± 18 23 Reference ID: 3516218 AUCinf T1/2 (h) (ng*h/ml) 14401.6 ± 95.6 ± 58.8 7307.8 (50.7) 56.7 ± 18.6 44.2 ± 15.2 (n=47) (26.7) (1-12) (35) (32.8) Table 9 Mean ± SD (%CV) Pharmacokinetic Parameters of netupitant and its metabolites after single dose administration of Akynzeo® under fasting condition in healthy subjects (NETU-10-12) Cmax (ng/ml) Netupitant (n=22) M1 M2 M3 596.4±233.0 (39.1) 43.7±12.4 (28.4) 202.2±97.3 (48.1) 81.8±37.9 (46.3) Tmax (h) AUCt (ng*h/ml) 17150±6122 (35.7) 4933±1452 12 (6-24) (29.4) 2076±929.1 4.5 (3-5.5) (44.8) 12 (4.5- 5348±2323 24) (43.4) 5 (4-8) AUCinf (ng*h/ml) 20039±8396 (41.9) 5886±2235 (38.0) 2254±945.3 (41.9) 5841±2654 (45.4) T1/2 (h) 101.2±52.8 (52.2) 82.2±37.0 (45.0) 48.9±45.7 (93.4) 65.6±29.1 (44.4) Table 10 Mean (± SD) Pharmacokinetic Parameters of Netupitant after single and multiple dose administration in healthy subjects (NP16601) Dose mg 100 Single dose (n=8) Cmax Tmax (ng/ml) (h) 111 ± 25.6 4.7 ± 0.71 (23) (15) 300 599 ± 228 (38) 5.5 ± 2.83 (51) 450 720 ± 255 (35) 5.4 ± 1.06 (20) AUC0-23 5 (ng*h/ml) 1360 ± 294 (22) 6400 ± 1700 (26) 9670 ± 3380 (35) Multiple doses (n=8) Cmax Tmax (ng/ml) (h) 269 ± 52.2 4.5 ± 0.93 (19) (21) 1060 ± 5.4 ± 2.94 202 (54) (19) 1790±770 (43) 7.1 ± 6.85 (96) AUC0-23 5 (ng*h/ml) 4160 ± 998 (24) 17100 ± 2850 (17) 28800 ± 13000 (45) AUCinf (ng*h/ml) 21400 (29.7) 93800 (32.6) 139000 (46.4) AF1 3.06 (12) 2.74 (12) 2.93 (14) 1 Accumulation Factor PK blood samples were collected up to 168 h post-last dose 2.2.5.3 How does the PK of the drug and its major active metabolites in healthy volunteers compare to that in patients? (NETU-10-09) Overall, the exposure to netupitant and its metabolites and palonosetron in cancer patients, in terms of Cmax, AUC0-t and AUC0-∞ values, were consistent with data reported in healthy subjects. Netupitant PK of netupitant was studied in cancer patients who are treated with moderate or highly emetogenic chemotherapy. In Study NETU-10-09, a single dose administration of combination 24 Reference ID: 3516218 (Netu 300 mg and Palo 0.5 mg) with selected chemotherapy agents (docetaxel, etoposide, or cyclophosphamide) to evaluate the pharmacokinetics of netupitant, its main metabolites and palonosetron along with the PK of the chemotherapeutic agents In a cross study comparison, PK parameters for netupitant in cancer patients were similar to that in healthy subjects (Table 11). The systemic exposure to netupitant in cancer patients were within the range observed in healthy subjects across multiple studies and seems to be independent of the chemotherapeutic regimen co-administered i.e. docetaxel, etoposide, or cyclophosphamide. Consistently the population PK analysis showed that PK for netupitant in cancer patients was similar to that in healthy subjects. Table 11 Pharmacokinetic Parameters (mean ± SD) of netupitant after single administration of combination (Netu+Palo) in cancer patients Healthy subjects1 (n=47) Patients with cancer2 Cmax (ng/mL) 434 ± 242 (56) 486±48.9 (51) Tmax3 (h) 5 (2-15) 4 (4-6) NP+Eto (n=12) 519±263.2 (51) 4 (3-8) NP+Cyc (n=10) 477±231.3 (48) 4.24 (2.1-5) NP+Doc (n=8) AUCt (ng*h/mL) 12321 ± 5210 (42) 14280 ± 4703 (33) 15220 ± 5956 (39) ± 13480 3560 (26) AUCi (ng*h/mL) 14402 ± 7308 (51) 16130 ± 4955 (31) (n=2) 18160±8296 (46) (n=9) 16440±4897 (30) (n=5) 1 Study NETU-09-07: Following single dose administration of FDC (Source: Listing 16.2.6.1.) Study NETU-10-09: Following single dose administration of FDC 3 Median (min-max) 2 In cancer patients, exposure to M1 and M3 relative to netupitant was similar to healthy subjects, accounting for 8-14% for Cmax and approximately 30-35% for AUC0-t (Table 12). Table 12 Mean Exposure Data to Metabolites M1, M2, M3 after Administration of 300 mg Netupitant to Cancer Patients (NETU-10-09) M1 Cmax Treatment: FDC capsule with: Docetaxel n AUC0-t ng/mL h·ng/mL 8 8 M2 AUC0-∞ Cmax AUC0-t AUC0-∞ Cmax AUC0-t ng/mL h·ng/m L 0a 25 Reference ID: 3516218 M3 8 AUC0 ng/mL h·ng/m L ng/m L h·ng/m L ng/mL 8 2 8 8 4 (N=8) Mean 36 4356 NA 361 4746 8527 64 4915 5946 CV (%) 32 41 NA 57 57 10 30 27 34 ratio M/P % 7 31 NA 74 29 53 13 34 37 Etoposide n 12 12 4 12 12 6 12 12 8 (N=12) Mean 41 4579 4203 219 2785 3719 74 5038 5294 CV (%) 34 34 44 55 42 41 44 31 32 8 30 23 42 15 20 14 33 29 n 10 10 4 10 10 4 10 10 6 Mean 40 4705 5993 215 2594 3061 68 4530 5821 CV (%) 32 25 18 28 28 30 58 37 33 8 35 36 45 16 19 14 34 35 ratio M/P Cyclophospham ide (N=10) ratio M/P NOTE: Ratio between Cmax and AUC of metabolites vs parent are also provided (M/P). For a better comparison among groups, AUC0-t is also reported as in some cases, the description of the terminal phase was not completely reliable. a not reliable results in this patient group. NA: not accountable. Digits were rounded. No significant influence of chemotherapeutics (doxorubicin, epirubicin, fluorouracil) on the disposition of netupitant and palonosetron was noted by population PK analysis performed for the subset of patients (n=117) during a phase 3 trial (NETU-10-02). Palonosetron In cross-study comparison, mean AUC and mean Cmax for palonosetron tended to be lower in cancer patients than in healthy subjects. PK of palonosetron was generally similar between healthy subjects and cancer patients (Table X). PK of palonosetron in cancer patients was similar to the previous observation (Table 13, Table 14). Table 13 Pharmacokinetic Parameters (mean±SD) of Palonosetron after single administration of combination (Netu+Palo) in cancer patients Healthy subjects1 (n=47) Patients with cancer2 NP+Doc (n=8) NP+Eto (n=12) NP+Cyc (n=10) Cmax (ng/mL) 1.53 ± 0.39 (26) Tmax3 (h) 5 (1-12) AUCt (ng*h/mL) 52.2 ± 18 (34) AUCi (ng*h/mL) 56.7 ± 18.6 (32.78) 1.16 ± 0.38 (33) 0.90 ± 0.35 (38) 0.85 ± 1.9 (22) 4.75 (1-12) 5.5 (0-12) 5 (2-7) 74.9 ± 31.8 (43) 43.8 ±11.7 (27) 40.4 ± 13.7 (34) 85.6 ±4.4 (51) 49.3 ±12.8 (26) 48.1 ±15.6 (32) 1 Study NETU-09-07: Following single dose FDC administration under fasting condition (Source: Listing 16.2.6.4.) 2 Study NETU-10-09: Following single dose administration of FDC administration 26 Reference ID: 3516218 3 Median (min-max) Table 14 From Aloxi capsule Package Insert The mean Cmax and AUCinf of palonosetron was approximately 30% and 65% higher, respectively in the docetaxel group than in the etoposide and cyclophosphamide groups (Figure 9). The reason for apparently higher systemic exposure to palonosetron in patients who received docetaxel is unclear. 2. 2.5.4 What are the characteristics of netupitant absorption? Measurable plasma netupitant concentrations were detected between 15 minutes and 3 hours after single dose oral studies. Plasma concentrations reached Cmax in approximately 5 hours. The absolute bioavailability was not adequately studied. Nevertheless, in a cross-study comparison, the total clearance and the volume of distribution were similar between oral and intravenous administration although somewhat higher after oral administration (Table 15). The exposure to metabolites was generally comparable with higher systemic exposure to M1 after oral than i.v. administration (Table 16). Of note there was greater than dose-proportional increase in the systemic exposure to netupitant from 100 mg to 300 mg in dose-ascending studies. Due to the cross-study comparison and the small number of subjects support the PK data, a reliable conclusion cannot be drawn from this comparison. Of note, after intravenous administration, infusion site thrombosis was noted in some subjects. According to the study report, the intravenous formulation for netupitant will not be further studied. 27 Reference ID: 3516218 Figure 9 Mean Palonosetron Plasma Concentrations –time after administration of FDC with chemotherapy – PK Population Excluding Site 93 Patients Best Available Copy Table 15 Mean (%CV) PK Parameters after single dose administration of oral or intravenous Netupitant at 100 mg in healthy subjects 1 Study RO16603: PK sampling up to 168 h post-dose NETU-11-01: intravenous infusion over 15 min; infusion site thrombosis occurred in 2 patients; PK sampling up to 120 h after start of infusion 2 28 Reference ID: 3516218 Table 16 Mean PK Parameters for metabolites of netupitant after single dose administration of oral or intravenous Netupitant at 100 mg in healthy subjects PK parameters Oral Netupitantl 1V. Netupitantz Mean (CV M1: metabolite Cmax 14.2 9.1 [pg/L] (22.9) (16.5) AUC(0-last) 978 744 [h'ng/L] (26.2) (130) 170 1224 [hung/L] (15.6) (20.8) M2: N?oxide metabolite Cmax-MZ 34.9 39.6 [pg/L] (21.3) (34.5) AUC(O-last) 279 419.7 [burg/L] (46.8) (34.6) AUC(0-int) 500 494 [bong/L] (23.2) (34.4) M3: OH-methyl metabolite Cmax 25 .2 23.5 lug/ll (24.2) (24.9) AUC(O-last) 1300 1464 [mg/L] (33.9) (34.7) AUC(0-int) 1570 I 712 [hug/L] (27.6) (33.7) 1Study R016603: PK sampling up to 168 post-dose 1-01: PK sampling up to 120 after start of infusion The possibility enterohepatic circulation of netupitant and metabolite M3 was suggested by multiple peaks in the plasma concentration-time pro?le (Figure 10. Figure 11). Figure 10 Mean netupitant concentration-time profile (NETU 07-20) no I ?11huge-The b2: LSD 2.50 PAZ. ESSETRCEI i; {.52 11:? b3: ?22 1' 152' - 1.53 13 :3 3.32 . est AvaIlable Copy Reference ID: 3516218 29 Figure 11 Mean metabolite M3 concentration-time pro?le (NETU 07-20) 150 15? m? t] rB-s?Th! [I'3.5: b3: ?22 :3 :czr292mr: t: is: 31.5: 3.: Picrzosrmor: :3 c.5: 3.31 1' BeSt Ava?able copy 2.2.5.5 What are the characteristics of drug distribution? Netupitant is extensively distributed into tissues and is highly bound to plasma protein Similarly. metabolites M1. M2. M3 plasma proteins ranged between 97.7 to 99%. In vitro Blood/Plasma ratios were 0.69 (netupitant and M2). 0.61 (M3) and 1.1 (MI) (Table 17) The apparent volume of distribution after administration of 300 mg netupitant ranged. on average. from approximately 800 to more than 2000 L. suggesting an extensive distribution out of the circulation. In cancer patients. the volmne of distribution did not change. The volume of distribution in the central compartment (V2) and in the peripheral compartment (V3) in cancer patients were estimated through a population PK model. The ?nal median V2 and V3 estimates accomrted for 486 and 1170 L. respectively. Inter- individual variability for V2 was moderate The protein binding was evaluated by equilibrium dialysis at and pH 7.4 after addition of l4C-labeled netupitant or its metabolites to hmnan plasma. Human plasma protein binding of netupitant is greater than 99.5% at drug concentration ranging ?om 10-1300 ng/ml and protein binding of its major metabolites (M1, M2 and M3) are greater than 97% at drug concentrations ranging from 100 to 2000 ng/mL. The protein binding and blood/plasma ratio was concentration independent over a concentration range which exceeds the maximum plasma concentrations expected in man. 30 Reference ID: 3516218 Table 17 Protein binding and blood/plasma ratio for netupitant and major metabolites Netupitant M1 (RO0681133) M2 (RO0713001) M3 (RO0731519) Plasma protein Binding Mean ± SD (tested concentration range) 99.67 ± 0.032 % (10-1300 ng/ml) 99.09 ± 0.06 (120-2540 ng/mL) 97.7 ± 0.1 (115-2500 ng/ml) 99.12 ± 0.05 (115-2000 ng/ml) blood/plasma ratio (λ) Mean ± SD (tested concentration range) 0.69 ± 0.01 (50- 1000 ng/ml) 1.1 ± 0.02 (125-2460 ng/ml) 0.69 ± 0.01 (74-2500 ng/ml) 0.61 ± 0.02 (118-2310 ng/ml) 2.2.5.7 What are the characteristics of drug metabolism? In vitro studies showed that the metabolism of netupitant to M1, M2 and M3 is mainly mediated by CYP3A4 and lesser extent by CYP2C9 and CYP2D6 (study NETU-13-21). Netupitant was shown to undergo extensive metabolism, forming both phase 1 and phase II metabolites. After oral administration, more than 30 metabolites were identified in fecal samples and 13 metabolites in urine samples. Three metabolite, M1, M2, and M3 were detected in plasma and measured in pharmacokinetics studies for netupitant (Figure 10). In later development phase, an additional metabolite, M4 was identified. In humans extent of exposure (AUC) data indicated that M1 has the highest exposure relative to the parent (35%), followed by M3 (29%). M2 and M4 corresponded to the 13% and 3% of the parent, respectively. Mean Cmax corresponded to 11-12% of parent netupitant for M1, 41-46% for M2, 15-16% for M3 and 6% for M4. In vitro binding studies showed that M1, M2, M3, and M4 bind to the human NK1 receptor. In vitro studies showed netupitant is metabolized mainly by CYP3A4 and to a lesser degree CYP2C9 and CYP2D6. In vitro CYP3A4 appears to metabolize netupitant to metabolites M1 and M2. 31 Reference ID: 3516218 Figure 12 The proposed metabolic pathways for netupitant and chemical structure of netupitant metabolites identified in human plasma Figure 13 Mean plasma concentrations-time profile for netupitant and its metabolites (NETU-07-01) * After single dose administration of 450 mg netupitant 2.2.5.8 What are the characteristics of drug excretion? Following oral administration, netupitant is mainly excreted via feces over prolonged period of time. Netupitant and its metabolites are primarily eliminated through hepatobiliary route. A single oral dose of [14C]-Netupitant (187-264 mg) as oral suspension was administered to 6 healthy male subjects in an ADME study. Blood, urine and feces were collected up to 336 h postdose (Day 15). 32 Reference ID: 3516218 Approximately 50% of the administered radioactive dose was recovered within 120 h post-dose and an average of 73% of administered radioactivity was recovered within 336 h: 70% from the feces and 4% from the urine. Thirteen phase I and II metabolites (glucuronic acid derivatives) were identified in the urine within 192 h collection time and about 30 metabolites were identified in the feces. Netupitant and metabolites M1 and M3 were not detected in the urine collected over 336 h. Excretion in urine over 192 h post-dose was mainly represented by metabolites accounting for approximately 3% of the radioactive administered dose. The contribution of renal excretion of unchanged netupitant to the total clearance is negligible. After single dose of 450 mg netupitant, unchanged netupitant was not detectable in most of urine sample collected over 120 hours from 18 healthy subjects. In 5 out of 18 subjects, netupitant was detectable in urine collected within 48 hours and the renal clearance of netupitant ranged 0.070.65 ml/min (4.2-39 ml/h) in those subjects. (NETU-06-27) Since the recovery of the radioactivity was less than 90% at 336 h, subjects were required to collect feces samples for an additional period (456 to 480 h) at home, and both fecal and urine samples for an additional period (672 to 696 h) in the clinic. Including the extrapolated values (based on the assumption that the excretion was proceeding at a steadily decreasing rate for the periods 336 to 456 h and 480 to 672 h), the total drug-related material excreted by 696 h post-dose via the feces was estimated to be 86.5%; a mean of 4.7% of drug-related material was estimated to have been excreted in the urine in the same time period (Figure 12). Figure 14 Recovery of of Radioactive Components in urine and feces after Administration of a Single Oral Dose of [14C]-Netupitant* *Values during the time from 336 to 456 h and from 480-672 h were obtained by extrapolation taking the mean value of recoveries estimated in the collection intervals just prior to and just after the missing collection period. 33 Reference ID: 3516218 Biliary excretion of netupitant was observed in animals. Netupitant is excreted into bile in bile duct cannulated rat and dog. In rats, the biliary excretion accounted for 12 to 40% of the dose (4 and 2 mg/kg, oral and IV, respectively) after 96 h post dosing and less than 1% of the dose was found in urine. The unchanged netupitant accounted for 27 to 40% of the total biliary drugrelated material and the main metabolites M1 (7-13%), M2 (5-12%), M3 (9-13%) and M8 (2.57%) were observed. (Report 1009719). In bile cannulated dogs treated with a single oral (6 mg/kg) and intravenous (2 mg/kg) administration the biliary excretion after 72 hours accounted for 30-39% and 59% of the oral and intravenous dose, respectively. The unmetabolized netupitant accounted for 2 to 4% of the total biliary drug-related material while metabolites M2 accounted for 30-43%. (Report 1009870) In some netupitant concentration-time profiles, a second peak was observed suggesting that netupitant may undergo the enterohepatic circulation. 2.2.5.9 Based on PK parameters, what is the degree of linearity or nonlinearity in the doseconcentration relationship? The plasma exposure to netupitant increased with dose in a slightly supra-proportional fashion at lower doses 100 mg to 300 mg, but showed dose- proportionality at higher doses from 300 mg to 450 mg (Table 18). Table 18 Mean (± SD) Pharmacokinetic Parameters of Netupitant after single and multiple dose administration in healthy subjects (NP16601) Dose Single dose (n=8) Multiple doses (n=8) Cmax AUC0-23 5 Cmax AUC0-23 5 mg (ng/ml) (ng*h/ml) (ng/ml) (ng*h/ml) 111 1360 269 4160 100 (23) (22) (19) (24) 599 6400 1060 17100 300 (38) (26) (19) (17) 720 9670 1790 28800 450 (35) (35) (43) (45) AUCinf (ng*h/ml) 21400 (29.7) 93800 (32.6) 139000 (46.4) 1 Accumulation Factor Similarly, when a single dose netupitant/palonosetron combination was administered at 200 mg/0.5 mg and 600 mg/1.5 mg combination, a dose-proportional increase in mean AUC and Cmax was observed for both netupitant and palonosetron (Table 19). 34 Reference ID: 3516218 Table 19 Mean (± SD) PK parameters for netupitant and palonosetron after coadministration of NETU 600 mg and PALO 1.5 mg in healthy subjects Dose Netupitant (n=48) Palonosetron (n=49) (mg) Cmax Tmax1 AUC0-47 5 Cmax Tmax1 Netu/palo (ng/ml) (h) (ng*h/ml) (ng/ml) (h) 4.1 4587 ± 6.2 200/0.5 253.9 ± 122 849 ±248.8 (2.1-10.2) 1939 (2.2-8.2) 5.2 14369 4.2 600/1.5 816.2 ±456.6 2648 ±596.8 (4.2-10.1) ±6720 (1.2-14.2) AUC0-47 5 (ng*h/ml) 23219 ±5905 69178 ±13978 1 Median (min-max) 2.2.5.10 Do the PK parameters change with time following chronic dosing? AKYNZEO® is developed for a single dose administration at 1 hour prior to the initiation of chemotherapy. The PK parameters did not significantly change after multiple doses. The median half-life and apparent clearance for netupitant was similar to that after single dose. The systemic exposure to netupitant exposure was approximately 3-fold higher compared to that after single dose in consistent with the long half-life. The degree of accumulation for metabolites M1 and M3 was greater than netupitant while lower degree of accumulation for metabolite M2 was noted compared to netupitant (Table 20). 2.2.5.1 What is the variability of PK parameters in volunteers and patients? Moderate variability of PK parameters of netupitant was observed with % CV of 25-60% in healthy subjects across studies. According to population PK analysis, CV% for clearance and the central volume of distribution was 65.4% and 43.5%, respectively in cancer patients showing similar degree of variability with those in healthy subjects. The CV% for other PK parameters was not estimated in the population PK analysis. There is no difference in pharmacokinetics for HV and patients. 35 Reference ID: 3516218 Table 20 Arithmetic Mean (%CV) PK Parameters of Netupitant and Metabolites After Single and Daily Treatment for 7 Days (NP16601) 2.3 Intrinsic Factors 2.3.1 What intrinsic factors influence PK and/or response and what is the impact of any differences in exposure on efficacy or safety responses? The effects of age, gender, and hepatic impairment on the systemic exposure were evaluated in dedicated PK studies as well as in a subset of patients during phase 3 trial by a population PK approach. Age In Study NETU-10-12 the effect of age on the pharmacokinetic parameters of netupitant and palonosetron (FDC) was evaluated by comparing 22 healthy adult subjects aged between 22 and 45 years with 12 healthy elderly subjects, aged between 66 and 79 years. In elderly subjects, the AUC0-∞, AUC0-tz, and Cmax increased by 25%, 13%, and 36% for netupitant and by 37%, 34%, 36 Reference ID: 3516218 and 10% for palonosetron. respectively compared to those in yomiger adults (Table 21. Table 22). Table 21 Comparison of Systemic Exposure to netupitant between Young and Elderly subjects after single dose administration of AKYNZEO Parameter Young Subiects (22?45 yr) Elderly Subiects (66?79 yr Meal (SD) Meal (SD) Cmax [ng/nlL] 22 596.4 (233) 12 880.8 (479.2) [ngh/mL] 22 17150 (6122) 12 19604 (6747) [ugh/mL] 22 20039 (8396) 12 24739 (9390) 22 2851 (1633) 12 4101 (5406) 22 20.5 (10.8) 12 18.7 (12.5) [11] 22 101.2 (52.8) 12 129.6 (72.7) Table 22 Comparison of PK parameters for netupitant and palonosetron after AKYNZEO administration between Healthy Young and Elderly subjects (A) Netupitant Pharmacokinetic Point estimate 90% Con?dence Parameter for Interval Cmax 136.36 95.87 - 193.96 113.42 87.66 - 146.75 124.91 95.29 - 163.75 (B) Palonosetron Pharmacokinetic Point estimate 90% Confidence Parameter for interval Cmax 110.44 95.96 - 127.11 133.81 114.28 - 156.68 136.89 117.44 - 159.56 *Point estimate (PE): ratio of geometlic means (Parameter for the elderly/Parameter for the young) Reference ID: 3516218 37 Gender In an exploratory analysis of pooled data of phase 1 PK studies (112 males and 41 females), a trend of 35% higher Cmax for netupitant in female and comparable AUCt for netupitant between males and females was noted (Table 23). Similarly to the previous observation, palonosetron Cmax and AUCT was 30% and 36% higher in females, respectively than in males. About 30% higher Cmax for netupitant was noted than in males. There was no significant difference for AUC of netupitant by gender. The safety of mildly increased Cmax to netupitant and palonosetron in females was studied in Study NETU-08-18 and NETU 10-29 in which 98% of patients were female patients with breast cancer. More male patients were included in NETU-07-07 (56.6% males vs. 43.4% females at 300 mg) while the overall number of male patients exposed to 300 mg netupitant is significantly low compared to the number of female patients. Table 23Comparison for mean systemic exposure to netupitant by gender with 300 mg netupitant in individual studies after administration T = test, R= reference Hepatic impairment The effect of hepatic impairment on PK of netupitant and palonosetron was studied after administration of administration of AKYNZEO (NETU-10-10). Currently no dosage adjustment is recommended for palonosetron by hepatic impairment based on the clinical experiences with higher doses while the mean AUC for palonosetron was 35% and 55% higher in patients with mild and moderate hepatic impairment, respectively compared to that in healthy subjects after single dose AKYNZEO administration (Table 24). 38 Reference ID: 3516218 The increase in the systemic exposure to netupitant and palonosetron in patients with hepatic impairment was observed (Table 25, Figure 13). The mean AUC of netupitant was 58% and 101% higher in patients with mild and moderate hepatic impairment, respectively than in healthy subjects. The Cmax of netupitant was about 30% higher in patients with mild and moderate hepatic impairment. Only two patients with severe hepatic impairment provided PK data. In one patient with severe hepatic impairment, Cmax and AUC of netupitant were about 2- and 6-fold higher, respectively while Cmax and AUC of palonosetron were about 2- higher than the mean for control group. Table 24 Geometric mean and ratio of PK parameters for netupitant in subjects with hepatic impairment and healthy subjects PK blood samples for netupitant were collected up to 240 hours post-dose Table 25 Geometric mean of PK parameters for palonosetron in subjects with hepatic impairment and healthy subjects PK blood samples for palonosetron were collected up to192 hours post-dose. Figure 15 Individual AUCinf for (A) netupitant and (B) palonosetron in patients with hepatic impairment and in healthy subjects (A) netupitant 39 Reference ID: 3516218 (B) palonosetron Reviewer’s comments: The sponsor calculated the ratio of PK parameters between subjects with hepatic impairment and matching control group i.e. one group for mild hepatic impairment and another group for moderate hepatic impairment. Upon review of the data, the demographic information such as age and gender was similar across control groups to different degree of hepatic impairment while the PK parameters for netupitant showed differences among controls due to the variability across groups. This variability between control groups confounded the evaluation of the effect of hepatic impairment on the PK of netupitant. Therefore PK parameters from patients with hepatic impairment were compared to the pooled control group. One healthy subject had a substantially high AUC for netupitant, that was similar to the highest AUC observed in a patient with severe hepatic impairment. The AUC was not considered reliable due to ~75% extrapolation for AUCi and excluded from the control group. Renal impairment In previous study, mild to moderate renal impairment did not significantly affect palonosetron pharmacokinetic parameters. The pharmacokinetics of neither palonosetron nor netupitant was studied in subjects with endstage renal disease. There was no dedicated PK study to evaluate effect of renal impairment on PK for netupitant. On the other hand, that no significant effects of mild and moderate renal impairment was noted in the population PK analysis. Please see the Pharmacometrics Review by Dr. Jingyu Yu for more details. A minor contribution of renal clearance to total clearance for netupitant was shown by minimal urinary excretion of unchanged netupitant in urine. About 4 % of administered dose was excreted in urine over 366 h in the ADME study and negligible amount of unchanged netupitant (<1% of administered dose) was detectable in urine samples from a subset of subjects after 450 mg netupitant dose. The incidence of TEAEs was analyzed by creatinine clearance in phase 3 trials. There was an increasing trend of incidence of TEAEs regardless of treatment as the creatinine clearance 40 Reference ID: 3516218 decreased (Table 26). Nevertheless the significant difference in the number of patients by renal function hampers a definitive conclusion. Briefly, per protocol patients with severe renal impairment or patients on dialysis were not included in the multi-cycle Phase 3 studies. The proportion of patients with normal renal function was 64.3% (1198/1862) while the proportion of patients with mild and moderate renal impairment was 31.1% (580/1862) and 4.3% (80/1862), respectively. Patients with moderate renal impairment tended to be older with mean age of 64.6 years compared to patients with normal and mild renal impairment with mean age of 51.9 and 58.5 years, respectively. Other demographic characteristics were similar among groups by renal function. The detailed review of safety by renal function is deferred to the clinical reviewer. Table 26 Incidence of TEAE in multi-cycle Phase 3 trials by creatinine clearance Renal function Normal (90 ml/min < CLcr) Mild (60 ml/min < CLcr < 90 ml/min) Moderate (30 ml/min < CLcr <60 ml/min) Treatment Netu/Palo (300/0.5) 89.5% (n/N= 598/668) 91.5% (280/317) 93.3% (42/45) Palo 0.5 mg 88.4% (421/476) 88.1% (192/218 ) 93.3% (28/30) Aprepitant/palo 92.6% (50/54 ) 88.9% (40/45 ) 100% (5/5) Source: Table 7.2.1 in Integrated Summary of Safety 2.3.2 Based upon what is known about exposure-response relationships and their variability, and the groups studied, what dosage regimen adjustments, if any, are recommended for each of these groups? Akynzeo® is proposed to be available as a fixed dose combination product consisted of 300 mg netupitant and 0.5 mg palonosetron. No other strengths will be available. As such the dosage adjustment for one component is not a feasible option. Currently no dosage adjustment is recommended for the approved palonosetron products based on organ impairment or other factors. 2.3.2.1 Elderly No significant need for dosage adjustment for elderly patients. 2.3.2.2 Pediatrics No studies were conducted in pediatric patients. A request for a has been submitted with this application. 2.3.2.3 Gender No need for dosage adjustment for gender. 41 Reference ID: 3516218 (b) (4) for pediatric studies 2.3.2.4 What are the covariates affecting the PK of netupitant based on population PK analysis? Please see the Pharmacometrics review by Dr. Jingyu Yu in Appendix for more detail. Based on the population PK analysis, there appears to be no effect of race, age and body weight on PK of netupitant. The population PK analysis also suggested that there appears to be no statistically significant effect of mild and moderate renal impairment on the clearance of netupitant. This is expected since renal pathway is a minor route of elimination for netupitant. The covariates for netupitant including body weight, BMI, age, race, smoking status, markers of cardiac, renal and liver function were evaluated for their potential influence on CL and volume of distribution (V2) in cancer patients in a population PK analysis performed during a comparative Phase 3 trial in 117 subjects in the FDC PK subgroup. A two- compartment base model with first order absorption adequately described the observed PK data of netupitant. The median netupitant apparent clearance was estimated to be 20.9 L/h and the volume of distribution to the central compartment was estimated to be 419 L. Based on sponsor’s analysis, none of the covariates had significant impact on the PK of netupitant based on population PK analysis. However, it is worth noting that for some intrinsic and extrinsic factors, population PK analysis is either supportive or limited. For example, only 4 male subjects were included in the population PK analysis as the patients enrolled in phase 3 trial is predominantly female patients with breast cancer. Therefore the effect of gender on PK cannot be evaluated in the population PK analysis. The impact of the smoking status, chemotherapy (doxorubicin, epirubicin, fluorouracil) and rescue medications on the PK of netupitant in combination with palonosetron were evaluated by population PK analysis. None of those factors appears to significantly influence the disposition of netupitant and palonosetron. However, it should be noted that definitive conclusions regarding these factors cannot be made as the population PK analysis may lack power to detect the effect of these factors due to the study design and/or insufficient PK sampling. 2.3.2.5 Renal Impairment Currently no dosage adjustment for palonosetron alone treatment in patients with renal impairment is not recommended. Given the minimal contribution of renal excretion to the total body clearance of netupitant and Akynzeo will be used as a single dose administration, the possibility of accumulation of netupitant in patients with renal impairment is minimal. However, because the PK and safety data is limited for netupitant in patients with moderate to severe renal impairment and patients on dialysis, Akynzeo should be used with caution in patients with severe renal impairment and with end-stage renal impairment. 2.3.2.6 Hepatic Impairment Currently dosage adjustment for palonosetron by hepatic impairment is not recommended. Because only limited information is available for use of netupitant in patients with severe hepatic impairment, Akynzeo should be used with caution in patients with severe hepatic impairment if the 42 Reference ID: 3516218 co-administration deemed necessary. Limited information is available for the systemic exposure for netupitant higher than the observed in patients with moderate renal impairment. The observed Cmax and AUC for netupitant and palonosetron in patients with mild and moderate hepatic impairment is lower or similar within the exposure at 600 mg in the tQT study (n=47). On the other hand in a multiple dose PK study with 450 mg once daily for 7 days (n=8), the Cmax and AUC for netupitant was 2-fold and > 5-fold higher than those after single dose administration of 300 mg. 2.3.2.7 What pregnancy and lactation use information is there in the application? PK were not evaluated in pregnant women or lactating females. Given the target patient populations are under chemotherapy and Akynzeo would be given as a single dose, it is unlikely that patients would be pregnant or lactating at the time of Akynzeo administration. Nevertheless because of the long half-life of netupitant and palonosetron, lactation should be avoided at least for a month after Akynzeo administration. 2.3.2.8. What other human factors are important to understanding the drug’s efficacy and safety? The patients enrolled in the clinical trials were stratified by gender as female gender is known to be more susceptible to emesis. The emetogenicity is considered to be dependent on chemotherapy regimen such as highly-emetogenic chemotherapy and moderately emetogenic chemotherapy. The categorization may be revised based on clinical observations. 2.4 Extrinsic Factors 2.4.1 What extrinsic factors influence dose- exposure and what is the impact of any differences in exposure on response? Coadministration of rifampin, a strong CYP3A4 inducer reduced the Cmax and AUC to netupitant component of AKYNZEO by 62% and 82%, respectively. In the dose-finding study, the combination of 100 mg netupitant or 200 mg netupitant with 0.5 mg palonosetron did not show significant difference from palonosetron monotherapy for the prevention of CINV during acute phase while statistically significant difference was demonstrated during delayed phase. The combination with 300 mg netupitant showed numerically higher CR rate than the combination with 200 mg netupitant during acute phase (98% vs. 92%) although the study was not designed to show the difference among doses. 2.4.2 Drug-Drug Interactions 2.4.2.1 Is there an in vitro basis to suspect in vivo drug-drug interactions? In in vitro studies, netupitant is a substrate and an inhibitor of CYP3A4. The sponsor has conducted follow-up in vivo studies with a strong CYP3A4 inhibitor ketoconazole, a CYP3A4 inducer rifampicin and a CYP3A4 substrate midazolam. 43 Reference ID: 3516218 In addition, netupitant is an inhibitor of P-gp and BCRP transporters. The sponsor conducted a follow-up in vivo study with P-gp substrate Digoxin and have shown that netupitant do not alter the exposure of digoxin significantly when administered concomitantly. However, netupitant’s potential interaction with BCRP was not evaluated in vivo. Since no significant in vivo inhibitory effect of netupitant on BCRP transporter is anticipated based on in vitro data with weak inhibition toward BCRP and negative in vivo inhibition data with P-gp substrate digoxin, we do not request an additional in vivo study to evaluate the potential of netupitant to inhibit BCRP. 2.4.2.2 Is the drug a substrate of CYP enzymes? Is metabolism influenced by genetics? The sponsor conducted two in-vitro studies to identify the enzyme responsible for netupitant metabolism (study 103832 and study NETU-13-21). It appear that the metabolism of netupitant to M1, M2 and M3 is mainly mediated by CYP3A4 and lesser extent by CYY2C9 and CYP2D6 (Table 27 and Figure 14). As netupitant is primarily metabolized by CYP3A4, the sponsor had conducted follow-up in vivo drug-drug interaction studies with a strong CYP3A4 inhibitor ketoconazole and a CYP3A4 inducer rifampicin. Since both CYP2C9 and CYP2D6 have polymorphism, metabolism of netupitant could be influenced by genetics. Study 103832: In the first study (study 103832), 10 μM netupitant was incubated with human hepatocytes for 24 hours and with human liver microsomes (HLM) for 20 minutes, and the supernatant was analyzed with HPLC to characterize the metabolic pathway of netupitant. Following the incubation of netupitant, both human hepatocytes and liver microsomes had resulted two metabolites of netupitant, M1 and M2. However, both test systems, hepatocytes and liver microsome, were not properly validated in terms of various CYP enzymes (both phase 1 and phase 2) prior to the use or have proper controls (positive or negative) during the incubations. Therefore, it is difficult to interpret the data from this metabolism study in hepatocytes and liver microsomes. The contribution of different microsomal CYP450 enzymes to the metabolism of netupitant (RO0673189) was studied by utilizing recombinant human enzymes. The enzymes were expressed in E. coli and isolated as a membrane fraction. The radiolabeled netupitant at 5 μM (1 µM for CYP2C9) was incubated with four of the major human CYP450 isoenzymes (CYP3A4, CYP2C9, 2C19 and 2D6) at 100 to 600 pmol CYP450/ml and the incubations were initiated by the addition of NADPH (1 mM). After the incubation for 30-60 min at 37°C, the reaction was terminated, and the supernatant was analyzed with HPLC. The results of this study suggested that CYP2C9, 2C19 and 2D6 do not catalyst the formation of any metabolite of netupitant while CYP3A4 appears to metabolize netupitant to the same metabolites (M1 and M2) that were observed when netupitant was incubated with human liver microsomes and hepatocytes. However, the sponsor did not evaluate the potential of CYP1A2, 2B6 and 2C8 to metabolize netupitant in this study. Study NETU-13-21 44 Reference ID: 3516218 In this second study, 10 μM netupitant was incubated with human liver microsomes (0.8 mg/ml) in the presence and absence of different selective CYP isoform inhibitors and with recombinant CYP 1A2, 2B6 and 2C8 (20 pmol P450) in pH 7.4 buffer for 60 minutes at 37°C in duplicates. Specific probe substrates for each enzyme were incubated as positive controls to validate the metabolic activities of the test systems used (human liver microsomes and cDNA expressed enzymes). Based on the results of this inhibition study in human microsome, it appear the metabolism of netupitant to M1, M2 and M3 is mainly mediated by CYP3A4 and lesser extent by CYP2C9 and CYP2D6. In addition, CYP1A2, CYP2B6, CYP2C8 and CYP2C19 do not appear to contribute to the metabolism of netupitant. Study in CYPIA2, 2B6 and 2C8 cDNA expressed enzymes further confirms that these enzymes do not contribute to netupitant metabolism. Table 27 Netupitant disappearance in Human Liver Microsomes and cDNA CYPs expressed systems in the presence and in the absence of CYP’s inhibitors 45 Reference ID: 3516218 Figure 16 Netupitant metabolites formation rate in HLM system 2.4.2.3 Is the drug an inhibitor and/or an inducer of CYP enzymes? Netupitant up to 20 μM and M1, M2 and M3 up to 2 μM are not considered to be inducers of CYP1A2, CY2C9, CYP2C19 and CY3A4/5 enzyme. The sponsor did not evaluate the potential of netupitant and its metabolites M1, M2 and M3 to induce CYP2B6. Based on in vitro study, netupitant and its metabolite M1 are considered to be CYP3A4 inhibitors. The sponsor did conduct a follow up in vivo study with CYP3A4 substrate midazolam. Netupitant did not inhibit CYP1A2, CYP2C19, and CYP2D6 in vitro. In vivo drug interactions via inhibition of CYP2B6, 2C8 and 2C9 at the clinical dose of 300 mg are unlikely based on weak inhibition of toward these enzymes in in vitro studies. M1 showed inhibition toward CYP 2B6, 2C8, 2D6, 3A4, and weak inhibition toward CYP 1A2, 2C9, 2C19 in in vitro studies. However, since Cmax/Ki >0.1 for only CYP3A4, in vivo drug interaction via M1 inhibition toward CYP enzyme is unlikely except for CYP3A4. M2 and M3 showed weak inhibition toward all major CYP enzymes. Since Cmax/Ki<0.1 for all enzymes, in vivo drug interaction via M2 and M3 inhibition toward CYP enzyme is unlikely. Induction (Study NETU-10-27): Hepatocytes from three different donors were incubated with 0.2, 2 and 20 μM netupitant or 0.02, 0.2 and 2 μM of M1, M2 and M3 or positive control inducers (omeprazole for CYP1A2 or rifampicin for CYP2C9, 2C19 and 3A4) for 72 hours at 37oC in duplicates. The exposure medium was refreshed every 24 hours. In addition, two wells were left untreated to determine the basal CYP1A2, CYP2AC9, CYP2C19 and CYP3A4 activities of the hepatocytes as negative control. At the end of the 72 hours of incubation period, the activities of target enzymes CYP1A2, CYP2C9, CYP2C19 and CYP3A4 were assessed by incubating the hepatocytes with model substrates (Phenacetin for CYP1A2, tolbutamide for CYP2C9, S-mephenytoin for CYP2C19 and 46 Reference ID: 3516218 midazolam for CYP3A4) for each target enzymes and measuring the appearance rate of their respective metabolites. Prior to the use, the hepatocytes were characterized by the supplier in respect to various phase I (CYP1A2, CYP3A4/5, CYP2B6, CYP2D6, CYP2C19) and phase II (glucuronidation and sulfation) enzyme activities. Netupitant, M1, M2 and M3 did not induce CYP1A2, CY2C9, CYP2C19 and CY3A4/5 enzyme activities when hepatocytes from three different human donors were treated with netupitant up to 20 μM concentration and M1, M2 and M3 up to 2 μM concentrations after 72 hours of incubation based on induction threshold of 40% of the positive control. Inhibition (Study 1003907): Human liver microsomal (pooled from 10 human livers) protein was incubated with netupitant (0, 0.5, 1, 10, and 100 µM) and the corresponding selective model substrates at 37oC in the presence of NADPH generating system for a specified duration of incubation time. No pre-incubation was carried out to assess the time-dependent inhibition. The enzymatic reactions were terminated by addition of methanol or acetonitrile. The inhibition potential of metabolites of netupitant (RO0673189), namely RO0681133 (M1) and RO0713001 (M2) were evaluated for CYP3A4 enzyme only. However, this study did not contain positive controls with known inhibitors to validate the test system regarding CYP enzymes activities. Nonetheless, the activity of CYP enzymes toward model substrates in the absence of netupitant as inhibitor was within the historical data observed. Table 28 Inhibition of CYP Enzyme by netupitant and its metabolites CYP450 isoenzyme CYP1A2 CYP2C9 Substrate used CYP2C19 CYP2D6 CYP3A4 CYP3A4 CYP3A4 CYP3A4 Tacrine Diclofenac Mephenytoin Bufuralol Midazolam Testosterone Nifedipine Simvastatin Substrate conc (μM) 25 5 32.8 40 5 20 20 3 HLM conc. (mg/ml) 0.5 0.1 1 1 0.1 0.075 0.2 0.02 Incubation Time (min) 12 5 30 30 10 20 10 5 >>100 22.6 ± 3 18.0 ± 6 >100 >>100 5.9 ± 1.0 1.7 ± 0.2 IC50 (μM) RO0673189 (netupitant) RO0681133 (M1) RO0713001 (M2) 12.0 ± 0.5 10.5 ± 0.8 1.2 ± 0.5 > 1 µM Table 29 Inhibition of CYP450 metabolism by netupitant; apparent Ki CYP450 isoenzyme CYP2C9 CYP3A4 CYP3A4 Substrate used Diclofenac Testosterone Midazolam Substrate conc.( μM) 2, 5, 10, 50 5, 10, 20 2, 5, 10, 50 HLM protein conc.(mg/ml) 0.1 0.075 1 Inhibitor conc. (μM) apparent Ki (μM) 0, 0.1, 0.5, 1, 5, 10 0, 0.2, 0.5, 1 0, 0.1, 0.5, 1, 5, 10 25.0 ± 7.4 1.1 ± 0.2 2.2 ± 0.6 Inhibition mechanism competitive competitive competitive 47 Reference ID: 3516218 Netupitant, at concentration 0-100 μM, did not inhibit enzymes CYP1A2, 2C19 and 2D6 (IC50 >100 μM). Netupitant had shown weak inhibition toward CYP2C9 with approximate IC50 value of 22.6 ± 3 μM. Further studies with different concentration of model substrate had shown that netupitant’s inhibition of CYP2C9 is through competitive inhibition with Ki value of 25 ± 7.4 μM. Since Cmax/Ki= 1.5 μM / 25 μM= 0.06<0.1, clinical in vivo relevance of this interaction with CYP2C9 is less likely. The inhibitory potential of netupitant for the CYP3A4 enzyme was evaluated with four different model CYP3A4 substrates, testosterone, midazolam, nifedipine and simvastatin. All of the model CYP3A4 substrates had demonstrated that netupitant is an inhibitor of CYP3A4 with IC50 value of 1.7-12 μM. Further studies with different concentrations of testosterone and midazolam had demonstrated that the CYP3A4 inhibition is a competitive inhibition with Ki value of 1.1 μM with testosterone and 2.2 μM with midazolam. The inhibition potential of netupitant metabolites, namely RO0681133 (M1) and RO0713001 (M2) were evaluated for CYP3A4 enzyme only with testosterone as the model substrate. RO0681133 (M1) appears to be an inhibitor of CYP3A4 with IC50 value of 1.2 μM. Due to solubility issue, RO0713001 (M2) was tested up to 1 μM, and notable inhibition was observed even at 1 μM. A follow-up in vivo study to evaluate the potential of netupitant to inhibit CYP3A4 is recommended for the following reasons: o Systemic exposure: Cmax/Ki = 1.5 μM /1.1μM = 1.4>0.1 o Gut exposure: [I]gut/Ki= 2074 μM / 1.1 μM = 1885>>>10 where [I]gut = dose/250 ml = 300 mg/250ml= 1.2 g/L. Inhibition (Study NETU-13-20): Human liver microsomal (pooled from 50 human livers) protein was incubated with test compound (netupitant, M1, M2 and M3) at 0.3, 1, 3, 10, 30 and 100 µM and the corresponding selective model substrates at 37oC in the presence of NADPH generating system for a specified duration of incubation time. No pre-incubation was carried out to assess the time-dependent inhibition(Table 30). The enzymatic reactions were terminated by addition of ice-cold acetonitrile. The human liver microsomes was characterized in respect CYP enzyme activities prior to the use. In addition, this study included appropriate positive controls with model CYP inhibitors of each CYP isozymes. Netupitant inhibition was evaluated towards CYP2B6 and CYP2C8. M1, M2 and M3 inhibition was evaluated towards the CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (two substrates). 48 Reference ID: 3516218 Table 30 Experimental conditions for CYP inhibition studies Netupitant showed weak inhibition toward both CYP2B6 and CYP2C8 with IC50 values of 33.39 μM and 50.4 μM, respectively. However, since Cmax/Ki are <0.1, no follow-up in vivo study is recommended (table 31). A metabolite, M1 showed inhibition toward CYP 2B6, 2C8, 2D6, 3A4, and weak inhibition toward CYP 1A2, 2C9, 2C19. However, since Cmax/Ki >0.1 for only CYP3A4, an in vivo study is recommended for CYP3A4. The sponsor had already conducted in vivo DDI study with netupitant concomitantly administered with CYP3A4 substrate midazolam. Metabolites, M2 and M3 showed weak inhibition toward all evaluated CYP enzymes. Since Cmax/Ki<0.1, no in vivo follow up study is needed. 49 Reference ID: 3516218 Table 31 [I]/Ki values of metabolites for CYP isoforms 2.4.2.4 Is the drug a substrate and/or an inhibitor of P-glycoprotein transport processes? Netupitant and its metabolites interaction with transporters were evaluated in vitro in Study NETU-06-13 (netupitant’s interaction with P-gp) and study NETU-12-81. Based on in vitro studies, parent drug netupitant is an inhibitor of P-gp and BCRP transporter. The sponsor had conducted a follow up in vivo study with digoxin to evaluate the P-gp inhibitory effect of netupitnat. Potential of netupitant being substrate of P-gp was not evaluated adequately. In addition, M2 is shown to be a substrate for P-gp. Based on in vitro data, in vivo interaction of netupitant as substrate for BCRP, OATP1B1, OATP1B3, and OCT1, or as inhibitor of BSEP, MRP2, OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 is unlikely. In addition, based on the in vitro data, in vivo interaction of three major metabolites, M1, M2 and M3 as substrates of BCRP, OATP1B1, OATP1B3, and OCT1, or as inhibitors of MDR1, BCRP, BSEP, MRP2, OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 are unlikely. 50 Reference ID: 3516218 Efflux Transporters: Substrate: The sponsor evaluated the potential of netupitant being a substrate for P-gp in ATPase activation assay, which suggested that netupitant may be a substrate for P-gp. However, the sponsor did not evaluate whether netupitant is substrate for P-gp on bi-directional transport assay system with net flux ratio information or evaluate the permeability of netupitant in the presence of potent P-gp inhibitor to predict the in vivo relevance of this interaction in this NDA application. Potential of M1, M2 and M3 being substrate for P-gp and potential of netupitant, M1, M2 and M3 being substrate for BCRP was evaluated in MDR1 or BCRP transfected MDCKII monolayer cultured cells (table 32 and table 33). Bidirectional transport through monolayers was determined by incubating of test compound at 3, 10 and 30 μM concentration with parental and MDR1/BCRP transfected MDCKII cell monolayers at 37 ± 1 °C. After the incubation, aliquots of samples were taken at 0, 15, 30, 60, 12 minutes from the receptor chambers to determine the amount of translocated test compound. The digoxin (5 μM) / prazosin (1 μM) efflux ratio was determined as a positive control for MDR1/BCRP function. As a follow-up, bidirectional transport of M2 in parental and MDR1 transfected MDCKII cells was determined in the presence and absence of the MDR1 inhibitor PSC833 to confirm the specificity of the transport in MDCKII-MDR1 cells. Table 32 Net Efflux Ratio from MDCKII-MDR1 studies for metabolites 51 Reference ID: 3516218 Table 33 Net Efflux Ratio From MDCKII-BCRP Studies for netupitant and its metabolites As the net flux ratio for M1 and M3 were below 2 at all concentrations, M1 and M3 are not substrate of MDR1. The net flux ratio of M2 for MDR1 was > 2 at all tested concentration. The sponsor further evaluated the potential for M2 being a substrate for MDR1 in presence of MDR1 inhibitor. Efflux of M2 in was further reduced in presence of MDR inhibitor suggesting that M2 is a substrate for MDR. Netupitant, M1, M2 and M3, are not substrates of BCRP transporter. Although the net flux ratio when corrected for parental cell are > 2 for under certain conditions, it appears that it was due to very low flux ratio in parental cells. Based on efflux ratio in BCRP transfected cells alone, none of the tested compounds are substrates of BCRP transporter as efflux ratio for all of them were less than 2 in BCRP transfected cell. Repeated experiments at 10 μM reconfirmed that netupitant, M1, M2 and M3, are not substrates of BCRP transporter as both efflux ratio in transfected cells alone and net efflux ratio when corrected for parental cells are <2 for all tested compounds. Inhibition: P-gp on Caco-2 monolayer cells: The sponsor evaluated the interaction of netupitant with P-gp transporter in 3 different assay methods, ATPase assay, Calcein Assay and bidirectional transporter assay on monolayer (Study 52 Reference ID: 3516218 Since as bidirectional permeability assay on monolayers is currently regarded as the defmitive assay for identifying P-gp substrates and inhibitors. this review only focused on the data from the bidirectional permeability assay on monolayers. Potential of netupitant being an inhibitor of P-gp was evaluated in Caco-2 cell where the bidirectional (AB and B-A) permeability of a model P-gp substrate 3H-digixin was evaluated in the presence of increasing concentration of netupitant (0.2. 1 5 uM) on Caco-2 cell line (on 24- well plate) at a?er 2 hours of incubation in duplicate. The paracellular permeability of the monolayer was assessed using l4C-mamritol (Papp(A/B) 2.13x106 cm/s). 60 uM Verapamil (known P-gp inhibitor) was included as the positive control (Table 34). Table 34 Apparent permeability (Papp) of 3H-digoxin in the apical-to-basolateral (A-B) and basolateral?tryapical direction in the presence of different concentrations of Netupitant . . A ical to Basolateral Basolateral to A ical . Pa: (1045cm/sec) Pm (1045cm/sec)p Ef?ux Ratio Control 0.87 25.32 29 60 uM Verapamil 2.98 4.07 1.4 Netupitant (0.2 uM) 1.25 29.73 23.8 Netupitant (1 uM) 1.07 26.23 24.5 Netupitant (5 uM) 2.8 13.24 4.7 Based on the result of this study. netupitant seems to inhibit P-gp at concentration dependent manner. However. IC50 value was not determined in this study. Therefore. relevance of this in vitro inhibitor interaction in in viva cannot be predicted. Thus. netupitant?s potential to inhibit P- gp in vivo at clinical dose carmot be ruled out. The sponsor did conduct a follow up in viva drug- drug interaction study with digoxin (a model P-gp substrate). MDRI, BCRP, BSEP and MRP2 on Vesicular Transport: The sponsor had used vesicular transport inhibition assay to evaluate the inhibition potential of ef?ux transporters BCRP, BSEP and MRP2 by netupitant and its metabolites. Vesicular transport assays were performed with inside-out membrane vesicles prepared from cells overexpressing human ABC transporters on 96-well plates. The netupitant. M1. M2. and M3 (at 0.01. 0.04. 0.12. 0.37. 1.11. 3.33. 10 and 30 uM) were incubated with membrane vesicle preparations (total protein: 50 rig/well or 25 rig/well in case of BCRP) and the probe substrate in triplicates. Incubations were carried out in the presence of ATP or AMP to distinguish between transporter-mediated uptake and passive diffusion into the vesicles. Reference inhibitors for each ef?ux transporters were included to serve as positive controls for inhibition (Table 35. Table 36) 53 Reference ID: 3516218 Table 35 Vesicular transport assay parameters Table 36 Efflux Transporters inhibition from vesicular transport inhibition assays • • • • Netupitant, M1, M2 and M3 do not inhibit MRP2 up to 30 μM concentration and thus IC50 values were not determined for MRP2 transporter. Netupitant, M2 and M3 slightly inhibited BSEP while M1 did not show any inhibition toward BSEP up to 30 μM concentration. Therefore, IC50 values could not be determined for BSEP transporter. Netupitant, M1, M2 and M3 inhibit BCRP in concentration dependent manner. o Netupitant: Cmax/IC50= (1-1.5 μM)/6 μM = (0.167-0.25)>0.1 o M1: Cmax/IC50=(0.07-0.09 μM)/8.6μM =(0.008-0.01)<0.1 o M2: Cmax/IC50= (0.17-0.58 μM)/22.6 μM=(0.0075-0.026) <0.1 o M3: Cmax /IC50 = (0.08-0.144 μM)/10.6 μM= (0.0075-0.013) <0.1 M1, M2 and M3 inhibit inhibits MDR1in concentration dependent manner. o Netupitant: was not evaluated in this study. o M1: Cmax/IC50=(0.07-0.09 μM)/4.95 =(0.014-0.018)<0.1 o M2: Cmax/IC50= (0.17-0.58 μM)/8.0 =(0.02125-0.0725)<0.1 o M3: Cmax /IC50 =( 0.08-0.144 μM)/5.35 = (0.014-0.027) <0.1 54 Reference ID: 3516218 MDR1 and BCRP on MDCKII Transfected Monolayer Cell: Inhibition potential of M1, M2 and M3 for MDR1 transporter and inhibition potential of netupitant, M1, M2 and M3 for BCRP transporter were evaluated in MDR1 or BCRP transfected MDCKII monolayer cultured cells. Bidirectional transport of model substrates for MDR1 (digoxin at 5 μM) and BCRP (prazosin 1uM) were determined in the presence and absence of netupitant, M1, M2 and M3 (10 and 30 μM) at 37 ± 1 °C after 60 minutes and 120 minutes incubation for prazosin and digoxin, respectively. The reference inhibitors PSC833 (10 μM) for MDR1 and Ko134 (1 μM) for BCRP were also included as positive controls (Table 37, Table 38). Table 37 Inhibition of MDR1 transporter by metabolites of netupitant from MDCKIIMDR1 studies Net ER (net efflux ratio) 55 Reference ID: 3516218 Table 38 Inhibition of BCRP transporter by netupitant and its metabolites from MDCKII-BCRP studies • M2 did not inhibit MDR1 and BCRP at both 10 μM and 30 μM, which is contrary to the vesicular transport inhibition assay result where M2 inhibited both MDR1 and BCRP in concentration dependent manner. Since bi-directional assay in monolayer is considered to be more reliable assay than the vesicular system, M2 is not considered as an inhibitor of MDR1 and BCRP. • M1 and M3 inhibited MDR1 in concentration dependent manner. However, IC50 values were not determined in this monolayer cell system. Based on rough estimate of IC50 56 Reference ID: 3516218 around 10 μM or based on the IC50 values from the vesicular system, an in vivo study is not needed for M1 and M3. • Netupitant, M1 and M3 inhibited BCRP in concentration dependent manner where no inhibitions were observed at 10 μM and inhibition was observed at 30 μM. However, IC50 values were not determined in this monolayer cell system. Since no significant Pgp inhibitory effect of netupitant was observed with Digixin in in-vivo where 5 μM netupitant have inhibited P-gp transporter in vitro, we do not anticipate a significant BCRP inhibitory effect of netupitan in vivo since netupitnat at 10 μM did not inhibit BCRP transporter in vitro. Uptake Transporters: Uptake transporters were evaluated using CHO cells or FlpIn293 cells stably expressing the respective uptake transporters (Table 39). Table 39 Experimental conditions for uptake transport assay Substrate: As netupitant and its metabolites are primarily eliminated through hepatobiliary route, the sponsor have evaluated the potential of netupitant and its metabolites M1, M2, and M3 being substrate for uptake transporters OATP1B1, OATP1B3 and OCT1 in CHO cells or FlpIn293 cells stably expressing the respective uptake transporters. The cellular uptake of netupitant, M1, M2, and M3 into cells was determined by incubating them at 1 and 10 μM concentrations with cells overexpressing the uptake transporters and control cells on 24-well plates at 37 ± 1 °C in pH 7.3 buffer for 2 and 20 minutes. In the positive controls, the sponsor did not evaluate the fold increase in uptake of model substrates (positive controls) in transfected cells compared to parental cell. However, the uptake of model substrates in absence and presence of model inhibitors of for these specific transporters were evaluated to validate the test system. Uptake of these model substrates were substantially inhibited in the presence of model inhibitors. 57 Reference ID: 3516218 Table 40 Fold increase in uptake in transfected cells compared to parental cell for uptake transporters for netupitant and its metabolites None of the test compound, netupitant, M1, M2 and M3 showed ≥ 2 fold increase in uptake in transfected cells compared to parental cell suggesting that netupitant, M1, M2 and M3 are not substrates for OATP1B1, OATP1B3 and OCT1 (Table 40). Inhibition: Potential of netupitant, M1, M2, and M3 being an inhibitor of OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 were evaluated by incubating them at 0.01, 0.04, 0.12, 0.37, 1.11, 3.33, 10 and 30 μM concentrations with cells stably expressing the those uptake transporters and the probe substrates on 96-well plate at 37 ± 1 °C in pH 7.4 buffer in triplicates. A reference inhibitor served as positive control for inhibition (Table 41). • OATP1B1: Netupitant, M1 and M3 showed weak inhibition toward OATP1B1, and thus, IC50 values could not be estimated. However, M2 did show some inhibition toward OATP1B1 with IC50 of >30 μM. Since total Cmax/IC50 = 0.58 μM / 30 μM = 0.02 < 0.1, a follow-up in vivo study is not needed. • OATP1B3: Netupitant and M1 showed weak inhibition toward OATP1B3 and IC50 values could not be estimated up to 30 μM. M2 and M3 inhibited OATP1B3 with IC50 values of 4.3 and 9.6 μM. Since Cmax/IC50 = 0.144 μM /9.6 μM = 0.015<0.1 for M3, an in vivo follow up study for to evaluate the inhibition potential of M3 toward OATP1B3 is not needed. Although total Cmax/IC50 = 0.58 μM /4.3 μM = 0.13 >0.1 for M2, R-value = 1+ (fu x I in,max/IC50) = 1.08 <1.25 and thus, in vivo study is not needed • OAT1: Netupitant, M1, M2 and M3 do not appear to inhibit OAT1 significantly up to 30 μM concentration and thus, IC50 values could not be determined. 58 Reference ID: 3516218 Table 41 Inhibition of uptake transporter by netupitant and its metabolites • OAT3: Netupitant, M1, M2 and M3 do not inhibit OAT3. • OCT2: Netupitant appears to inhibit OCT2 in concentration dependent manner with IC50 value of 22.3 μM while M1, M2 and M3 did not show significant inhibition toward OCT2. Since Cmax/IC50 = (1-1.5 μM)/22.3 μM = (0.045-0.07) < 0.1, in vivo follow up study is not needed. • OCT1: Netupitant, M1, M2 and M3 all appear to inhibit OCT1 in concentration dependent manner. o Netupitant: Cmax/IC50 = (1-1.5 μM) /7.9 μM = (0.13-0.19) >0.1 o M1: Cmax/IC50 = 0.07-0.09 μM /19 μM = (0.0037-0.0047)<0.1 o M2: Cmax/IC50 = (0.17-0.58 μM )/7.4 μM = (0.023-0.078)<0.1 o M3 Cmax/IC50 = 0.08-0.144 μM /4.4 μM =(0.018-0.033) <0.1 59 Reference ID: 3516218 For netupitant, although Cmax/IC50 =0.19 for OCT1, it is not substantially larger than 0.1. Since Cmax/IC50 <0.1 for OCT2, and OCT1 and OCT2 have overlapping substrate specificities, we do not anticipate a significant in-vivo OCT1 interaction for netupitant. Induction: Potential of netupitant and its metabolites to induce transporters were not evaluated in this NDA submission. Potential of netupitant and its metabolites to induce P-gp transporter do not need to be evaluated since it has already been shown that netupitant and its metabolites do not induce CYP3A4 in in vitro study NETU-10-27. 2.4.2.5 Are there other metabolic/transporter pathways that may be important? The sponsor did not explore the following potential metabolic/transporter that may be important: • • The potential of netupiant being a substrate for P-gp was not evaluated. The potential of netupitant and its metabolites M1, M2 and M3 to induce CYP2B6 were not explored. 2.4.2.6 Does the label specify co-administration of another drug and, if so, has the interaction potential between these drugs been evaluated? As a combination product, netupitant and palonosetron are to be co-administered. In addition, the efficacy of AKYNZEO was evaluated as a combination therapy with dexamethasone. Interaction between netupitant and palonosetron There was no significant PK drug interaction between netupitant and palonosetron. Concomitant administration of a single dose netupitant 450 mg and a single dose palonosetron 0.75 mg did not significantly affect the PK of each other (Table 42). These results are consistent with in vitro study results for different major metabolic enzyme i.e.CYP3A4 and CYP2D6 for netupitant and palonosetron, respectively and lack of significant inhibitory effects on CYP3A4 by palonosetron and CYP2D6 by netupitant. The proposed clinical dose for the combination product is netupitant 300 mg and palonosetron 0.5 mg and no significant PK interaction is expected based on these results. These results also indicate that the contribution of palonosetron to the efficacy in the combination with netupitant is expected to be similar with that of palonosetron alone treatment. Table 42 PK Parameters during the 3 Treatment Periods, with Netupitant Alone (450 mg), with Netupitant in Combination with Palonosetron (450/0.75 mg) and with Palonosetron Alone (0.75mg) 60 Reference ID: 3516218 Interaction with dexamethasone For CINV associated with highly emetogenic chemotherapy, dexamethasone (Dexa) is typically used as a multi-day regimen of 20 mg on Day 1 and 8 mg BID on Days 2-4 for adults. For CINV associated with moderately emetogenic chemotherapy, dexamethasone is typically used as a single dose of 20 mg on Day 1 only for adults. The effect of netupitant on dexamethasone PK was studied by co-administering netupitant on Day 1 with dexamethasone multi-day regimen (20 mg on Day 1, followed by 8 mg b.i.d. on Days 2-4). The effect of netupitant was studied at doses of 100 mg, 300 mg, and 450 mg and PK of dexamethasone was evaluated on Days 1, 2 and 4. The systemic exposure to dexamethasone was increased in a netupitant dose-dependent manner (Figure 15). The mean AUC0-24 was 1.5, 1.7, and 1.8-fold higher with co-administration of 100, 300, and 450 mg, respectively compared to that without netupitant. Notably the inhibitory effect of netupitant on CYP3A4 lasted till 4 days after single dose administration of 300 mg netupitant indicated by the 2.4-fold higher AUC84-∞ to dexamethasone. After co-administration of 300 mg netupitant Dexamethasone Cmax was increased: up to 1.2-fold increase on Day 1, and 1.7 fold increase on Day 2 and Day 4. The Cmin was about 3-4 fold higher over 4 days with concomitant netupitant 300 mg (Table 43,Table 44). Combined with this study results and no effects of palonosetron on CYP3A4 in vitro, the doses for dexamethasone regimen with netupitant and palonosetron was reduced to 12 mg from 20 mg on Day 1 and to 8 mg QD to 8 mg BID on Days 2-4 compared to the dexamethasone regimen for palonosetron alone treatment in Phase 2 and 3 trials. 61 Reference ID: 3516218 Figure 17 Mean plasma concentration-time profile for dexamethasone by netupitant dose Table 43 Mean (SD) Pharmacokinetic Parameters for Dexamethasone after Administration with single dose Netupitant on Day 1 1 Tmax : median (min, max) Per the Agency’s request, the sponsor estimated [I]/Ki for CYP3A4 inhibition by netupitant and its metabolites, mainly M1 beyond Day 4 11. 11 Response to the Information Request dated May 21, 2014 62 Reference ID: 3516218 PK samples for netupitant and its metabolites were collected up to 120 h and plasma concentrations beyond the last observed concentrations were extrapolated 12. In this study the median half-lives for netupitant and metabolite M1 were of 47 h and 68 h and was estimated relatively shorter than those in other studies. For example the mean half-life of 70-100 h was estimated for netupitant and M1 in other studies. Table 44 Mean Ratio PK parameters for Dexamethasone with and without concomitant 300 mg netupitant To assess the potential inhibitory effects on CYP3A4 over time, [I]/Ki values were computed for netupitant and these metabolites individually then added to calculate the total [I]/Ki at given time point. The mean total [I]/Ki ranged 0.0134-0.167 (ranged 0.089-0.285) on Day 4 when the AUC of dexamethasone was still 2-fold higher than the control. The mean total [I]/Ki decreased to below 0.1 on Day 6 and was 0.093 (0.049- 0.210) at 140 h post-dose. This estimation suggests that drug interaction via CYP3A4 inhibition by netupitant is less likely on Day 6 but cannot be ruled out (Table 45, Figure 16). Table 45 Mean ± SD of [I]/Ki (min-max) for netupitant and its metabolites Time(h) post-dose 84 120 140 Netupitant M1 M2* M3* Total 0.054 ± 0.018 (0.03-0.089) 0.038 ± 0.015 (0.020-0.080) 0.029 0.106 ± 0.035 (0.060-0.180) 0.074 ± 0.031 (0.041-0.157) 0.0613 0 0.007 ± 0.003 (0.003-0.013) 0.004 ± 0.002 (0.002-0.010) 0.003 0.167 ± 0.053 (0.097-0.283) 0.117 ± 0.048 (0.064-0.248) 0.093 ± 0.042 (0.049-0.210) * 0 0 In vitro studies indicated that M2 and M3 are not inhibitors of CYP3A4 12 Extrapolations were made using the equation 63 Reference ID: 3516218 Figure 18 Mean [I]/Ki values-time profile (A) for netupitant and its metabolites M1, M2 and M3 after administration of netupitant 300 mg plus dexamethasone , (B) the sum of [I]/Ki BEST AVAILABLE COPY (A) (B)x Interaction with chemotherapy Potential interactions between netupitant/palonosetron combination and chemotherapeutics were studied with docetaxel, etoposide, and cyclophosphamide in cancer patients. Within each group, all patients received an intravenous (IV) administration of 1 of the 3 chemotherapeutic agents (docetaxel, etoposide or cyclophosphamide) for 2 consecutive cycles; Day 1 of the 2 treatment periods was separated by at least 3 weeks. The patients received a single oral dose of AKYNZEO during either the first or the second treatment period and oral palonosetron 0.5 mg (Aloxi®, reference IMP) in the alternate period. Docetaxel and etoposide 13 are metabolized primarily by CYP3A4, and cyclophosphamide is metabolized by multiple CYP enzymes including CYP3A4. In Study 08-08, the clinical efficacy of FDA was studied in patients who received anthracycline and cyclophosphamide based chemotherapy. The dosage of chemotherapeutic agents varied among patients: docetaxel, 75 to 100 mg/m2; etoposide, 35 to 100 mg/m2; cyclophosphamide, 500 to 1000 mg/m2 ; however, was consistent between two treatment periods (Table 46,Table 47). Docetaxel 13 Kawashiro et al. (1998) A study on the metabolism of etoposide and possible interactions with antitumor or supporting agents by human liver microsomes, JPET 286(3):1294 64 Reference ID: 3516218 With netupitant/palonosetron combination, docetaxel exposure was approximately 37% higher for AUC0-t and 50% for Cmax than the exposure only with palonosetron. Etoposide The AUC0-t in the FDC period was approximately 21% higher than that in the reference period, while Cmax and AUC0-∞values were similar for both treatment periods. Cyclophosphamide When co-administered with FDC, the mean AUC and Cmax for cyclophosphamide were 19% and 27% higher, respectively compared to those after co-administration with palo alone. This suggests that there may be a minimal drug-drug interaction between IV cyclophosphamide and netupitant administered in the form of FDC with palonosetron. Table 46 Docetaxel, Etoposide and Cyclophosphamide Plasma Exposure in Co-administration either with Netupitant /Palonosetron (FDC, test) or Palonosetron Alone (Reference)* *PK samples were collected up to 24, 36, 48 hr post-dose for etoposide, cyclophosphamide, and docetaxel, respectively. 65 Reference ID: 3516218 Table 47 Mean ratio of PK parameters for chemotherapy after co-administration with FDC or oral Aloxi *Because n=2, the SE and the 90% CI could not be calculated SE = Standard error; FDC = netupitant/palonosetron fixed dose combination; CI = Confidence interval CYP3A4 substrates Midazolam and erythromycin (NP16599) When administered concomitantly with oral netupitant 300 mg, the exposure of CYP3A4 substrate was increased (Cmax and AUC were approximately 92 and 56% higher for erythromycin while Cmax and AUC were 36% and 126% higher for midazolam) (Table 48). Co-administration with midazolam and erythromycin did not affect the exposure to netupitant. Based on about 2 fold increase in midazolam’s systemic exposure, netupitant at 300 mg is considered as a moderate inhibitor of CYP3A4 in vivo. Oral contraceptive: ethinylestradiol and levonorgestrel The potential effects of FDC on PK of an oral contraceptive (Mycrocynon®: a fixed dose combination of 0.03 mg ethinylestradiol and 0.15 mg levonorgestrel) were studied after a single dose administration of OC with and without AKYNZEO in healthy female subjects (n=24). For bioanalytical assay, two tablets of Mycrocynon® were administered although the approved dose is one tablet of Mycrocynon®. When given with AKYNZEO the mean Cmax and AUC0-∞ of Ethinylestradiol was 5% and 16% higher, respectively. For the levonorgesterel component, there was no effect of the FDC on levonorgestrel Cmax, while exposure parameters (AUC0-∞ and AUClast) were increased by approximately 40% (Table 49). 66 Reference ID: 3516218 Table 48 Erythromycin and Midazolam Exposure (Means) with and without Netupitant (300 mg) Table 49 PK Parameters (mean ±SD) for Ethinylestradiol and Levonorgestrel after Oral Administration of Microgynon® with and without AKYNZEO 67 Reference ID: 3516218 2.4.2.7 Are there any in vivo drug-drug interaction studies that indicate the exposure is different when drugs are co-administered? Drug Interaction Studies With strong CYP3A4 inhibitor Ketoconazole Single dose AKYNZEO was administered with ketoconazole following once daily administration of 400 mg ketoconazole for 12 days and the PK of netupitant and palonosetron were compared to that after administration of AKYNZEO alone (NETU-10-11). Netupitant Co-administration with ketoconazole, a strong CYP3A4 inhibitor with AKYNZEO increased mean Cmax by 1.3-fold and mean AUC by 2.4-fold when compared to the administration of AKYNZEO alone. Mean Cmax and AUC were lower for all three metabolites after coadministration of ketoconazole (Table 50,Table 51). Metabolites of netupitant M1 Concomitant ketoconazole delayed the median Tmax for M1 from 12 h to 96 h. The average metabolite to parent ratio for M1 based on AUCinf was 24.9% with ketoconazole and 30.3% without ketoconazole. M2 Concomitant ketoconazole did not affect the median Tmax for M2 (Tmax was 5.5 h for both treatments). The average metabolite to parent ratio for M2 based on AUCinf was 6.37% with ketoconazole and 12.1% without ketoconazole. M3 With ketoconazole the median Tmax for M3 was delayed from 12 h to 24 h. The average metabolite to parent ratio for M3 based on AUCinf was 15.1% with ketoconazole compared with 28.1% without ketoconazole. Palonosetron Concomitant ketoconazole did not affect the pharmacokinetics of palonosetron. 68 Reference ID: 3516218 Table 50 Mean (±SD) PK Parameters after Oral Administration of Netupitant/Palonosetron (300 mg/0.5 mg) with and without Ketoconazole (400 mg q.d.) (NETU-10-11) Table 51 Mean (±SD) PK parameters of metabolites of netupitant with and without ketoconazole Source: In-Text Tables 11.4-3, 11.4-5, 11.4-7 in Study Report of NETU-10-11 a n=9; bn=17; cn=16 69 Reference ID: 3516218 Strong CYP3A4 inducer Rifampicin Single dose FDC was administered with rifampicin following once daily administration of 600 mg rifampicin for 17 days and PK of netupitant and palonosetron were compared to that after administration of FDC alone (NETU-10-11). Co-administration of rifampicin, a strong CYP3A4 inducer rifampicin with netupitant/palonosetron FDC decreased the mean Cmax and AUC0-∞ by 2.6, and 5.9 fold, respectively. Co-administration of rifampicin decreased the mean AUC for palonosetron by 20% (Table 52). Table 52 Pharmacokinetic Parameters (Mean±SD) after Administration of AKYNZEO with and without Rifampicin (600 mg q.d.) (NETU-10-11) Metabolites of netupitant Concomitant administration with rifampicin increased the systemic exposure to M2 but decreased the systemic exposure to M1 and M3 suggesting that M2 is the major metabolite formed by CYP3A4 while M1 and M3 are further metabolized by CYP3A4 or other enzymes inducible by rifampicin (Table 53). 70 Reference ID: 3516218 Table 53 Mean (±SD; CV) PK parameters of metabolites of netupitant with and without Rifampicin Source: In-Text Tables 11.4-4, 11.4-6, 11.4-8 in Study Report of NETU-10-11 a n=16; b n=18; c n=11 P-glycoprotein Digoxin In vitro studies showed that netupitant interacts with P-glycoprotein (P-gp) resulting in a concentration-dependent modulation of digoxin transport. Therefore, an in vivo drug interaction study with digoxin, a P-gp substrate was conducted to assess the effects of netupitant on the pharmacokinetics of digoxin at steady-state in healthy volunteers (n=16; NETU-07-01). Digoxin was administered once daily 0.25 mg digoxin for 11 consecutive days [Days 2-12] following loading dose of 0.75 mg digoxin on Day 1 and a single dose 450 mg netupitant was administered on Day 8. The systemic exposure to digoxin on Day 6 and Day 8 was compared. The PK and urinary excretion of digoxin was similar in the presence and absence of netupitant (Table 54, Table 55) This study results indicate that when netupitant was co-administered with digoxin simultaneously, it does not significantly affect the PK and overall absorption of digoxin at steady-state. Table 54 Mean PK parameters for Digoxin in the Absence and Presence of Netupitant (NETU07-01) Tmax: Median (min - max) 71 Reference ID: 3516218 Table 55 Mean (SD) urinary excretion of digoxin (N=16) Reviewer’s comments: This study demonstrated that no significant effects of netupitant on digoxin absorption and urinary excretion when netupitant and digoxin were concurrently administered. In this study, the median Tmax for digoxin was 1 h when netupitant concentration is about 80% lower than the mean Cmax (mean Cmax for netupitant was 755 mcg/L and mean concentration at 1 hour was 121 mcg/L) and the median Tmax for netupitant was 4 h. No significant effects on the urinary excretion of digoxin suggest that the effect of netupitant on the P-gp on the kidney was not significant. 2.4.2.9 Is there a known mechanistic basis for pharmacodynamic drug-drug interactions, if any? The efficacy of AKYNZEO is based on the indirect pharmacodynamics drug-drug interactions. The binding of serotonin and NK1 released upon administration of emetogenic chemotherapy is proposed to be blocked by AKYNZEO. 2.4.2.10 Are there any unresolved questions related to metabolism, active metabolites, metabolic drug interactions or protein binding? Single dose netupitant could increase the systemic exposure to dexamethasone administered by 2 fold on 3 days after single dose administration. The inhibitory effect was not studied beyond 4 days after administration of netupitant while estimated to last for at least 6 days based on [I]/Ki values. 2.4.3 What issues related to dose, dosing regimens or administration are unresolved, and represent significant omissions? None. 2.5 General Biopharmaceutics 2.5.1 What are the solubility and the permeability of netupitant? 72 Reference ID: 3516218 The permeability of netupitant was determined Parallel Artificial Membrane Permeation Assay (PAMPA) and CACO-2 cell line. With the PAMPA model, the permeability of netupitant was determined to be 1.1 x 10-6 cm/s (10.6 nm/s) and 2.5 x 10-6 cm/s (24.6 nm/s) at concentrations of 10 and 50 μM, respectively (NETU-1026). As controls, the reference compounds [3H]-propranolol (high permeability) and sulfasalazine (low permeability) were included in the assay. However, netupitant had very high, about 65-70% non-specific binding in this study. In addition, it appears that netupitant did not dissolve well in the buffer used in the experiment, and therefore, the actual concentration of netupitant that is exposed at the donor side is much lower than what is stated theoretically. With Caco-2 model, permeability of [14C]Netupitant at three concentrations (1, 10, and 100 uM) were evaluated from apical side to the basolateral side (A→B) and from the basolateral side to the apical side (B→A) on Caco-2 cells in triplicate wells and was repeated on two different days. As controls of monolayer integrity, the reference compounds [3H]-propranolol (high permeability) and [3H]-mannitol (low permeability) were included in the assay. However, in this study, netupitant had very high, about 30-90% non-specific binding. In addition, it appears that netupitant did not dissolve well in the buffer used in the experiment, and therefore, the actual concentration of netupitant that is exposed at the donor side is much lower than what is stated theoretically. Furthermore, the apparent permeability was calculated using the final donor concentration at the end of the incubation instead of initial donor concentration. Table 56 Permeability of [14C]Netupitant at the initial concentrations Co of 1, 10, and 100 μM n.a.: not applicable. Could not be determined since no detectable [ 14C] Netupitant was present in receiver compartment Both of these permeability studies are hard to interpret as both studies had very high non-specific binding and actual concentration in donor side is significantly different than the theoretical concentration. In addition, the suitability of Caco-2 cell method was not evaluated with sufficient number of model drugs, and the expression of P-gp transporter on Caco-2 cell was not characterized with a model substrate. 2.5.2 What is the relative bioavailability of the proposed to-be-marketed formulation to the pivotal clinical trial? The review of bioequivalence study is deferred to the biopharmaceutics review in the ONDQA. 73 Reference ID: 3516218 The to-be-marketed formulation was used in phase 3 trials but the sponsor proposes to change the manufacturing site for marketing. In addition, extemporaneous combinations of netupitant and Aloxi was used for the phase 2 dose-finding study which establishes the contribution of netupitant to the combination product as well as the efficacy of the combination for the prevention of CINV associated with cisplatin-based chemotherapy (HEC). Two pivotal BE studies were conducted to bridge the manufacturing site change and between the extemporaneous formulation and the to-be-marketed formulation. One study was to bridge the (b) (4) Phase 2 formulation (extemporaneous combination of capsules containing netupitant plus Aloxi® softgel administered simultaneously) and the Phase 3 formulation (FDC containing three 100 mg netupitant intermediate tablets and one 0.5 mg palonosetron softgel) in study NETU-09-07. The palonosetron softgel in the FDC is different from the approved Aloxi oral (b) (4) softgel for the size of capsule (Table 57). Bioequivalence was established between the phase 2 formulation and the phase 3 formulation and between two FDCs with the same formulations manufactured at 2 different manufacturing sites: (b) (4) HBP (test formulation, Phase 3/proposed commercial material) and (reference formulation, Phase 3 material). Both FDCs contained Intermediate netupitant tablets and a palonosetron softgel (NETU-11-02) (Table 58). Table 57 Bioequivalence between extemporaneous combination used in phase 2 trial and FDC formulation used in phase 3 trial (NETU-09-07) 74 Reference ID: 3516218 Table 58 Bioequivalence between FDC formulations manufactured at different sites 1- 02) Netupitant Geometric mean ratio Parameter 90% CI Cmax 92.72% 86.41 99.50% 93.93% 89.35 98.74% 92.62% 87.34 98.22% Palonosetron Parameter 90% Cl Cmax 102.36% 100.38 - 104.37% 101.11% 99.32 102.94% 101.08% 99.23 102.96% Test: FDC (Helsinn Birex manufacturer) Reference: FDC 2.5.3 What is the effect of food on the bioavailability (BA) of the drug from the dosage form? About 17% increase in systemic exposure to netupitant by a high fat meal was observed while a high fat meal did not affect the PK of palonosetron. NETU-10-12: The food effect study was conducted after administration of a single dose of PDC in 24 healthy male and female subjects. 011 Day 1 of each treatment period. a single oral dose of the FDC of 300 mg netupitant and 0.5 mg palonosetron was administered. The drug was administered 30 min after start of a high fat meall4 following 10 hour overnight fast. In this study the high fat. high-caloric breakfast led to a delay in absorption of netupitant. There was an increase in systemic exposure of about 16% for and about 18% for and Cmax (Table 59.Table 60). For palonosetron. the systemic exposure was not signi?cantly affected by a high fat meal (Table 61). 14 The content of the high-fat, high-caloric breakfast followed the recommendations given in the FDA guidance ?Food Effect Bioavailability and Fed Bioequivalence Studies?. The breakfast contained 150 protein calories, 250 carbohydrate calories and 500 to 600 fat calories resulting in a total caloric content of about 945 kcal. 75 Reference ID: 3516218 Table 59 Comparison of Netupitant Cmax and Exposure Values between Fasted and Fed Healthy Subjects Table 60 Effect of food on netupitant PK (n=22) Table 61 Effect of food on palonosetron PK (n=22) 2.6 Analytical Section 2.6.1 How the active moieties are identified and measured in the plasma/urine in the clinical pharmacology and biopharmaceutics studies? Akynzeo® contains two active ingredients, netupitant and palonosetron. The concentrations of netupitant and palonosetron in human plasma were determined using validated liquid chromatography mass spectrometry (HPLC/MS/MS) methods. 76 Reference ID: 3516218 Netupitant and its metabolites were quantified by validated LC/MS and LC/MS/MS methods, using stable label internal standards (IS) for each analyte. Partial validations were conducted throughout the development program to account for changes in assay method including extraction method, the change to a 96 well-plate format and selectivity in presence of palonosetron. Bioanalytical assay method for palonosetron was based on the previously established method used for studies supporting the approval of Aloxi. The method was further validated to account for the change to a 96 well-plate format and selectivity in presence of netupitant. Bioanalytical assay for co-administered medications During the development of the combination program, several other drugs were also analyzed in clinical trials. Corresponding assays were developed and validated with acceptable accuracy and precision. Midazolam (NP16599) Midazolam was isolated from plasma by liquid/liquid extraction and determined by LC-MS/MS. The limits of quantification were 0.100 ng/mL for all assay batches. The inter-day precision of the assay was below 3.8% (CV). The inter- day accuracy of the assay was better than 95.3%. Erythromycin (NP16599) Erythromycin was isolated from plasma by liquid/liquid extraction and determined by LCMS/MS. The limit of quantification was 20.0 ng/mL for all assay batches. The inter-day precision of the assay was below 7.2% (CV). The inter-day accuracy of the assay was between 93.6% and 108.1%. Dexamethasone (NETU-06-07) Dexamethasone was isolated from plasma through liquid/liquid extraction and measured by LCMS/MS. The original method by which the LLOQ was determined to be 1.06 μg/L (precision=8.77%, accuracy =-3.03%) was partially validated further to determine LLOQ at the concentration of 1.004 μg/L (n=6, precision = 13.45%; accuracy = -4.28%). The total precision for dexamethasone in human plasma was in the range from 6.02% (at 95.393 μg/L) to 6.98% (at 2.687 μg/L). The accuracy for dexamethasone was better than 8%. The presence of netupitant did not disturb the recovery. The acceptance criteria (precision and accuracy <15%) were met for all analytes. Digoxin (NETU-07-01) Digoxin was measured in plasma and urine using liquid/liquid extraction from plasma and a validated LC/MS/MS method. The inter-day precision for digoxin in human plasma was in the range from 5.27% (at 3.9 mg/L) to 9.65% (at 2.522 mg/L). The inter-day accuracy for digoxin was better than -4%. The inter-day precision for digoxin in human urine was in the range from 6.53% (38.997 mg/L) to 8.77% (25.220 mg/L). The accuracy for digoxin in urine was better than 4%. 77 Reference ID: 3516218 Ethinylestradiol/levonorgestrel (NETU-10-08) For the analysis of ethinylestradiol and levonorgestrel in plasma, liquid/liquid extraction and a validated LC-MS/MS method was used. The LLOQ was 5.111 pg/mL for ethinylestradiol and 0.492 ng/mL for levonorgestrel. Calibration ranges were 5.1-230 pg/mL for ethinylestradiol and 0.492 – 22.123 ng/mL for levonorgestrel. Precision was better than 10% and 5 for ethinylestradiol and levonorgestrel respectively and accuracy was better than 5% and 3% for ethinylestradiol and levonorgestrel, respectively. Docetaxel (NETU-10-09) Docetaxel was measured in human plasma using solid-liquid extraction and a validated LC/MS/MS assay. The calibration range was 1-500 ng/mL and the LLOQ was 1 ng/mL. Interassay accuracy (in the QC range) ranged from 1.6% to 5.2% and inter-day precision ranged from 3.1% to 4.9%. At the LLOQ, inter-day accuracy was 3.9%, and inter-day precision was 5.7%. Etoposide (NETU-10-09) Etoposide was measured in human plasma using a validated LC- MS/MS assay, after protein precipitation. The inter-day accuracy (in the QC range) ranged from 1.8% to 5.7% and inter-day precision ranged from 3.2% to 5.0%. At the LLOQ, inter-day accuracy was 1.5%, and inter-day precision was 7.2%. Cyclophosphamide (NETU-10-09) Cyclophosphamide was measured in human plasma LC/MS/MS assay, after protein precipitation. The calibration range was 0.10 -50.0 ng/mL and the LLOQ was 100 ng/mL. The inter-day accuracy (in the QC range) ranged from -1.5% to 5.2% and inter-day precision ranged from 1.3% to 2.1%. At the LLOQ, inter-day accuracy was -0.7%, and inter-day precision was 6.2%. 2.6.2 Which metabolites have been selected for analysis and why? NETUPITANT Three oxidative metabolites (M1, M2, and M3) were isolated from an in vitro incubation of netupitant with recombinant human CYP3A4. A fourth metabolite was identified during ADME study (NETU-09-21). In vitro all the metabolites showed binding affinity to human NK1 receptor. The exposure to metabolites M1, M2, and M3 resulted >10% of the parent drug exposure, in term of AUC0-t., although only M3 resulted >10% of the total radioactivity exposure. M4 was not observed in previous preclinical study, but identified later, during human mass balance study, and quantified in study NETU-11-23 accounting 3% of the parent drug exposure, in term of AUC0-t. Although active, this metabolite was; therefore, considered of negligible clinical relevance. PALONOSETRON Bioanalytical methods for palonosetron and its metabolites quantitation developed during clinical development of netupitant/palonosetron FDC, were based on previously validated methods for palonosetron during clinical development as single agent. The lowest limit of quantitation was 50 pg/mL for palonosetron and M4, and 10 or 50 pg/mL for M9, depending on the study. 78 Reference ID: 3516218 2.6.3 What is the range of the standard curve? What are the lower and upper limits of quantification (LLOQ/ULOQ)? What is the accuracy, precision and selectivity at these limits? Validation of the bioanalytical methods performance used for the determination of concentrations of netupitant and palonosetron in plasma are presented in Table 62 and Table 63. Table 62 Bioanalytical Method Validation Analytical Parameters Analytical Range Between-batch Precision (%) Between-batch Accuracy (%) Within-batch Precision (%) Within-batch Accuracy (%) Recovery (%) Freeze-thaw Stability LQC (three cycles) (%) Freeze-thaw Stability HQC (three cycles) (%) Freezer Stability LQC (34 months, -70°C) (%) Freezer Stability HQC (34 months, -70°C) (%) *- 34 Months at -70°C Netupitant Palonosetron 2 to 500 ng/mL 45 to 1500 pg/mL 2.92 to 3.62 2.2% to 5.6% 0.41 to 1.92 0.0% to 1.8% 1.45 to 4.88 1.1% to 7.2% -1.58 to 3.56 0.1% to 3.3% 62.0 95.6 6.28 -1.7 0.74 2.1 * 5.50 -11.48** -0.43* -14.90** ** - 22 Months at -20°C The bioanalytical method is acceptable for the determination of concentrations of netupitant and palonosetron from the plasma samples. In all the methods, calibration curve fitting was obtained by least-square linear regression analysis of weighted analyte concentration (1/X2) versus peak area of the analyte/IS (Y). The approach followed was to re-assay approximately 5-10% of the entire PK study samples both for netupitant and/or metabolites and palonosetron. Two samples of each concentration time profile were re-analyzed: one sample around Cmax and another sample with a concentration > LLOQ. At least 67% of all re-analyzed incurred samples had not to deviate by more than ±20% of their original concentration. The methods developed for netupitant and metabolites, as well palonosetron, metabolites, and other co- administered drugs, were selective enough to generate reliable data. Indeed, the interference between the analytes and the other drugs, or endogenous substances, was within the acceptance criteria established (defined as 20% of the LLOQ analyte response or <5% of the IS response) in all the experiments conducted. The precision and accuracy of netupitant and metabolites, as well as palonosetron and metabolites, in the presence of other drugs, and vice versa, was not affected and proved to be within the 15% acceptance criteria established. The precision and accuracy of netupitant and 79 Reference ID: 3516218 metabolites, as well as palonosetron in the presence of 5% of lysed blood or up to 50% of a standard hyperlipidemic matrix, was within the 15% acceptance criteria established. Table 63 Bioanalytical Method Validation for netupitant and its metabolites in plasma and urine 80 Reference ID: 3516218 3 Major Labeling Recommendations 1) Add a statement “Avoid use in patients who are already on CYP3A4 inducers” in Section 7. 2) Add a statement about the duration of CYP3A4 inhibitory effects after single dose administration of AKYNZEO in Section 7. 3) Add the subheading of “Drug Interactions” and “Specific Population” in Section 12.3 and move detailed PK study results from Sections 7 and 8. 4) Detailed labeling recommendations will be conveyed to the sponsor during the labeling negotiation. 81 Reference ID: 3516218 4 Appendices 4.1 Table of Clinical Pharmacology Studies BAIBE study 5* Mdm' ?ae ?it-pf.- An etplontm'y Randomized open- Single P0 18 HVs am 61-) relative BA study label. thteeway crossover to Netupitant 450 mg sodium Age 24-59 evaluate bioavailability of two dodecyl sulfate (SDS) di??etent foamulations (SDS and capsule fonmlation and SE capsules). and to evaluate the nctupitant 450 mg sucrose SE foundation with food ester (SE) capmle ?omnlation 1-23 Conparative Comparison between three Single P0 24 HVs (24 M) bioavailability fonnulations with different Age 218-47 study dissolutions pto?les. FDC 300 mg"0.5 mg capsule with standard Randomized open labeL 3? dissolution ucanmnt. 6- sequence. 3-paiod crossover study. FDC 300 mgi?0.5 mg capsule with slow dissolution and extmporaneous netupitant 300 mg suspension plus palonosetron 0.50 mg so?gel NETU-08-12 Pilot bioemtivalence BE of di??etent formulations Single P0 8 (8M) healthy subjects study (?nal FDC and mmponneous) (19-45 yrs) FDC 300 mg'0.5 mg Randomized. open label. single- capsules dose. 2 petiod. two-square crossover. pilot study vs. Netupitant 2 150 mg capsules plus Palonosetion 0.5 mg softgel given as extemporaneous contination NETU-09-07 BE study BE of different ?mmulations( Single P0 50 W5 (26F. 24M) FDC and extenporanemis) PK tiom 47 subjects 300 mg?0.5 mg 241') 19-45 yrs. Randomized open label single capsules dose. 2 petiod. two-sequence cmssova. pilot study. vs. Nempitant 2 150 mg capsules plus Palonosetnon 0.5 mg so?gel given as attenporaneous combination 1-02 BE study Bioeqmvalence study SinglePO 88 W5 (19F, 69M) . between FDC capsules with FDC 300 mg mg 82 conpleted. same formulation ptomcedby manufactutedby PKpopulation?xnetupitmt: two di??eient matmfactums: 0(4) 82 subjects 17?) PK Helsinn Bitex Pharnnoeuticals population for palonosetron? Commercial vs. 79 subjects (63 Product) and 161-) (Pluse 3 andlatephase FDC). FDC300mg/05mg manufactuted by HBP Reference ID: 3516218 82 Studies with Netupirant alone . . . Dose and [huge Form Subjects Characteristic Study Emily Glue-cure and Desagn [range and meaniSD] Mass balance ADME Study ?fringe PD 6 HUS 5 completed) study Liana balance and metabolic oral suspension 390' mg (EM. {If} pro?le {1101111113} @053} 32?56 2.21 {60 oil} Impact of PK'Eafery drug? interaction trial Single PD Nerupitant 3m 20 HVS (RUM, oetupirant on ?re with and rug age 30:1,: pharmacolaochcs midazolana 1 . . of midazolam and madman; rang *0 51ml?; mm? 590mg drug Evaluation of Food and Age arm trial of 319%?? P0 Fm 12 11? the e??ects of netupimnt: ,age 33?44(11) food and age on Food E?ect; netupitant 30-0 Age effect: 5 H'v 5 (car. the PK of mg {3x151} mg caps) age rid-T2 nettrpiranr 115 received active drug 2 received placebo Randonnzed. double? Multiple P0 33 Hit-'5 {3 3M, ?Fj Multiple blind, placebo-controlled, Placebo. netupitant 100. 3B HVS age 31?44 yrs. 3 ascending dose multiple ascending dose in two 3111]. 450 mg 55-68 (E) PK shady in parts {young and elderly 25 subjects received active healthy young and subjects): PE?safety in healthy drug; 1 placebo and lelderly elderly volimteers Young and elderly volunteers dropped out. Apoinorphine Randomized' . double? blind 3:52:30 32 HUS {3'3 1 Net?upitaot 300 mg Nempitaat 450 mg 24 received active thug 1E) ?Pm-503 Single ascending Randomized doublobhdd, 5mg?: PD dose studv placebo? controlled single? Placebo Wempitanr l? . mg age 19-43 0f oetupirant ESEZIUI . ?20? PK safety ?1 Nempitant 313 111g received active drug-1E} 3' "1 eers Newpitantl?? ing placebo Nerupitant 3131] mg Newpitaot 450 mg Reference ID: 3516218 83 Studies with Netupitant alone Reference ID: 3516218 . . . Bose and Dosage Form Subjects Characteristic Said} Study Objecme and Desagn (range and ?mm PKInteraction Randomized, open 3?period Treatment A: 26 H'lis bem? crossover {lib-'1, 1 If} Nehtpitant and Latin Square (subjects {14 M. 11 P) age Oral ?ont Day 2 to Illa}r 4) 13?12 were treated and Dexamethasone included in at least one Regimen: population set. Dexametliasone [as in group A.) plus Netupitnnt 101] mg on D'aj,r 1 Treatment C: Dexametliasone [as in group A) plus Nehtpitnnt 300 mg on Illa].r 1 Treatment D: Dexamethasone (as in group plus Nentpitnnt 450 mg on Day]. Receptor Single?dose: randomized, open? ?5er PO ?5 completed (5M, occupancy study 1313,31 PET 5mm,- Nempitant 100 mg 2 per each dose level using PET - . - - i . 20?25 mt-eshgatmg the degree of Netti itant 450 5?3 occupancy of receptors in the human brain after single oral doses in Support dose selection for Phase 2 trials PK interaction PIC-safety drug?interaction trial '3er P0 16 H'v's (SM. 31:} r31 between with Diggxm: Netupitant 1945 yes nempitant and 451] mg on Day 3 digoxin Bigot-tin 0.25 mg loading dose 330.5 mg on Dayl. followed bf,? {125 mg for 11 consecutive Days 84 Studies with netupitant and palonosetron combination Reference ID: 3516218 aways Study Objective and Design ?mg? Fm" Drug Interaction Randomized. open? Single PD 13 HF between nempitant label. single dose. 3 period (PM. and Palonosecron. study to evaluate the PK Nempitant 450 mg 18?36 interaction between netupitant and palonosetron in healthy Netupitant 450 mg volunteers Palonosetron 0.75 mg Palonosetron 0. NEW-0120 Thorough QT study: Randomized double- blind Single PO .200 {1065.41, 94?} {196 (except Placebo completed} double?dummy, parallel Age :19?45 group placebo and open? netnpitant 100 mg label positive controlled smdy palonosetron 0.5 mg to investigate possible ECG effects of netupitant and netnpitant 500 mg palonosetron palonosetron 1.50 mg moxi?oxacin 400 mg NEW-1002 Population Population PK study SinglePD Nempitant analysis: 11? PK study NEW-1002. Subgroup from 113$}. 55 yrs? (29-75); Phase 3 study Group 1? oral ??mpita?t-?p310?0 Bet?F011 Race: 101 Caucasian. 16 {3 00 mg] Asian. dexamethasone BW: 11* {34 125BMI: 2143* (14.?241 .14) 1191112 Group oral palonosetron 0.50 mg (Aloxi) and oral Palonosetron analysis: 113 dexaniethasone (SM. 113?} 55 yrs? {2945}: 20 mg on Day 1. Race: 103 Caucasian 16 Asian. Group 1 only netupitant and palonosetron BW: measurements were Ely-ll: 2145* (14.12?11.14) included in the analysis. leg-m2 *median Drug?Interaction Open. randomizeti two?way Single P0 .24 HVs (0M. 24F) trial with oral crossoer trial to evaluate the 19?10 contraceptives: effect 05mg two cthinylestradiol and tablets of Microgynon? leyonorgestrel in healthy {30 ug ethinylestradiol 4.- female subjects 150 pg leuonorgestrel per tablet} vs. Two tablets of ItdicrogymortlEI 85 Studies with netupitant and palonosetron combination Reference ID: 3516218 . . . Dose and Dosage Subjects Characteristic Study Study Objectrve and Desngn Tomi (range and mearetSD) DDI between Single?dose. open? label, Single PO Cancer patients and chenro two period Nempitant'Palonosetron FDC Docetaxel Group: 3 cancer [docetatreletoposide 300 mg {1.5mg patients Sill-81 yrs) Randonnzed crossover drug DocetatrehEtoposidei? interaction stud}; of?re Etoposide Group: 13 cancer oral netupitant-?palonosetron patients {1 1M, 22??3 yrs) FDC on the PK of docetaxel. vs. Palonosetron 0.5mg etoposide or cvclophosphamide Docetavel-?Etoposidei Cyclophosphamide Group: in cancer patients. lerclophosphanride 10 cancer patients UM. 91:; 33-69 yrs) Hepatic impairment Single center. open labeL one Single P0 In totalz3o Patients {26 men, period PK stud},r in patients with 3110 mg-' 0.5 mg 10 women) [39?111 years old) different stages of hepatic impairment 3 pts. with mild hepatic Impairment 3 pts. with moderate hepatic Impairment 2 pts. with severe hepatic impairment drug? interaction Open randomized two?group: Single P0 35 (31M study with two?way crossover stud},r to 3110 mg {1.5 mg PKpopulation: lcetoconazole and evaluate the e?ect of plus Ketoconazole group: 1? rifarnpicin: concomitant administration Ketoconazole subjects 33?55 of ketoconazole or rifampicin__ 400 mg i; 13 consecutive days 1.15,) on the PK of netupitant and or palonosetron Rifampicin 600mg 9; 11? Rifampicin group: 13 subjects consecutive day?s 3 3?55 yrs) Food and Age effect Open randomized coo?way. Single P0 35 {221191. 14F) trial with cross?over study to investigate PKpopulation in cross-over the effect of food (comparison 3110 mg-?tLS mg part: 22 H?v's [22-45 years) fasted and fed condition) with one parallel group of elderly: PK population in the parallel subjects to investigate the part: 12 H?v's [do?F9 years). effect of age [comparison elderly versus younger subjects in the fasted group). Gender effects on Post-hoc analysis report to Information in stud},r Pooled analysis in 153 HF PK using pooled PK evaluate the effect of gender 18?503; data on in?vivo palonosetron and 02 and 112 41 netupitant phannacolonetic 11?3 3 profiles? Netupitant: 330 pooled PK profiles in M: 115 pooled PK pro?les in Palonosetron: 293 pooled PK profiles in M: 112 pooled PK profiles in 86 4.2 Pharmacometric Review OFFICE OF CLINICAL PHARMACOLOGY: PHARMACOMETRIC REVIEW 1 0F FINDINGS 1.1 Key Review Questions The purpose of this review is to address the following key questions. W?hat are the covariates affecting the PK of uetupitant based on population PK analysis? No statistically significant covariates were identified in population PK analysis. A two- compartment base model with first order absorption adequately described the observed PK data of netupitant. The median netupitant apparent clearance was estimated to be 20.9 [lb and the volume of distribution was estimated to be 419 Based on sponsor?s analysis, none of the covariates had significant impact on the PK of netupitant based on population PK analysis (Table 1). However. it is worth noting that for some intrinsic and extrinsic factors, dedicated studies were available and therefore population PK analysis is supportive. For gender. only 4 male subjects were included in the population PK analysis. therefore the effect of gender on PK should be derived primarily based on the dedicated studies. lior hepatic impairment. a dedicated study was conducted to evaluate the effect of hepatic impairment on PK. Based on the population PK analysis. there appears to be no effect of race and body weight on PK of netupitant. In addition there was no dedicated renal impairment study conducted for netupitant. Based on population PK analysis, there appears to be no statistically significant effect of mild and moderate renal impairment on the clearance of netupitant. This is expected since renal pathway is a minor route of elimination for netupitant. For drug-drug interactions. the data from dedicated clinical pharmacological studies were available to evaluate the effect of drug interactions on netupitant PK. The impact of the smoking status. chemotherapy (donorubicin. cpirubicin. lluorouracil) and rescue medications on the PK of nctupitant in combination with palonosetron were evaluated by population PK analysis. None of those factors appears to significantly in?uence the disposition of netupitant and palonosetron. However. it should be noted that definitive conclusions regarding these factors cannot be made as the population PK analysis may lack power to detect the effect of these factors due to the study design andfor insufficient PK sampling. 87 Reference ID: 3516218 netu pita nt (n=1 1 7) Commie N=t 17 Continuous I?hrfabie: Medim (range) Age (years) 55 {29-715) Body mass index (kg. m1] 27.43 (143241.74) Body weight {kg} 71 (34 ?125) Baseline ALT 18(6-105) Baseline AST 19 {9 90] Baseline alkaline phosphatase (TU-L) (20-1192) Baseline total hilimhin (penal-L) 6 (l 13) Baseline albumin [g 44 {29-52} Baseline cmatim'ne clearance 10%] (3t 150} Baselinenenn'ophilcmim?oyl) 4(15 -9.4) Cotegon?raf Thimble: Count Sex {females'males} Race (Caucasaam?ASian) 113 4 {4.323) Table 1. Demographics and baseline characteristics in population PK analysis for 16 95 (31.2: 6 75 {3511.511 (0.993} Tobacc usage ECOG performance status (0:1 Chemotherapmnic rem (Cyclophosphamide and T4 Doxmnhicm' Cyclophosphamide and Epiruhticin) rating qrclophosphmae 11? (1009:.) Taking doxmubicin [63 .2913} Taking epuuhicin $9 Taking ?umowacil 44 Not taking rescue modication?- acute phase 110 Not taking rescue medication?. delayed phase Not taking rescue medicanoo". overall phase 101 (3121-3) 102 (3121 ?Among patents talang rescue. all but one tool: metocloprannde Source: Sponsor?s data analysis report for study Page 37 1.1.2 What are the exposure?response relationship for efficacy and safety? A formal assessment of exposure-response relationship could not he made due the limited PK data collected in the clinical studies as onl},r 117r patients out of 7'26 in FDC arm in the study had PK samples. 1.2 Recommendations The application is acceptable from phannacometrics perspective. Following are the recommendations: No dose adjustment required based on race and body weight No dose adjustment required based on age (29-75 years) I No dose adjustment required for mild or moderate renal impairment 1.3 Label Statements See section 3 in clinical phannacology review. Page 2 of2 88 Reference ID: 3516218 4.3 IRT-QT team review (For detailed review, please see the original review dated 1/20/2010) 89 Reference ID: 3516218 1.2 OVERALL Sits-ni-IARY or thmxos The sponsor used as their primary co1rection method. Based on our evaluation for different correction methods. we believe corrects RR more suf?ciently than in this study: therefore. FDA analysis results based on are suggested for the labeling. No significant prolongation effect of nempitantfpalonosetron (therapeutic dose 200 mgf0.50 mg and supratherapeutic dose 600 mg? 1.50 mg} was detected in this TQT study. The largest upper bounds of the 2-sided 90% CI for the mean difference between two netnpitants?palouosetron dose and placebo groups were below 10 ms. the threshold for regulatory concern as described in ICH E14 guidelines. The largest lower bound of the two-sided 90% CI for the for moxi?oxacin was greater than 5 ms: however. the moxi?oxacin profile is missing a rising phase {Figm'e 9): therefore. we would like to evaluate moxi?oxacm induced effect at 15 minutes and 30 minutes post-dose as well. In this double-blind. randomized. parallel- group study. 200 healthy subjects received 200 mg netupitantr? 0.50 mg palonosetron. 600 mg nempitant? .50 mg palonosetron. placebo. and a single dose of moxi?oxacin 400 mg. Overall of findings is presented in Table 1. Table 1: The Point Estimates and the 90% CIs Corresponding to the Largest Upper Bounds for netupitant?palonosetl'on (200 1ngf0.5 ] mg and 600 Ingf1.50 mg) and the Largest Lower Bound for Moxi?oxaein (FDA Analysis) Treatment Time (hour) anTcF (ms) 90% (ms) Netupitanta?palonosetron . 14 4.4 1.5. TB (200 rug/0.50 mg) ?1 Netupitantr?palonosetron . (600 mgfl.50 mg) 16 '9 9'1) Moxifloxacm 400 mg* 4 13.2 (10.1. 16.3] Multiple endpoint adjustment was not applied. The largest lower bound after Bonferroni adjustment for 5 timepoints l. 2. 4. 5.. and 5 hours] was 9.2 ms. The supratherapeutic dose (netupitant 600 mgr?palonosetron 1.50 mg) produces mean netupitant and palonosetron (7mm values 32-fold higher than the mean (?max for the therapeutic dose [netupitant 200 mgjpalonosetron 0.50 mg}. The expected high clinical exposm?e scenario for netupitant is unknown at this time. Hepatic impailment may decrease netupitant?s clearance as hepatic metabolism is the route of metabolism. However. exposure data in patients with hepatic impairment is not available. The accmnulation of netupitant is 2.16-3.06 after 7 days dosing. so the 600 mg supratherapeutic dose in this study may not cover high clinical exposure with cln'onic dosing and hepatic impairment. Intrinsic and extrinsic factors have not been shown to signi?cantly increase palonosetron exposin?e. so the 1.50 mg supratherapeutic dose is expected to cover the high clinical exposure scenario. 90 Reference ID: 3516218 4.3 OCP Filing Form Office of Clinical Pharmacology Nat-v Drug App/icoiim?i and Review Form .4 the .S'utimis sin? Information Information Number 205-118 Brand Name Aks'nzeo OCP Division (I. II. IV. V) Generic Same Nerupitantipalonosctron Medical Division DC IEP Drug Class Anti-cmctics OCP Reviewer Ins-colt Kim, Pl'l.D. Indication?) Prevention of acute and delayed Dilara Jappar, with initial and repeat courses ofllighlg.? emetogenic chemotherapy and moderatelr emetogenic chemotheraps' OCP Team Leader Lee? Dosage Form fixed dose combination capsule Pharmaco metrics Reviewer Jingyu "Jerry" Yu. Dosing Regimen 60 min prior to initiation of chemotherapy Leader Nilin llehl'nlra, PILD. Route ol'AilnIinislruliun ll'Ilral Date of Submission 9.118013 Sponsor Helsinn Estimated Due Date of OCP Review 5123012014 Priority Classification 5 Medical Division Due Date 5F30i201~l Due Date 9I'26i201-i (Yin- Pimrm. and Biopharm. Infornmriml if included Sumher of Number of Critical lComments If any at ?ling studies studies submitted reviewed TY PE Table of Contents: prevent and tut?t'ieient to locate reports?. tahlcc, data. etc. Tabular Lia-ling (IL-HI Iluman Studies- J: IIPK Sunlnlan' :c Labeling .1: Reference Iiinunulylicnl and Analytical .1: I2 'l'wn active ingredients in plasma and urine Drugs used in DDI studies 1. Clinical Pharmacologv 11am- balance: it 1 {netupitant} leazt'me characterization: 1 ii [003831 Bloodiplasma ratio: 1: 2 #1006047 {parent drug] #1010383 (metabolites) Plasma protein binding: 3: Same study as- #10060-t'i {parent drug] ratio #1010388 (metabolites-J Pharmacokinetice Phase - I Health} 1Volunteers- ailluleduxe: ii 1 Kl'l??lli multiple dose: 1: 1 1?1 Patients? ailtgle dose: .1: NI'Z'I'l'Jll-ll'i' from it I'll}! multiple (lime: Dose proportionalitt' - [stating - non-fasting sinale tie-ye: :c fag-tine non?fastimz multiple close: file name". 5_Clinical Pharmacology and Biopharmaccmics Filing formiChecklist for Dr Reference ID: 3516218 91 CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS FILING FORMICHECKLIST FOR NDAIBLA 01' Supplement Drug-drug inlernetien studies - [n-i'ii'e effects on primary dine: SETH-1041 iketeeonazele and rifurnpirin on I?ll?) effects of primal? drug: {netupitnnl rm niillnzulnnl :uul (netupitnnt on llnlunmeirnn} {nelnpitnnt nu (nelupilnnt un ilignxin] [netupitant on (FDIC on em] SETH-10409 0n the nmlhernpv in patients] In-riu'o: Sullpupulntiun studieh - eilmiein': gender: NEPA-13-11 pooled nnalyais Ill .3 sludies In evuluule gentler PK ped_inn'ies: ue?nuiew: renal nnpniunent: llepntle inqminnent: l-?D - Piliihe 2: Plume 3: PALD-J .V Ill?29 Phase 1 and or 2. pl'ooi'et'ceneept: challenge study} study] NEIL-0110 Mime 3 eliltienl ninl: Pullulnlinn - Data rich: Dan 9 spame: (pnpulnlinn PK from Phase 5 trial} II. Alimlule IJinm?uiluliilih? Relnlire Ilinm?uiluhilih? - nu Inmm ?31' reference: alternate feu'nnllalien as reference: Formulation development I I8 Bieequivale nee studiee - design: single lilulli dune: for pale and between phase 2 lurmulnlinn and plume 5 flirmulnl'mn] NEIL-11432 for manufacturing hiie change) i?eplienle desinn: Hiilnle mulli clme: Food-drug interaction studies (netnpiln 111?} ltl- I 2 ?in-wniver requexl limed nn class [lissnlutiun study In Hnlunte nlenhnl inlluee? (lune?dumping File name: 5 Clinical and Biepharmaeeuties Filing Penn-"Checklist for NDA BIA m' Supplement Reference ID: 3516218 92 CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS FILING FOR or Supplement Ill. Other Studies studies Pediatric Studv Plan Total Number of Studies On initial review of the application for ?ling: (?ontent Parameter Criteria for Refusal to File (RTF) I Yes No . Comment Data I Has the applicant submitted bioequivalenee data comparing to- be-marketed produet(s) and those used in the pivotal clinical trials? 2 Has the applicant provided metabolism and drug-drug interaction information? 3 Has the sponsor submitted broavailability data satisfying the (JR requirements? 4 Did the sponsor submit data to allow the evaluation of the validity of the analytical assay? 5 Has a rationale for dose selection been submitted? 6 Is the clinical pharmacology and biophannaceutics section of the NDA organi7ed. indexed and paginated in a manner to allow substantive review to begin? 7 Is the clinical pharmacology and section of the NDA legible so that a substantive review can begin? 8 Is the electronic submission searchable, does it have appropriate anddothe hyperlinks ?ark? - Criteria for Assessing?Quality of an NBA (Preliminary Assessment of Quality) to Are the data sets. as requested during pie-submission discussions. submittedin the appropriate format . . If applicable. are the phannacogenomic data sets submitted in the appropriate fonnat? Studies and Analyses ll Is the appropriate pharmacokinetic information submitted? 12 Has the applicant made an appropriate attempt to detemiine reasonable dose individualization strategies for this product appropriately designed and analyzed dose-ranging or pivotal studies)? 13 Are the appropriate exposure-response (for desired and undesired effects) analyses conducted and submitted as described in the Exposure-Response guidance? l-l Is there an adequate attempt by the applicant to use exposure- response relationships in order to assess the need for dose adjustments for intrinsic/extrinsic factors that might atTect the phamtacokinetic or phannaeodynamies'? Fixed dose combination product File name: 5_Clinieal Pharmacology and Biopharmaeeutics Filing Fonn/Checklist for or Supplement 090808 93 Reference ID: 3516218 CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS FILING FOR or Supplement 15 Are the pediatric exclusivity studies adequately designed to PSP to request :1 demonstrate effectiveness. if the drug is indeed effective? 16 Did the applicant submit all the pediatric exclusivity data. as described in the 17 Is there adequate infonnation on the phannacokinetics and exposure-response in the clinical phamtacology section of the label? GeneralAre the clinical pharmacology and biophannaceutics studies of appropriate design and breadth of investigation to meet basic requirements for approvabilitlof this product? 19 Was the translation (ofstudy reports or other study information) from another language needed and provided in this submission? IS THE CLINICAL PHARMACOLOGY SECTION OF THE APPLICATION Yes If the is not fileable from the clinical phannacology perspective. state the reasons and provide comments to be sent to the Applicant. Please identify and list any potential review issues to be forwarded to the Applicant for the 74-day letter. 0 Please provide the assay validation for cardiac troponin levels (cTnI). If such infonnation is already submitted. please guide the reviewer to the location ofthe information. For Study NETU-07-20, please extract data for lTlt)Xil]0X8CIlL placebo at 15 minute, 30 minute post-dose and the corresponding baseline for us to evaluate. Upon the review of the thorough QT study. IRT-QT review team found that the moxilloxacin profile was missing the rising phase. The maximum moxi?oxacin induced ddQ'I?cF effect appeared almost at the first available time point. which was 1 hr after dose. Therefore we request additional analysis to understand what happened before hour 1. Insook Kim. 10/29/13 Reviewing Clinical Phannacologist Date Sue-Chill Lcc. 10/29/13 Team Leader/Supervisor Date 94 Reference ID: 3516218 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------INSOOK KIM 05/30/2014 DILARA JAPPAR 05/30/2014 JINGYU YU 05/30/2014 NITIN MEHROTRA 05/30/2014 SUE CHIH H LEE 05/30/2014 Reference ID: 3516218