CENTER FOR DRUG EVALUATION AND RESEARCH APPLICATION NUMBER: 205677Orig1s000 PHARMACOLOGY REVIEW(S) Tertiary Pharmacology Review By: Paul C. Brown, Ph.D., ODE Associate Director for Pharmacology and Toxicology, OND IO NDA: 205677 Submission date: 5/31/2013 Drug: tasimelteon Applicant: Vanda Pharmaceuticals Inc. Indication: Treatment of Non-24-hour disorder in the totally blind Reviewing Division: Division of Neurology Products Discussion: The pharmacology/toxicology reviewer and supervisor found the nonclinical information submitted for tasimelteon to be adequate to support approval of this NDA for the indication listed above. Tasimelteon is a melatonin receptor agonist. This is an existing Established Pharmacologic Class and is acceptable for labeling. Two-year carcinogenicity studies of tasimelteon were conducted in mice and rats. The executive carcinogenicity assessment committee found the studies to be acceptable. There were no drug-related neoplasms in mice. Liver adenomas and carcinomas were increased in male rats and liver adenomas were increased in female rats. Uterine and cervical squamous cell carcinomas and uterine endometrial adenocarcinomas were also increased in rats. These tumors only occurred at doses that were substantially higher than the human dose on a mg/m2 basis. Reproductive and developmental studies in rat and rabbits identified some effects on the dam, fetuses and offspring. These effects are described in the proposed labeling and it is noted that the effects only occurred at doses that were substantially higher than the human dose on a mg/m2 basis. Conclusions: I concur that this application can be approved from the pharmacology/toxicology perspective. 1 Reference ID: 3441725 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------PAUL C BROWN 01/24/2014 Reference ID: 3441725 MEMORANDUM DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service Food and Drug Administration ________________________________________________________________________ Division of Neurology Products (HFD-120) Center for Drug Evaluation and Research Date: November 12, 2013 From: Lois M. Freed, Ph.D. Supervisory Pharmacologist Subject: NDA 205-677 (tasimelteon; Hetlioz) ________________________________________________________________________ NDA 205-677 was submitted by the sponsor (Vanda Pharmaceuticals Inc.) on May 30, 2013 for tasimelteon as treatment for non-24-hour disorder in the totally bind individual. The application was filed and given a Priority classification (Filing Communication letter, dated 7/29/2013), with a user fee goal date of January 31, 2014. The only nonclinical request was for “…a signed and dated Pathology Report for the pivotal 6month toxicity study in rats (Study TAJ0007-98348)…,” which was submitted to the NDA on August 20, 2013 (SDN 11). The sponsor conducted a full battery of nonclinical studies in support of clinical development (under IND 54776) and marketing approval. These studies were reviewed in detail by Dr. Melissa K. Banks-Muckenfuss and were found to be adequate to support approval of the NDA, with appropriate labeling (Pharmacology/Toxicology NDA Review and Evaluation, NDA 205-677, Melissa K. Banks-Muckenfuss, Ph.D., 10/30/2013). Comments and Recommendations Pharmacology: Tasimelteon is a melatonin MT1 and MT2 receptor agonist, with high in vitro binding affinity for both receptors, similar to that of melatonin. Three major circulating metabolites were identified in human plasma following oral administration: M9, M12, and M13. In humans, plasma levels of M9 and M12 are higher (1.3- and 1.8fold, respectively), whereas those of M13 are similar, to those of the parent compound. All three major human metabolites exhibited in vitro binding affinity for the MT1 and MT2 receptors. The data (Ki in nM) are summarized in the following table. 1 Reference ID: 3405615 COMPOUND MT, tasinrelteorr 0.30-0.35 0.07-0.17 M9 1180 71.9 M12 136 10.8 M13 4 0.92 melatorrin? 0.01-0.33 0.11-0.25 Icalculated based on data from Hardeland er (11. Expert Opin Invesfig Drugs 2010 (4) (4) In monosed labeling the snonsor has stated (0) (4) The data provided by the sponsor establish tasimelteon as a melatonin and MT2 agonist. The sponsor also conducted three nonclirrical studies in Long-Evans hooded rats to assess the ?chronobiotic? properties (or adjustment of the timing of internal circadian of tasimelteon. In Study 52254, groups of rats were kept for 3-6 weeks either in a room in which the lights-on period was room in which the lights- on period was 7 pm to 7 am. Two group of rats housed in the 7 pm-to-7 am lights-on room for 3 weeks were transferred to the reverse light-dark cycle for 2 days and then sacri?ced. In one of these two groups, animals received three subcutaneous (SC) injections of melatonin (1 mg/kg) or tasimelteon (1 or 5 mg/kg) shortly before transfer (shortly before lights on) and shortly after the begirming of the lights-off period on the next two days; these animals were sacri?ced after two days exposure to the reversed light-dark cycle. At sacri?ce in all groups, hippocampal slices were prepared and electrical activity in the suprachiasmatic nuclei (SCN) was recorded. The high dose of tasimelteon delayed the peak SC activity by 1.3 hrs, similar to melatonin; the low dose of tasimelteon had no effect. In Study 52274, the chronobiotic effects of melatonin and tasimelteon were tested in an animal model of acute phase shifting. Rats were maintained rmder a 12-hr light/ 12-hr dark cycle for ~2 weeks, rmtil the lighting conditions and the onset of wheel-rrmning were Animals were then placed in continuous darkness for the remainder of the experiment. After two weeks in total darkness, animals received a single SC injection of vehicle, tasimelteon (0.01, 0.1, 1.0, or 5.0 mg/kg), or melatonin (0.1 or 1.0 mg/kg) two horu's prior to each animal?s anticipated onset of wheel-rurming. Melatonin advanced the onset of wheel-running at both doses, in a dose-related manner. Reference ID: 3405615 Tasimelteon advanced the onset of wheel-running at 1.0 and 5.0 mg/kg, with a slightly greater effect at 1.0 mg/kg; the lower doses were without effect. In Study 52273, rats were maintained under light/dark conditions similar to those used in Study 52274. In this experiment, however, after two weeks in total darkness, animals received SC injections of vehicle, melatonin (1.0 mg/kg), or tasimelteon (0.01, 0.1, 1.0, or 5.0 mg/kg) daily for 66 days. The data were expressed as the percentage of animals entrained. Entrainment was considered established when (1) the onset of wheel-running was “locked to injection time” and (2) “free-running” (endogenous) rhythm was reestablished after the end of the dosing period. Melatonin induced entrainment in 100% of treated animals. For tasimelteon, the % of animals entrained was 11.3, 25, 91, and 100% at 0.01, 0.1, 1.0, and 5.0 mg/kg, respectively. The sponsor considered the results of Studies 52254, 52274, and 52273 as evidence that tasimelteon, like melatonin, exhibits chronobiotic properties. The results obtained with melatonin are consistent with the results of published studies (cf. Arendt J, Skene DJ. Sleep Med 9:25-29, 2005). However, the study reports do not provide sufficient data to be (b) (4) considered definitive evidence of a chronobiotic effect for tasimelteon Toxicology: The pivotal oral toxicity studies of tasimelteon were conducted in SpragueDawley rat and cynomolgus monkey, with duration of dosing up to 6 months (0, 5, 50, 500 mg/kg/day) and 1 year (0, 3, 20, 150 mg/kg/day), respectively. The primary target organs were the CNS (convulsions) in both rat and monkey, and liver (centrilobular hypertrophy, focal necrosis), kidney (chronic progressive nephropathy, interstitial macrophages, hyaline droplets and pigment in cortical tubules, cortical tubular dilatation), and spleen (hemosiderosis, extramedullary hematopoiesis) in rat. A doserelated increase in pica was observed in the 6-month rat study (males and females), a behavior thought to reflect a compound’s emetogenic potential in this species which cannot vomit (Takeda N et al. Pharm Biochem Behav 45:817-821, 1993; Yamamoto K et al. Eur J Pharmacol 554:34-39, 2007); increased emesis was observed in monkey. These studies provided an adequate assessment of tasimelteon in both species, and the major human metabolites in one or both species. (Plasma exposures for M9, M12, and M13 in the 1-year monkey study were estimated based on data from a single-dose PK study.) Reproductive and developmental toxicology: Studies were conducted in rat (fertility and early embryonic development [0, 5, 50, 500 mg/kg], embryofetal development [0, 5, 50, 500 mg/kg], and pre/postnatal development [0, 50, 150, 450 mg/kg]) and rabbit (embryofetal development [0, 5, 30, 200 mg/kg]). These studies identified disruption of the estrus cycle (rat), embryolethality, reduced fetal body weight, and delayed fetal development (rabbit), and persistent reductions in pup body weight, delayed physical development, and impaired performance on learning and memory tasks (rat) as tasimelteon-related effects. These findings are consistent with a Pregnancy Category C, as recommended by the sponsor and Dr. Banks-Muckenfuss. It is of note, however, that these findings were primarily observed at the highest doses tested, with the highest noeffect doses generally providing substantial safety margins for humans based on plasma 3 Reference ID: 3405615 levels of parent compound and metabolites M9, M12, and M13 in rat or body surface area (mg/m2) comparisons for rabbit. (In rabbit, plasma exposure data were not available for parent compound at the highest no-effect dose or for M9, M12, or M13 at any dose.) Carcinogenicity: Lifetime carcinogenicity studies were conducted in CD-1 mouse (0, 30, 100, 300 mg/kg/day) and Sprague-Dawley rat (0, 20, 100, 250 mg/kg/day). The results of these studies were reviewed by the Division and the Executive CAC (Executive CAC Meeting Minutes, dated 10/16/2013). Both studies were considered to be acceptable, and adequately assessed the carcinogenic potential of parent compound (both species) and metabolites M9, M12, and M13 (rat). (Plasma exposure data were not available for M9, M12, or M13 in mouse.) There were no drug-related increases in neoplasms in the mouse study. In rat, the following neoplasms were identified as drug-related: uterus (endometrial adenocarcinoma at the HD), uterus and cervix (squamous cell carcinoma at the HD), and liver (adenoma in MDF and HDF; adenoma and carcinoma combined in MDM and HDM). The positive findings in uterus were statistically significant, whereas those in other organs were identified as drug-related based on the incidences exceeding historical control ranges. Genetic toxicology: The genotoxic potential of tasimelteon was tested in a standard battery of studies: Ames assay, in vitro cytogenetic assay in primary human lymphocytes, and in vivo micronucleus assay in rat (conducted as part of the 1-month oral toxicity study]). The Ames and the in vivo micronucleus assays were found to be adequately conducted and negative, both with and without metabolic activation. In the in vitro cytogenetics assay, tasimelteon was positive (2-fold increase in % of cells with structural aberrations) in the absence of metabolic activation, at the highest concentration (HC; 200 µg/mL) tested (24-hr exposure); at this concentration, there was a decrease in mitotic index (MI) of approximately 50%. Tasimelteon was also positive at the HC (800 µg/mL) tested (5-hr exposure) in the presence of S9; however, this concentration was excessively cytotoxic (74% decrease in MI). With the 5-hr exposure (+S9), the next lower concentrated tested (400 µg/mL) was associated with a MI similar to control. Dr. BanksMuckenfuss noted that concentrations between 400 and 800 µg/mL should have been tested under these conditions (5-hr exposure, +S9) and considered the results of the assay to be “equivocal/positive” for clastogenicity. While, Dr. Banks-Muckenfuss’ conclusions are reasonable regarding the in vitro cytogenetic assay, the increases in % of cells with structural aberrations were ≤2-fold, even in the presence of maximum recommended or clearly excessive cytotoxicity. Overall, the data suggest a lack of genotoxic potential for tasimelteon. In addition to the genotoxicity studies for tasimelteon, the sponsor conducted a separate evaluation of the genotoxic potential of metabolite M11, a minor circulating metabolite in humans. M11 was negative in an Ames assay, but was positive in an in vitro cytogenetic assay in CHO cells. In the in vitro cytogenetic assay, M11 was reproducibly positive in the presence, but not in the absence, of metabolic activation at concentrations not consistently associated with excessive cytotoxicity. (The sponsor stated, however, that the high M11 concentrations associated with positive findings exceeded the maximum concentration recommended in ICH guidance [ICH S2(R1), 2012]). The reason for the 4 Reference ID: 3405615 sponsor?s concern regarding M11 is unclear. While M11 was undetectable in rat plasma, plasma M11 levels estimated to have been achieved in the mouse carcinogenicity study are 1.1-2.2 times that in hmnans at the recommended daily dose of 20 mg. Therefore, the carcinogenic potential of this minor human metabolite has been adequately assessed. Impurities: Twelve process (drug substance) impurities were identi?ed by the sponsor. The speci?cation limits for these impmities are set such the total daily dose of all 12 impurities combined would be Therefore, these impurities are adequately controlled. However, in addition to these impurities, the MC review team identi?ed two potentially genotoxic impm'ities mm which would not be adequately controlled if determined to be genotoxic. The results of an FDA QSAR evaluation resulted in a positive prediction (by all three software programs) for Salmonella mutagenicity for M0 but not for (5x4) An information request was sent to the sponsor (letter dated 9/20/2013) asking for additional information on (W) In submission SDN0020 (10/10/2013), the sponsor proposed a speci?cation limit of NMT GM) for this impurity, and stated that ?the total am01mt of potential genotoxic impurities is NMT If confnmed by the MC review team, these limits would be acceptable, ?'om a nonclinical standpoint. Recommendations: The nonclinical studies of tasimelteon submitted by the sponsor support approval of the NDA for the proposed indication, with appropriate labeling. Since tasimelteon has been granted orphan drug status, the sponsor is exempt from the requirement (under PREA) to assess safety and effectiveness in the pediatric population; therefore, no nonclinical postmarketing requirements are recommended. Additional labeling recommendations will be commlmicated separately. APPEARS THIS WAY ON ORIGINAL Reference ID: 3405615 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------LOIS M FREED 11/12/2013 Reference ID: 3405615 DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION CENTER FOR DRUG EVALUATION AND RESEARCH PHARMACOLOGY/TOXICOLOGY NDA REVIEW AND EVALUATION Application number: Supporting document/s: 205-677 NDA 205-677: SDN1; SDN11; SDN16 Applicant’s letter date: NDA 205-677: 05/31/13; 8/20/13; 9/17/13 CDER stamp date: NDA 205-677: 05/30/13; 8/20/13; 9/17/13 Product: tasimelteon, proposed tradename Hetlioz (VEC-162, BMS-214778) Indication: Applicant: Review Division: Reviewer: Supervisor/Team Leader: Acting Division Director: Project Manager: Treatment of Non-24-Hour Disorder in the Totally Blind Vanda Pharmaceuticals Inc. DNP, HFD-120 Melissa K. Banks-Muckenfuss, Ph.D. Lois M. Freed, Ph.D. Eric P. Bastings, M.D. Cathleen Michaloski, BSN, MPH Disclaimer Except as specifically identified, all data and information discussed below and necessary for approval of NDA 205-677 are owned by Vanda Pharmaceuticals or are data for which Vanda Pharmaceuticals has obtained a written right of reference. Any information or data necessary for approval of NDA 205-677 that Vanda Pharmaceuticals does not own or have a written right to reference constitutes one of the following: (1) published literature, or (2) a prior FDA finding of safety or effectiveness for a listed drug, as reflected in the drug’s approved labeling. Any data or information described or referenced below from reviews or publicly available summaries of a previously approved application are for descriptive purposes only and are not relied upon for approval of NDA 205-677. 1 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. TABLE OF CONTENTS EXECUTIVE SUMMARY ................................................................................................ 4  1.1  1.2  1.3  2  INTRODUCTION .................................................................................................... 4  BRIEF DISCUSSION OF NONCLINICAL FINDINGS ...................................................... 4  RECOMMENDATIONS ............................................................................................ 6  DRUG INFORMATION ............................................................................................ 9  2.1  2.2  2.3  2.4  2.5  2.6  2.7  3  DRUG ................................................................................................................. 9  RELEVANT INDS, NDAS, BLAS AND DMFS ......................................................... 10  DRUG FORMULATION ......................................................................................... 10  COMMENTS ON NOVEL EXCIPIENTS ..................................................................... 10  COMMENTS ON IMPURITIES/DEGRADANTS OF CONCERN ....................................... 10  PROPOSED CLINICAL POPULATION AND DOSING REGIMEN .................................... 12  REGULATORY BACKGROUND .............................................................................. 12  STUDIES SUBMITTED .......................................................................................... 13  3.1  3.2  3.3  4  STUDIES REVIEWED ........................................................................................... 13  STUDIES NOT REVIEWED ................................................................................... 16  PREVIOUS REVIEWS REFERENCED...................................................................... 16  PHARMACOLOGY ................................................................................................ 17  4.1  4.2  4.3  5  PRIMARY PHARMACOLOGY ................................................................................. 17  SECONDARY PHARMACOLOGY ............................................................................ 19  SAFETY PHARMACOLOGY ................................................................................... 19  PHARMACOKINETICS/ADME/TOXICOKINETICS .............................................. 19  5.1  6  PK/ADME ........................................................................................................ 19  GENERAL TOXICOLOGY ..................................................................................... 36  6.1  6.2  7  SINGLE-DOSE TOXICITY ..................................................................................... 36  REPEAT-DOSE TOXICITY .................................................................................... 36  GENETIC TOXICOLOGY ...................................................................................... 61  7.2  7.4   IN VITRO ASSAYS IN MAMMALIAN CELLS .............................................................. 62  OTHER GENETIC TOXICITY STUDIES.................................................................... 64  8  CARCINOGENICITY ............................................................................................. 75  9  REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY .............................. 118  9.1  9.2  9.3  FERTILITY AND EARLY EMBRYONIC DEVELOPMENT ............................................. 118  EMBRYONIC FETAL DEVELOPMENT ................................................................... 126  PRENATAL AND POSTNATAL DEVELOPMENT ....................................................... 142  10  SPECIAL TOXICOLOGY STUDIES ................................................................. 167  11  INTEGRATED SUMMARY AND SAFETY EVALUATION ............................... 173  2 Reference ID: 3399188 NDA 205-677 12  Melissa K. Banks-Muckenfuss, Ph.D. APPENDIX/ATTACHMENTS ........................................................................... 184  APPENDIX 1: LITERATURE REFERENCES CITED ............................................................. 184  APPENDIX 2: HISTOPATHOLOGY INVENTORY ................................................................. 186  3 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Executive Summary 1.1 Introduction Tasimelteon is a melatonin (MT1 and MT2) receptor agonist. The development program has spanned greater than a decade, and the proposed indication has evolved (e.g., insomnia, circadian rhythm disorders, eastward-bound jet lag). The development of the compound was discontinued, with several toxicology studies in progress and terminated early, and was restarted several years later by the current sponsor. The mechanism of action appears similar to marketed drug ramelteon (the EPC will be the same), although the observed toxicities differ slightly. On 1/19/10, the sponsor was granted an orphan drug designation for tasimelteon for the treatment of Non-24-hour Sleep-Wake Disorder in Blind Individuals With No Light Perception. 1.2 Brief Discussion of Nonclinical Findings Tasimelteon showed affinity at melatonin (MT1 and/or MT2) receptors, with slightly greater affinity at MT2 receptors; tasimelteon’s metabolites showed at least a 10-fold lower affinity at melatonin receptors. In an early in vitro receptor binding study, tasimelteon was shown to have affinity similar to melatonin at MT1 receptors and greater than melatonin at MT2 receptors. Tasimelteon did not show affinity at other targets in a receptor binding screen. The primary target organs of tasimelteon toxicity include the: CNS, liver, kidney, and reproductive organs. Some alterations were also observed in spleen and heart, as well as the hematologic and endocrine systems (i.e., thyroid, pituitary, and adrenal glands). Generally, slight reductions in weight and sometimes food consumption were observed across the studies. CNS signs including convulsions (clonic and/or tonic), tremors, loss of righting reflex, flaccidity, ataxia, and/or labored breathing were observed at high doses in studies in rats, mice, and monkeys. Liver was clearly a target organ in most of the toxicological species used; increased liver enzymes (as well as cholesterol and/or triglycerides), increased liver weight and size, histopathological alterations (i.e., centrilobular to panlobular hepatocellular hypertrophy, increased hepatocellular mitoses, bile duct hyperplasia, cystic degeneration, hepatocyte vacuolation, regenerative hyperplasia, centrilobular fatty change, focal pigmented macrophages, pigment in hepatocytes, focal hepatocyte necrosis, and/or periportal/serosal/subserosal fibrosis), and tumors were observed. Kidney toxicity was observed in rats (i.e., increased kidney weight; exacerbated CPN; minimal to slight cortical tubules with hyaline droplets; minimal to moderate cortical tubular pigment; interstitial pigmented macrophages; slight cortical tubular dilation; pelvic dilatation; interstitial inflammation; and/or simple tubular hyperplasia), with minimal evidence in mice and monkeys. Changes in reproductive organs were observed in rats (e.g., decreased prostate weight; testicular interstitial cell hyperplasia; altered estrus cycling; increased ovary weight; ovarian follicular cysts; uterine dilatation; uterine adenocarcinoma; uterine squamous cell carcinoma; uterine cervix epithelial hyperplasia and/or keratinization; and uterine cervix squamous cell carcinoma), mice (e.g., decreased prostate weight) and monkeys (e.g., decreased prostate, seminal vesicle, and testes weights; lack of corpora lutea in the ovary). Slight 4 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. reductions (5-15%) in red blood cell parameters (and slight increases in platelets) were observed in rats and monkeys. In rat spleen, hemosiderosis and extramedullary hematopoiesis were observed; the sponsor believed these findings reflected an increased rate of RBC removal from peripheral blood leading to a "compensatory" response. A few effects were observed among the studies in the thyroid (e.g., increased weight; follicular cysts, atrophy, and/or hypertrophy) and pituitary (e.g., focal hyperplasia). The no adverse effect levels for the general toxicity studies were within the clinical range; however, effect doses were approximately 20 times the recommended human dose (RHD) of 20 mg on a mg/m2 basis. Tasimelteon was negative in an in vitro Ames assay and an in vivo rat micronucleus assay; however, an in vitro chromosomal aberration assay demonstrated clastogenic effects. Tasimelteon metabolite M11 (a non-major human circulating metabolite that has poor coverage in the animal species) was also negative in an in vitro Ames assay but was clastogenic in an in vitro chromosomal aberration assay in CHO cells. Two year carcinogenicity bioassays were conducted in rats and mice. In rats, tumors were observed in the liver (≥ MD; adenomas in F and adenomas and carcinomas in males), uterus (endometrial adenocarcinomas, HDF), and uterus and cervix (squamous cell carcinomas, HDF). The relevance of these tumors to humans is unknown. Tumors were not observed at approximately 10 times the RHD, on a mg/m2 basis. No drugrelated neoplasms were reported in mice. As suggested in the toxicity studies (e.g., effects on reproductive organs, such as a slightly increased incidence of absent corpora lutea in the rat carcinogenicity study and the lack of corpora lutea in the ovaries of female monkeys in the chronic toxicity study), effects on female rat fertility and fetal development in rats and rabbits (into adulthood in rats) were demonstrated in the reproductive toxicology studies. Irregular estrus cycles, slightly increased infertile pairings, and slight reductions in fertility parameters were observed at MD and HD in females; the no-effect dose was approximately 2.5 the RHD on a mg/m2 basis. The embryofetal development (EFD) studies in rats and rabbits demonstrated some embryofetal toxicity. Drug-related slight delays in development and decreased fetal body weights were observed at MD and HD in rats and rabbits; rabbits also demonstrated an increase in abortions and slightly increased post-implantation losses (late resorptions) at MD and HD. Maternal toxicity was also observed (i.e., clinical signs, and slight reductions in body weight and food consumption) in the dams and does at HD. The no-adverse-effect doses in rats and rabbits were approximately 25 and 30 times the RHD on a mg/m2 basis. In the pre/postnatal development study (dosing the dams throughout gestation and lactation), clear toxicity in the F1 generation was demonstrated as developmental delays and a reduced growth rate postpartum and into adulthood. The gestation period was very slightly increased in all VEC-162-treated dams, and the live birth index was slightly reduced at HD. Although average pup birth weights were similar (<5% difference; provided as the average of the litter averages for each sex), HD pups demonstrated reduced (~15%) body weights throughout lactation and into adulthood. Sexual maturation was achieved at a similar age (PND) but at a lower body weight in males, but was delayed in females. Other developmental milestones (e.g., righting responses) were similarly delayed. Neurobehavioral testing 5 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. (i.e., Morris Water maze) demonstrated clear deficits in males (i.e., trial times, sector entries, and failed trials) but not females, when tested at PND 31/32; when re-tested at PND 47/49, no consistent differences were reported. A few effects on F1 generation reproduction were observed. Reductions in body weight continued in the adult female F1 generation during gestation, and slight effects on fertility were observed at HD. The F1 generation was exposed to VEC-162, M9, M12 and M14 (as measured on LD4), although to a much lesser extent than the dams. Limited F0 maternal toxicity was demonstrated (i.e., slightly [~3-5%] reduced body weights during gestation at MD and/or HD, and during lactation in all VEC-162-treated dams). The no-effect dose was approximately 2.5 times the RHD on a mg/m2 basis. 1.3 1.3.1 Recommendations Approvability From a Pharmacology/Toxicology perspective, the application is recommended for approval. While most of the observed toxicities occurred at exposures exceeding those anticipated at the RHD or would be monitorable in humans (e.g., liver enzyme increases), the carcinogenic and reproductive effects are considerable. Taking into account the orphan population and toxicities identified for similar drugs (e.g., tumors and malformations observed with ramelteon), the identified toxicities do not preclude approval but should be fully disclosed in the labeling. Of particular importance are: the tumors in the liver, uterus, and uterine cervix, the altered cyclicity observed in female rats and suggested in female monkeys (and potential effects on fertility in humans), and the persistent effects on growth of offspring exposed during gestation and lactation (i.e., exposed in utero in humans). 1.3.2 Additional Non Clinical Recommendations Please see suggested labeling. 1.3.3 Suggested Labeling Final labeling requires discussion among the review team members, as well as with the sponsor. This suggested labeling is provided in case the application moves to approval, and represents the reviewer's current thoughts, but does not reflect final label wording. Highlights: __________________ _________________ INDICATIONS AND USAGE Hetlioz is a melatonin receptor agonist indicated for the treatment of Non-24-Hour Disorder (Non-24) in the totally blind. (1) _______________  _______________ USE IN SPECIFIC POPULATIONS Pregnancy: Based on animal data, may cause fetal harm. (8.1) 1. Indications and Usage 6 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Hetlioz is a melatonin receptor agonist indicated for the treatment of Non-24-Hour Disorder (Non-24) in the totally blind. 8.1 Pregnancy Pregnancy Category There are no ade uate and well-controlled studies of Hetlioz in re nant women. In animals, Hetlioz should be used during pregnancy only if the potential bene?t justi?es the potential risks. In pregnant rats administered tasimelteon at oral doses of 5, 50, or 500 /k Ida durin the eriod or ano enesis and food consumption. The was approximately 30 times the RHD on a mg/m2 basis. Oral administration of tasimelteon 50, 150, or 450 Ida to rats throughout and lactation resulted in The no-effect dose for was approximately times the RHD on a mg/m2 basis. 8.3 Nursing mothers It is not known whether Hetlioz is secreted into human milk, ?Because many drugs are excre In 0 uman ml Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, 12.1 Mechanism of action The mechanism by which tasimelteon exerts its therapeutic effect in Non-24 Hour Disorder is unknown. Tasimelteon's at MT1 and MT2 receptors are OUQ 06 V60 "1 13.1 Carcinogenesis, mutagenesis, Impairment of fertility Carcinogenesis Mutagenesis Impairment of Fertility Reference ID: 3399188 NDA 205-677 2 Drug Information 2.1 Drug CAS Registry Number CAS Name Generic Name Code Name Chemical Name Melissa K. Banks-Muckenfuss, 609799-22?6 Propanamide, tasimelteon (INN, USAN) VEC-162 iVandai, BMS-214778 (BMS), (1 ro-4- ro-1 -benzofuran-4- (1 R, propionamide derivative of Molecular Formula/Molecular Weight 245.32 Structure or Biochemical Description Q30 0 Pharmacologic Class Reference ID: 33991 88 melatonin receptor agonist NDA 205-677 Melissa K. Banks-Muckenfuss, 2.2 Relevant INDs, NDAs, BLAs and DMFs IND 54,776, the associated IND for the treatment of circadian sleep disorders IND 112, 702, MDD (DPP) 2.3 Drug Formulation See sponsor's Table 3.2.P.1-2, below. Table 3.2.P.1-2: Composition of Tasimelteon 20-mg Capsules Component Reference to ?eight (mg) standard capsule composition per capsule - ., 1 (m Tasunelteon drug substance Vanda (bectiou 20.00 3.18.4.1) Lactose NF . . 09(4) cellulose . . . . 09(4) Collordal silicon diox1de roscarmellose sodium (19(4) Magnesium stearate (5X4) . . . \vA-v) Size 1. dark blue opaque. hard gelatin capsules printed with 20 mg" in white' Total capsule weight 376.00 1 Weight is adjusted for ?a assay of drug substance. 2 Capsules used for stability and clinical batches were printed with mm but validation and commercial batches will have 20 mgg?ginted in white. 2.4 Comments on Novel Excipients In section 3.2.P.4, the sponsor stated that there are no novel excipients; all excipients used in the manufacture of tasimelteon 20-mg capsules are commonly used USP and/or NF compendial excipients. 2.5 Comments on Impurities/Degradants of Concern The sponsor identi?ed a number of potential organic impurities. The sponsor provided Table 3.2.8.3.2-1 to describe 12 impurities (see below); of these, ?m were identi?ed as potentially genotoxic compounds by the sponsor. The sponsor stated that these compounds are controlled at a total combined speci?cation of levels, which does not exceed at the clinical dose of 20 mg (see sponsor's Table The CMC 1 0 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. reviewer (see review by Dr. Kambhampati) indicated that all potential genotoxins but (b) (4) two were adequately controlled. Those two compounds were referred to FDA’s (b) (4) Computational Toxicology group, which identified as a potential genotoxin. The CMC reviewer has requested additional information from the sponsor. (b) (4) 11 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. (b) (4) 2.6 Proposed Clinical Population and Dosing Regimen Hetlioz (20 mg tasimelteon capsules) is proposed for the treatment of Non-24-Hour Disorder in the totally blind, a circadian rhythm disorder that occurs when individuals are “unable to entrain (synchronize) their endogenous master body clock to the 24-hour day-night cycle.” Tasimelteon was granted an orphan drug designation, for the treatment of “Non-24-Hour Sleep-Wake Disorder in Blind Individuals with No Light Perception.” 2.7 Regulatory Background The sponsor has previously proposed tasimelteon for the following indications: the treatment of Insomnia, Circadian Rhythm Sleep Disorders, and Jet Lag Disorder due to eastward travel. While being developed by BMS, tasimelteon (then known as BMA214778) development was discontinued; the ongoing nonclinical studies were terminated early (e.g., the 6-month rat toxicity study, the 12-month monkey toxicity study, the fertility and early embryonic development study). Vanda Pharmaceuticals purchased the development program and again began to develop tasimelteon. The sponsor hired CROs to complete and finalize the nonclinical studies that had been terminated early, which is unusual; however, the studies have the GLP and QA statements (note that the report of the 6-month rat toxicity study did not contain a separate, signed pathology report; however, it was provided upon request). 12 Reference ID: 3399188 NDA 205-677 3 Studies Submitted Melissa K. Banks-Muckenfuss, 3.1 Studies Reviewed Ova-view Text Article: bimbo Spades-nil? We! Dustin-elm MW GI) Study Mile- Ceqlm N-hu Repeat-dose toxitity WSW 0n! savage 26 weeks 0. 50. $00 Yes M. Mt. Vernon. TAJ0007 W500 (98348) Cynomolgus Nasogastric 1 year 0. 3. 20. 150? Yes Monkey in?ation (or on! savage when mes-my) My Pliny hum NA NA 0 to 200 udnl (69) YO: .10 to!? we 89); 4 dose levels 353! Salmonella NA NA 0 to 5.000 pig/plat (5 Yes OWM. to 6 dose levels per TA98, TAIOO, assay) TA153S. TA1537. and Escherichia colt WPZ um ChineseHamsux NA NA 0 m5,000 (Sto Yes Ovary Cells 8 dose levels pet may) . . . (him 0, Sam, 300 Yes GI) BR WCDO (hi 104 weeks 0, E, 100, 250 Yes (sums BR Reproductive and (level-punts] my Far?lio? and mrly anbryonic devalopman (50) Oral savage Females: ewes; o. s. 20?. 599? Yes BMS. New TAJ0008 IGS BR hem puny-ml 7 NJ (99009) days after mung units: 4 weeks be?le ?rst pa'n'ng and mu] m?on Embryo-feral development (SD) 0.21 savage 10 days (Days a o, 5, 50.5.91? Yes BMS, New 99005 IGS BR through 15 of Btunswick. NJ gestation) may On! gavage 13 days (Days 7 0, 130.295? Yes BMS. New 99001 through 19 of Bumswick. NJ gestation) 1 3 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, 031m Fszy6ahr 0,50,150,450 No 1001' Indian (SD) Gal ah: 0,3,150,450 Y6 matingtoDayZOof hc?ion Incal'l'olennce BovmeCm NA NA 0.20% Yes GuinenPigl Intudmml (1d) Won: 0, 2.5% Yes Dunln'n/Hmley hjectionand mm (0.1 ml id), 0, 50% (0.4mm; 1' Ipplicliou challenge:15%, 30% 1mm: (02mlhaim02? (02mlmpiul); 069:5an NA NA NA NA No the elevenmice NOA?wasnotooncmsivcly established ?1 M11 development. haltoxicityatanydosc 1? hem, toxicityatanydose. Bristol-M Co y; will Numerous pharmacology and pharmacokinetics studies were brie?y or fully reviewed, including those listed below (not exhaustive). 14 Reference ID: 33991 88 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Orem' Test Antc' le: tasunel' teon andits inatn' metabolnes? Type of Study Test System Method Testing Fadity Study of Number Allin. Primary Pharaacedynanics A?nity and ef?cacy oftasimelteon at the human NIH-3T3 Cells In Vm EMS Pharmaceutical Research 52253 melatonin receptors Institute Wallingford. CT Assessment of the interaction" of tasnnelteon? with a Radio ligand In Vmo (4). 52 l86 comprehensive pand ofteceptors. handin' sites and bindin? and enzymes enzyme assays Extended receptor bniding pro?le of tastmelteon Radioh' ?gand In Vim . 00(4) 1095880 Binding Assays (5) (4). Amity of tasitnelteo' and metabolites M9. M11. M13, Ram pm In mm . AA82606 M12 Ml-l at the human melatonin and Assays 00(4) recqatots Further chamctenza' tion ofdae af?nity and e??cacy of Radio lipnd In Vm'o 0X4) M85237 tasimelteon and metabolttes' MD. and MD at the Binding? Assays 00(4), Innianmelatonin' MT. Prinher characterization of the af?niry and ef?cacy of Radioligand In Vzrro (4). tasimelteon and metabolites 3112 and M14 at the human Binding Assays (5N4) melatonin MT. and Ml'geceptors A?nityande?cacy oftnetabolite M3 atthe Radioligand In Pm 00(4), A813313 melatonin MT. and mireceptors Binding Assays (5X4) Extended receptor binding pto?le of metabolites M9, Radio ligand In 7m (5) (4), M9807 5 M11. and M13 Binding' Assays Extended receptor binding pro?le of metabolites 3112 Radioligznd In Firm (5) (4). W243 and MN Binding Assays (4) Extaided receptor pro?le of metabolite 313 Radio ligand In 7m 0) (4) Bmding' Assays (4) Tasimelteon activity in an a who model of chronobiotic Rat s.c. EMS Phamaacentical Research 52254 modulation Institute, Wallingfortl. CT Tasimelteon ability to shi? the onset ofrunmng' -wheel Rat s.c. BMS Pharmaceutical Research 52 274 activity Institute. Walling?otd. CT Tasimelteon ability to entrain wheel activity Rat s.c. BMS Phannaceutical Research 52 273 Institute. Wallingford, CT Safety Pharmacology (by Ef?ects of tasitnehaeon on cloned potassium Human Embryonic In Win 070227 WBO KidneyCells Ef?ects of tasitnelteon on action potentials in isolated Rabbit cardiac In PM 70228 W30 rabbit cardiac Pnrkinje ?bets th?ng?e ?beis Mass balance ?ollomng? smgle? otal or iv. adminis' nation and nssne' Rat Oral and i (5) (4) 178214778003 distribution mum smgle' oral nation of ["Cl-tasnnelteon? (mo tats' In an? serum of ["Cl-tasnnel? teon in 2mm monkey. In via-u NA EMS-FRI 910073823 rat and mouse 1 5 NDA 205-677 Melissa K. Banks-Muckenfuss, (4) Metabolis- Single?dose study With of ta51melteon in rats Rat 1.v. 3267 7 71 Single-dose study with oral of in rats' Rat Oral Metabolite radio-profiling and identi?cation after single oral dose of In vr?rro NA 16 ?C]-tasimelteon to rats? Single-dose study with oral admrnistran'on of in Monkey Oral monkeys' Metabolite radio-pro?ling and identi?cation after single oral dose of In vino NA XBL07815 no monkeysm Repeated-oral dose plums: okmeu'c study with tasimelteon in rats? Rat Oral TM 0014 Repeated-dose pharmacokineuc study With tasunelneon in pregnant Rabbit Oral 0016 rabbits Repeaud-oral dose pharmacoldnetic and)? with tasinnlteon tn unce' Mouse Gal TAJ 0018 of tadnnheon mtabohte In mouse plasma Mouse Gal 3:31.09628 Excretion Mass balance following Single oral or i v. administration and tissue Rat Oral and i v. 178 214178 003 distribution following single oral administration of in rats' Platinum-ed; Drug Interactions Evaluation of tasmlteon as an inducer of liver microsomal Rat. (x Oral XTOGZOOS cytochrome P450 and UDP glncuronosyhransferase expression "w 1 This study was conducted tn compliance with Good Laboratory Practice regulations Biomalysis portion of Study Biomalysis portion of Study 2 (4) BMS-PRI- Bristol-Myers Squibb Pharmaceutical Research Institute; High-performance liquid chomtogrqahy?mass spectrometry mass spectronaetry. UPLC-MS MS- ultra-performance liquid cinematography/Hans spectrometry mass spectromem: armyi?ble. NR - Not reported. - unravenous. UDP - Undme (4) 3.2 Studies Not Reviewed Studies previously reviewed are not reviewed in full here; a few studies previously reviewed are brie?y described. 3.3 Previous Reviews Referenced Dr. A. Atrakchi review, dated 3/7/00 Meeting Minutes, dated 8/23/99 Dr. A. Atrakchi review, dated 2/19/98 Dr. A. Atrakchi review, dated 1/16/98 Meeting Minutes, dated 10/16/13 1 6 Reference ID: 3399188 NDA 205-677 4 Pharmacology 4.1 Primary Pharmacology Melissa K. Banks-Muckenfuss, Ph.D. Tasimelteon (also known as BMS-214778 and VEC-162) and its metabolites were evaluated in a number of receptor binding screens. Tasimelteon was shown to be a full agonist at melatonin receptors MT1 and MT2 (IC50s of 0.35 nM and 0.17 nM, respectively, and pKis of 9.5 and 9.8, respectively). These values were similar to melatonin at MT1 and greater than melatonin at MT2. Tasimelteon also demonstrated potent, concentration-dependent inhibition of forskolin-stimulated cAMP accumulation (EC50 at MT1= 0.74 nM; EC50 at MT2= 1 nM). Later studies with tasimelteon and metabolite M13 showed nanomolar affinity at human melatonin receptor MT1; tasimelteon and metabolites M11, M12, M13, and M14 showed nanomolar affinity at human melatonin receptor MT2, although the affinity of tasimelteon was considerably greater than those of the metabolites (see excerpt from the sponsor’s submission, below). Metabolite M3 showed low affinity at human melatonin receptors MT1 and MT2 (see excerpt from the sponsor’s submission below). 17 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In the clinical pharmacology summary, the sponsor provided the following summary table of binding affinities at melatonin receptors for tasimelteon, and metabolites M3, M9, M11, M12, M13, and M14. There appeared to be an error in the Ki for M12 at MT2 (corrected below by the reviewer). The role of tasimelteon (BMS-214778) as a “chronobiotoic” was investigated in one ex vivo and two in vivo studies. In the ex vivo study, suprachiasmatic nucleus (SCN) electrical activity was recorded from brain slices taken from rats administered melatonin (1 mg/kg) or tasimelteon (1 and 5 mg/kg) the day prior to and for 2 days following a reversed light-dark cycle (i.e., light onset delayed by 12 hr); SCN electrical activity rhythms shifted significantly faster in the slices taken from melatonin- tasimelteontreated animals than in slices from the vehicle-treated animals. In an in vivo study, tasimelteon (1.0 and 5.0 mg/kg SC) and melatonin (0.1 and 1.0 mg/kg SC) advanced the onset of running-wheel activity in rats housed in constant darkness in a model of acute phase shifting of locomotor activity in rats. In a second in vivo study, tasimelteon (1.0 and 5.0 mg/kg SC; 66 daily injections) and melatonin (1.0 mg/kg SC) were reported to “synchronize” the “free-running” locomotor rhythms so that the onset of activity coincided with the injection time in a model of entrainment of “free-running” activity rhythms in rats. All rats treated with 5.0 mg/kg tasimelteon and 90% of those treated with 1.0 mg/kg entrained to the injection time; the ED50 for the tasimelteon entrainment effect was 0.21 mg/kg. 18 Reference ID: 3399188 NDA 205-677 4.2 Melissa K. Banks-Muckenfuss, Ph.D. Secondary Pharmacology Tasimelteon and its metabolites were evaluated in a number of receptor binding screens. At concentrations up to 10 µM, no other binding was observed. MT3 receptor binding was not assessed. 4.3 Safety Pharmacology The sponsor did not conduct the standard battery of safety pharmacology studies. CNS and respiratory safety pharmacology studies were not conducted, and the cardiovascular safety pharmacology study in dogs was conducted pre-2000 and not under GLP. A few other cardiovascular safety pharmacology studies were conducted, including GLP hERG and action potential duration assays. The hemodynamic effects of tasimelteon were investigated in anesthetized and conscious dogs after IV administration (non-GLP study). In anesthetized dogs, tasimelteon induced a 14% reduction in mean arterial blood pressure (MABP) at 3.0 mg/kg, with a steady reduction (maximum 17%) in heart rate (HR) over the dose range tested (0.1 – 3.0 mg/kg). In conscious dogs, no changes in MABP, HR, or ECG (including QT) were reported to result from tasimelteon administration. Tasimelteon (maximum concentration tested 100 µM) inhibited hERG current by 14.0 ± 2.2% at 100 µM; an IC50 was not calculated. In an in vitro isolated rabbit cardiac Purkinje fiber action potential duration assay, tasimelteon produced statistically significant shortening of the APD90 after exposure to 100 µM tasimelteon at 1 and 0.5 second basic cycle lengths; no statistically significant changes in resting membrane potential, action potential amplitude, or the maximum rate of depolarization were reported. The maximum rate of depolarization was reduced slightly, but this difference did not reach statistical significance. In vitro non-GLP studies in rat anterior cerebral artery (ACA) and isolated rat caudal artery (CA) showed that, with lower potency than melatonin, tasimelteon was vasoconstrictive in pressurized (60 mm Hg, constant) ACA and CA in a concentrationdependent manner (EC50 estimated to be 500 nM and 180 nM, respectively). 5 Pharmacokinetics/ADME/Toxicokinetics 5.1 PK/ADME Many PK/ADME studies were conducted by the sponsor during the development of BMS-214778/VEC-162/tasimelteon. Where newer studies were available to provide data, the older studies were not reviewed since the understanding of tasimelteon’s ADME has evolved extensively over the last 5 years. Analytical A GLP validation of an LC-MS/MS method for the measurement of tasimelteon in the plasma of rats (0.2- 100 ng/mL in a volume of 200 µL; study TAJ0005) and mice (21000 ng/mL in a volume of 100 µL; TAJ0004) was conducted. Only an older (1999) 19 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. validation of an LC-MS/MS method for the measurement of BMS-214778 in the plasma of monkeys (2- 1000 ng/mL in a volume of 15 µL; 910074952) was provided. GLP validation of an LC-MS/MS method for the measurement of tasimelteon, M9, M12, M13 (all 5- 5000 ng/mL in a volume of 25 µL), and M14 (all 5.438- 5438 ng/mL in a volume of 25 µL; 208-1104) in rat plasma was conducted. GLP validation of an LC-MS/MS method for the measurement of metabolite M3 (5- 5000 ng/mL in a volume of 25 µL; 208-1213) in rat plasma was conducted. A partial (from human plasma) GLP validation of an LC-MS/MS method for the measurement of tasimelteon and M11 in rabbit plasma (0.3- 300 ng/mL in a volume of 100 µL; study 208-1103) was conducted. Two partial (from human plasma) GLP validations of an LC-MS/MS and a UPLC method for the measurement of tasimelteon (5- 5000 ng/mL in a volume of 25 µL) and metabolite M11 in mouse plasma (1- 1000 ng/mL in a volume of 25 µL; 208-1204 and 208-1214) was conducted. ADME Generally, orally administered tasimelteon is rapidly absorbed, widely distributed, and has a relatively short half-life. Following oral administration of [14C]-tasimelteon (BMS214778) to rats, plasma levels of radioactivity declines through 120 hr and were not detected by 168 hr post-dose. The tissues with the highest mean concentrations (in order) were stomach, small intestine, liver, kidney, plasma, large intestine, blood, and adrenals; the only tissues still showing radioactivity after 168 hr were eyes, liver, thyroid, lungs, and kidneys. Based on the long half-life of radioactivity in the eyes (126 hr) and longer half-life in pigmented versus non-pigmented skin (15.4 vs. 7.6 hr) following dosing of [14C]-tasimelteon (BMS-214778), binding of tasimelteon to melanin was suggested. Terminal half-life of the radioactivity was also extended in lung (66 hr), kidneys (49 hr), liver (47 hr), adrenal (40 hr),and stomach (22 hr). The half-life of elimination of the radioactivity from brain was approximately one hour. Moderate, concentration-dependent serum protein binding was observed in human, monkey, rat, and mouse. See the sponsor’s summary table, below. 20 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Tasimelteon was shown to induce liver enzymes in rats (STUDY ?"0062005- Amended report; liver tissue taken from rats in the TK arm of the carcinogenicity bioassay); after administration of 100 and 250 mg/kg tasimelteon for 26 weeks, dose-dependent increases in cytochrome b5, cytochrome P450, and NADPH cytochrome reductase activities were observed in male and female rats. Although only NADPH-cytochrome reductase activity was increased more than 2-fold, a 30-40% induction was observed for the cytochrome b5 and cytochrome P450 enzymes. Tasimelteon was identi?ed as a moderate phenobarbital-type inducer of (17-36% as effective as phenobarbital in 7-pentoxyresorufin O-dealkylation and 70-100% as effective as phenobarbital in testosterone 16b-hydroxylation), a moderate isoniazid-type inducer (70-80% as effective as isoniazid at inducing CYP2E1), and a mild dexamethasone-type inducer (20-70% as effective as dexamethasone at inducing In addition to Phase I enzymes, tasimelteon was shown to induce Phase II UGT enzymes and UGT2B2). See excerpt from the sponsor?s report regarding the demonstrated effects on CYP P450 enzymes, following. Dosing of rats with tasimelteon caused dose-dependent increases in 7-pentoxyresoru?n 0-dealkylation in both male and female rats (up to .2400% and 1800%. respectively). The increases were statistically signi?cant at the 100 ingn'kgx'day and 250 mgr?kgl?day dosage groups. Similarly. tasimelteon caused statistically signi?cant dose-dependent increases in the activity of following enzymes in both male and female rats; testosterone l6B-hydroxylation up to 2500% and 1100%, respectively). 4-nitrophenol hydroxylation (CYP2E1. up to 160% and 87%. respectively). testosterone 68-hydroxylation up to 260% and 640%. respectively). launc acid (CYP4A1-3. up to 49% at 30%. respectively), 4-methylumbelliferone glucuronidation (up to 130% and 130%. respectively). thyroxine glucuronidation (up to 110% and 84%. respectively) and triiodothyronine glucuronidation (up to 100% and 45%. respectively) when compared to the vehicle control. The sponsor also conducted several in vitro evaluations of tasimelteon and its metabolites as an inducer and inhibitor of human cytochrome P450 enzymes. These studies are summarized below. The sponsor indicated that hepatic enzyme induction of CYPs 1A2 and 286 is not expected in humans at the RHD. Tasimelteon up to concentrations of 326 uM) and its metabolites (M9, M11, M12, M13, or up to 5 uM each) did not impact P-gp function in Caco-2 cells, indicating that the compounds are not P-gp inhibitors at the tested concentrations. Protocol ?"0063014: In vitro Evaluation of VEC-162 as an lnducer of Cytochrome P450 Expression in Cultured Human Hepatoc?es Conducted by dated 3/28/07 [non-GLP] Drug: VEC-162, lot number 3049118, batch number 800153250, 99.3% purity; 0.1% dimethyl sulfoxide (vehicle) Three preparations of cultured hepatocytes were treated once daily for three consecutive days with 0, 1, 10 or 100 11M VEC-162. VEC-162 had little or no-effect on CYP1A2 activity. VEC-162 caused concentration-dependent increases in CYP2C8 activity (up to 4.43-fold, and activity (up to 2.27- fold, which were accompanied by increases in CYP2C8 and CYP3A4 protein levels. VEC-162 was up to 60.3% and 83.2% as effective as rifampin at inducing CYP2C8 and activity, 21 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, respectively. VEC-162 caused concentration-dependent reductions in CYP2C9 activity (as much as 70.8%, and CYP2C19 activity (as much as 64.9%, compared to vehicle. Decreased CYP2C9 activity was not associated with a corresponding decrease in the level of CYP2C9 protein. (The lack of a specific antibody precluded an assessment of CYP2C19 protein levels.) Protocol vgc-1sg: In Vitro Evaluation of vgc-16g as an Inhibitor of Human C?ochrome P450 Enzymes Conducted by dated 8/23/06 [non-GLP] Drug: VEC-162, lot 800153250, 99.3% purity (HPLC) VEC-162 caused direct inhibition of CYP2C19 (IC50 80 nM) and, to a lesser extent, CYP1A2, CYP2C8, CYP2C9, CYP2D6, and (using testosterone 68- hydroxylation and midazolam There was little to no evidence of time- dependent inhibition by VEC-162 of any of the CYP enzymes evaluated. See the sponsor's summary table, below. Vanda Pharmaceuticals Con?dential Page 17 33335016 vac?162 Table 3: Summary ofResults: In Vitro Evaluation as an Inhibitor ofHuman CYP Enxymes Direct inhibition Metabolism-dependent inhibiticn Zero-minute pie?amt: 30-minute pie-incubation Evidence of Maximum intn'bitiou Maximum inhibition Enzyme CYP Reaction to? govt) at 100 nM (pM) at 100 (V0) inhibition" CYP1A2 Phenacetin 0-deethylation 100 nM 30 l00 uM 29 little or no Amodiaquine N-dealkylation 28 100 11M 13 little or no Diclofcnac 4'-hydroxylation 100 11M 12 100 26 little or no 4'-hydroxylation 30 24 55 68 25 61 little or no CYP2D6 Dextromethorphan O-demethylation 100 l7 100 um 1.2 little or no Testosterone 100 pM 34 100 11M 35 little or no Testosterone repeat? 100 32 100 38 little or no Midazolam l'-hydroxylation 100 nM 28 100 uM 34 little or no Notes Values were calculated using the average data obtained from duplicates for each incubation condition. The values were calculated using For those CYP enzymes where an actual value was obtained the value is followed by the standard deviation. Maximum inhibition is calculated using the following formula and data for the highest concentration of test article for which usable data were collected (results ate rounded to two signi?cant ?gures): Maximum inhibition (Vt) 100% Percent solvent control (see Appendix 4). Time-dependent inhibition was determined by comparison of lC? values with and without pie-incubation and by visual inspection of the plot. Experiment was repeated because the metabolism-dependent positive control did not show inhibition after a pie-incubation. NA Not applicable 6 Study In Vitro Evaluation of Tasimelteon and Metabolites M9, M12I and M13 as Inducers of Cytochrome P450 Expression in Cultured Human Hepatocytes Conducted by dated 2/20/13 [non-GLP] The effects of tasimelteon and/or its most abundant metabolites (M9, M12, and M13) on the expression of cytochrome P450 (CYP) 1A2 and 286 enzymes were evaluated. Tasimelteon?s potential for inducing CYP1A2 was previously evaluated (Study ?(0063014), and was not included in this study. At concentrations similar to the sponsor-reported "average human maximum plasma concentrations" of 1 to 2.5 uM (Cm.x at 20 mg), neither tasimelteon nor its metabolites appeared to induce CYP1A2 or however, at higher concentrations (10x to 250x higher) tasimelteon and metabolites M12 and M13 induced CYP2B6 activity (6x, 10x, and 5x for 100 nM tasimelteon, M12, and 13, respectively) and levels (21x, 16x, and 6x for 100 nM tasimelteon, M12, and 13, respectively). There was a slight suggestion of M12 activity 22 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. (3-5x changes at 100 M) on CYP1A2 activity and mRNA. See the sponsor's summary Figure 9, below. (b) (4) Study 12A024: In Vitro Evaluation of M9, M12, and M13 as Potential Inhibitors of Cytochrome P450 (CYP) Enzymes in Human Liver Microsomes (b) (4) Conducted by dated 2/19/13 [non-GLP] The ability of three tasimelteon metabolites M9, M12, and M13 to inhibit, in vitro, the major CYP enzymes in human liver microsomes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) was evaluated. M12 showed some effects on CYPs 2C9 and 2C19 (see summary table from the sponsor's report, below). 23 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. (b) (4) STUDY NUMBER: 10VNDAP1R1: P-gp Interaction Assessment of the Customer's Test Compound (VEC-162) and Metabolites (b) (4) Conducted by dated 9/6/11 [non-GLP] The potential of VEC-162 and its metabolites (M9, M11, M12, M13, and M14) to be Pgp substrates and inhibitors was assessed in Caco-2 cell monolayers. The efflux ratios of all test compounds were less than 2 at the tested concentrations, indicating that the compounds are not P-gp substrates. The presence of VEC-162 (up to concentrations of 326 μM) and M9, M11, M12, M13, or M14 (up to 5 μM each) did not impact P-gp function in Caco-2 cells, indicating that the compounds are not P-gp inhibitors at the tested concentrations. The maximum concentrations tested were based on the tolerability of the Caco-2 monolayers. In particular, the understanding of the metabolism of tasimelteon has been evolving over the last several years. Knowledge about the number, identity, and relative abundance of in vivo VEC-162 metabolites in humans and the relevant toxicologic species has changed considerably since the beginning of the drug’s development, and much of the data have been obtained relatively recently. Notably, complete steady state metabolism data are available for rat (tasimelteon and M11 only for mouse), but only single dose metabolism data were provided for monkey. Tasimelteon and M11 exposures were determined at only 200 mg/kg PO in rabbit. Although several metabolites will be discussed, this review will focus on interspecies comparisons of the only the major human circulating metabolites (M9, M12, and M13, identified by FDA’s Clinical Biopharmaceutics staff). 24 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In study PHZ00011 (and addendum study 07816), a mass balance study of a single 14 25 mg/kg [ C]-VEC-162 dose administered PO by oral gavage to male SpragueDawley rats demonstrated that the highest mean radioactivity in plasma was observed at 1 hr post-dose, and mean concentrations of radioactivity decreased to below the quantifiable limit within 72 hr. Elimination of radioactivity in urine and feces accounted for 75.31% and 21.64% of the dose, respectively, through 72 hours after dosing. Approximately 91% of the dose was eliminated by 24 hours after dosing. Cage residue samples contained a combined total of 2.62% of the dose, and carcasses contained a mean total of 0.63% of the dose. Tasimelteon and human metabolites M3, M9, M12, M13, and M14 were confirmed in rat plasma. It is noted that M11 was not observed to be present in rat plasma. See the sponsor’s summary Table S-2 below. (b) (4) In an IV study (Study 8267772), tasimelteon was administered to male Sprague-Dawley rats in a single dose of 0.25 to 5 mg/kg. For tasimelteon, the observed clearance was high (34-75 mL/min/kg) and the volume of distribution greatly exceeded total blood volume (1240-2070 mL/kg; approximately 6-10x total blood volume, cf. Derelanko, 2000). The observed elimination half-lives were short (means ranging from 0.25- 0.69 hr). Exposure was generally linear (based on AUC), but a potential for enterohepatic recirculation at the 5 mg/kg dose was noted (based on the apparent second increase in plasma concentrations at 12 hr). Metabolites M9, M12, M13, and M14 demonstrated short Tmax (0.08- 0.5 hr). The observed half-lives for all metabolites were short. A doseresponsive, but not dose–proportional, increase in Cmax and AUC was reported for all metabolites (see the sponsor’s summary figures, below). 25 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Figure 1: Mean Concentrations of tasimelteon and Metabolites in Plasma Following Intrarenous Administration of 0.25 rug-"kg tasimelteon to Male Sprague Dawley Rats Tasimelteon Time {hr} Figure 2: Mean Concentrations of tasimelteon and lletabolites in Plasma Following Intravenous Administration of 0.50 rug-"kg tasimelteon to Male Sprague Hartley Rats 1000' ?109. +Tasimelteon ~Time [hr] Figure 3: llean Concentrations oftasilnelteon and lletabolites in Plasma Following Intravenous Administration of 1.0 lug-"kg tasimelteon to Male Sprague Dau?ley Rats 1000' Tasimelteon M9 1110Time {hr} 26 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Steady state plasma exposure to tasimelteon (and metabolites M9, M12, M13, and M14) was investigated in Sprague-Dawley rats (3/sex/gp) following 29 days of daily oral gavage administration of 25, 10, 250, or 500 mg/kg VEC-162 (GLP, with the exception of M3 data, Study TAJ0014). PK data for M3 were unvalidated. Tasimelteon was tolerated at doses up to 250 mg/kg; underactive behavior, reduced body temperature (as judged by coolness to touch), partially closed eyelid(s), unsteady gait, irregular breathing (F), piloerection (F), abnormal gait (F), and uncoordinated unsteady gait (F) were observed at 500 mg/kg. The rate and extent of systemic exposures to tasimelteon and the characterized metabolites were generally non-linear and dose-dependent. Usually, the AUC24 values were lower than predicted based on a linear relationship. Accumulation was suggested for tasimelteon (500 mg/kg; 1.5x in F only), M9 (250 and 500 mg/kg; 1.5x and ~3x, respectively) and M13 (250 and 500 mg/kg; 2x and 4.5x, respectively, in F only). (Accumulation for M3 in females ranged from 2x to 29x.) Summary PK data for tasimelteon, M9, M12, and M13 are presented below from the sponsor’s submission, as well as metabolite ratios (metabolite AUC/ tasimelteon AUC) for M9, M12, and M13. (For reference, sponsor-provided human AUC plasma exposures to tasimelteon, M9, M12, and M13 at the RHD of 20 mg were 411.4, 379.4, 655.2, and 393.7 ng*hr/mL, respectively.) 27 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Table 16 Pharmacokinet?ic parameters derived from plasma tasimelteon concentrations in male rats administered tasimelteon by oral gavage over 29 days at nominal doses of 25, 100, 250 and 500 mg/kg/day Nominal Animal number Day (Iii-L) (lit-1) (lit) (DUEL) (HM) (BEN-L) (MI) 01) 25 1 3350 4 BLQ 18400 0.5825 1.2 3790 2 BLQ 12700 0.6327 1.1 25 2 4200 4 BLQ 29500 1.0382 0.7 2990 2 BLQ 11200 0. 5092 1.4 25 3 4870 0.5 BLQ 17100 0.3855 1.8 2080 2 BLQ 8010 0.4352 1.6 4140 4' - 21700 0.6687 1.0? 2950 2' - 10600 0.5257 1.3' Sd 760 - 6810 0.3348 860 - 2400 0.0998 100 4 17700 4 5.90 121?? 0.4834 1.4 3160 0.5 BLQ 13000 0.7895 0.9 100 5 7520 8 BLQ 91000 8030 0.5 BLQ 26100 100 6 8610 8 BLQ 75800 4620 8 BLQ 34700 Mm 11300 8? - 95900 - - 5270 0.5' - 24600 - - Sd 56w - 23000 2500 - 10900 250 7 12100 6 215 163?? 0.2601 2.7 5280 6 11.8 41900 50 8 20700 0.5 3220 216?? 0.085 8.1 10600 4 6.7 94000 0.4529 1. 5 250 9 15100 6 34.2 168?? 0.3867 1.8 12700 8 5.12 72500 Mun 16000 6' 1160 182010 0.2441 28" 9530 6' 7.87 69500 - - Sd 4400 1790 29000 0.1512 3820 3.49 26200 500 10 21400 0.5 5020 1131000 13390 2 6.43 73800 0.43117 1.6 500 11 25800 1 6880 219M 14400 8 165 195000 0.2957 2.3 500 12 22800 0.5 14460 2211!? 25300 0.5 12.8 129000 0.423 1 1.6 Mm 23300 0.5' 9050 207M - - 16000 2? 61.4 133000 0.3858 1.9 Sd 22(1) 4710 23000 8600 89.7 61000 0.0784 . Modianfot rm Calculated as inllmean Mom at! Sd: calculated ?om mn?lcdvalucs Table 17 Pharmacokinetic parameters derived from plasma taslmelteon concentrations in female rats administered taslmelteon by oral garage over 29 days at nominal doses 0125, 100. 250 and 500 mg/kg/day Ali-d ?-156: Day 1 Day 29 (3:0 1.. cu Avcu 11 1.. cu 1113c? 1; (nu-L) (I) (awn-L) M) o) 0) (nah-L) (new-L) (1m 00 25 13 4670 2 BLQ 20100 1520 0.5 BLQ 3000 1.0442 0.7 25 14 6100 0.5 BLQ 24000 5 7190 1 BLQ 20300 0.6373 1.1 25 15 10100 0.5 BLQ 17200 0 8773 4260 2 BLQ 13600 0.9974 0. 7 Man 6090 0.5' . 20400 . . 4320 1' . 12600 0.8896 0.0? sa 2000 - 3400 2830 . 3200 0.2204 100 16 17600 0.5 1060 106000 0.0975 7.1 10400 0.5 BLQ 34700 5 100 17 10000 0.5 277 106000 02103 3 3 15500 0 5 13142 07000 0.1592 4.4 100 14 12600 4 BLQ 104000 5 11400 0 5 3110 45200 0.4122 1.7 Man 13700 0.5- 446 105000 0.1539 45' 15400 0.5- - 72300 0.2857 24? 56 3500 550 1000 4000 - 23500 - 250 19 19000 05 1540 152000 0.0931 7.4 15300 0 BLQ 199000 5 5 250 20 14300 0.5 3900 133000 10400 0.5 5.39 127000 5 250 21 37900 0.5 7060 238000 5 5 10100 0.5 7.59 132000 5 5 Man 24000 0.5' 4190 191000 - - 12100 0.5' 4.33 153000 - - $6 12300 2770 35000 3200 3.91 40200 500 22 22500 0.5 4040 136000 16000 0 5 4030 171000 500 23 17200 0.5 8690 147000 2600 0.5 74.8 214000 500 24 26100 0.5 5890 195000 35700 0.5 616 355000 5 Man 21900 0.5' 6210 159000 - - 24400 0.5' 1570 247000 - - so 4500 2340 31000 10000 2140 96300 Memmfotl?.? Cakulacdaslt?nnank 28 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Table 18 Pbarmacokine?c parameters derived from plasma concentrations in male rats administered tasimelteon by oral gavage over 29 days at nominal doses of 25, 100, 250 and 500 mg/kg/day Nominal Animal number Day 1 Day 29 (mg) 1.,x cu 41:0,; 1 1, 1-, cu 18 (Ila/ml) (11) (I181 Illa) (nah/Ilia) (us/Illa) (us/IL) (1?11) (11) 25 1 1150 0.5 BIQ 3220 0.3919 1.8 1460 0.5 BLQ 3270 0.4147 1.7 25 2 1440 0.5 BLQ 35 20 0.3501 2.0 895 0.5 9.19 3330 0.1440 4.8 25 3 1620 0.5 BLQ 2840 0.3778 1.8 715 0.5 BLQ 2700 0.4298 1.6 Mean 1400 0.51 - 3190 0.3733 1.9? 1020 0.5I - 3100 0.3295 2.1" Sd 240 - 340 0.0213 390 - 350 0.1608 100 4 1580 0.5 39.8 8360 0.1623 4.3 2830 0.5 BLQ 6550 0.3222 2.2 100 5 1230 0.5 5.02 61 10 0.2867 2.4 1970 0.5 9.81 6080 0.2156 3.2 100 6 3280 0.5 342 12000 0.2031 3.4 2750 0.5 8.76 14100 0.3078 2.3 Man 2030 0.5' 26.3 20 0.2174 3.2b 2520 0.5' 5.39 8910 0.2819 2.5" $6 1100 18.7 2970 0.0634 480 0.74 4500 0.0578 250 7 1890 0.5 239 12300 2420 1 12.5 16100 0.2839 2.4 250 8 4000 0.5 603 18600 20 0.5 30.9 21300 0.2427 250 9 1670 0.5 81.8 93 30 0.1092 6.3 2800 8 19.3 21100 0.2941 -. Man 2520 0.5? 308 13400 - - 2950 1' 20.9 19500 0.2736 2.5" $61 1290 267 4700 610 9.3 2900 0.0272 500 10 4770 0.5 1180 18 100 4930 0.5 36.6 37900 0.2786 2.5 500 11 4150 0.5 544 15500 1250 50100 500 12 2810 0.5 756 13300 10300 0.5 152 41(00 0.1708 4.1 Mm 3910 0.5? 827 15600 8740 0.5' 480 43000 0.2247 $11 1000 32-1 2400 3320 670 6300 Median for 1"m Calculaxedas ln2"meank Could notbe reliably estim?ed from the data Means and calculated ?10m rounded values Table 19 Pharmacokinetic parameters derived from plasma M9 concentrations in female rats administered tasimelteon by oral gavage over 29 days at nominal doses of 25, 100, 250 and 500 mg/kg/day Nominal An'nd number Day (981ml) (Its/ml) (0811/le (Uh) (981ml) (I901) (ugh/mid) (10) 25 13 1350 05 BLQ 3430 04127 1.7 349 05 BDQ 1160 25 14 2130 0 5 BLQ 4290 2063 2 3 2830 1 BLQ 7200 0 2493 2 8 25 15 3570 05 103 4180 1930 05 215 4290 Mean 2350 0 5' - 3970 0 3545 2 0 1700 0 5" - 4220 - - Sd 1130 - 470 1260 - 3090 100 16 1980 0 5 182 9260 2490 5 8 39 9950 02603 2 7 100 17 2550 0.5 689 8850 0.1187 5.8 2300 0.5 5.71 10100 0.2981 2.3 100 18 3140 05 102 10300 0.2425 2.9 2910 05 BLQ 70 0.2006 35 Mean 2560 0.53 87.0 9470 0.1806 38? 2570 0.5? 4.70 9510 0.2530 2.7? Sd 580 87.3 750 310 4.29 901 0.0492 250 19 4210 0 5 329 15300 0.0263 26 5 3780 5 441 27500 250 20 2590 0.5 512 12800 9850 0 49.5 22600 0.1852 3.7 250 21 7530 0.5 802 23700 2650 0.5 53.6 23900 0.2176 3.2 Mean 4780 0.5I 548 17300 - - 5430 49.1 24700 0.2014 3.4b Sd 2520 239 5700 3870 4.8 2500 0.0229 500 22 4500 0.5 651 14300 3970 0.5 1480 44300 0.0226 30.7 500 23 2240 0.5 517 11400 8470 0.5 225 44000 500 24 5170 0.5 719 16500 4010 0.5 703 51300 Mean 3970 0.5' 629 14100 . 5480 0.51 803 46500 . . Sci 1540 103 2600 2590 633 4100 Median for (?altulated Could not be reliably estimated tom the data Mans and calculated ?om totmded values 29 Reference ID: 3399188 BEST AVAILABLE COPY NDA 205-677 Melissa K. Banks-Muckenfuss, Table 20 Pharmacoklnetlc parameters derived from plasma concentrations In male rats administered taslmelteon by oral gavage over 29 days at nominal doses of 25. 100, 250 and 500 Nominal Anhnl and)" Day 1 Day (3kg(will) (W) (ISM-IL) (MI) (9) (Bull) 0) (um) (MI) 25 1 3230 4 EU) 23100 0.6863 1.0 4510 2 EU) 21W 0.7044 1.0 25 2 4100 6 BLQ 35000 0.2959 2.3 5520 2 BLQ 369m 0.3001 2.3 25 3 3330 2 BLQ 23000 0.5518 1.3 3540 2 BLQ 22000 0.5756 1.2 Mean 3550 4' - 27000 0 5113 14? 4520 2I - 26600 05267 1. $0 480 - 6900 0.1983 990 - 8900 02065 100 4 13000 4 552 1580?) 7320 0.5 810 41900 0.4972 1.4 100 5 8620 12 BLQ 1280M 8610 0.5 BLQ 62600 0.5997 .2 100 6 8320 12 118 11301!) 7530 8 765 85300 04-472 1 5 Mean 9980 12' 57 7 1330?) - - 7820 0 5" - 63300 0 5147 3" Sc! 2620 59.0 23000 690 - 21710 0.0777 250 7 13900 12 1750 2100(1) 10200 2 BLQ 91400 250 8 18800 12 12500 30501!) 17900 4 BLQ 38000 250 9 14800 12 624 2150?) 22600 8 BLQ 148000 Mean 15800 12? 4960 2430?) - - 16900 4' - 156000 - St! 2 6560 53000 6300 69000 500 10 14500 12 13300 27901!) 16600 2 8w 232000 500 11 17800 24 moo 335000 19200 12 927 296000 500 12 18900 24 189m 2840?) 24000 0 5 775 245000 0 3445 2 0 Mean 17100 24' 16700 2990?) - - 19900 2' 335 258000 - Sc! 2300 3000 31000 3800 514 34000 Medaanfot Tm 1' Calculatedas W1: Meals and 860 calcuhrd ?om mnmitd walucs Table 21 Pharmacoldnetic parameters derived from plasma 3112 concentrations In female rats administered tasimelteon by oral garage over 29 days at nominal doses of 25. 100. 250 and 500 No-hal All-? Day 1 DI: 29 (:30 c_ cu Arc(It?ll-a) (I) (lg/IL) (U5) (ll) (U5) 0) 25 13 6630 4 BLQ 52500 0.4420 1.6 5970 4 BLQ 24W 0. 7804 0.9 25 14 6680 8 BLQ 87400 10200 4 BLQ mm 0 2883 2 4 25 15 7390 2 BLQ 51000 0.4652 1 5 9220 2 BLQ 52100 0 6606 10 Man 6900 4' - 63600 0.4536 1 5" 8460 4' - 51300 0 5764 12? Sd 430 - 20600 2210 2 0.1566 100 16 19700 12 11100 336000 163m 6 11.3 2231!? 0 4753 1.5 100 17 13010 12 10500 230000 13300 4 81.0 186000 100 18 13900 8 167 187000 0.2930 2.4 14700 4 BLQ 142000 0 1819 3.8 Mean 15500 12? 7260 251000 14M 4? 184W 0.3286 2.15 $6 3600 6150 77000 1500 - 41000 250 19 26200 12 14700 418?!) 20500 2 8 46 317W 250 20 19500 24 19500 375?? 211W 2 10.8 276?? 250 21 24200 3 21500 445W 162m 12 47.9 Mean 23300 12' 18600 413W - - 19300 12' 22.39 280030 - - Sd 3400 3500 351110 2700 22 13 36W 500 22 16700 24 16700 3220(1) 21700 12 11200 380W 500 23 24100 24 24100 398% 25W 12 426 377W 500 24 19100 24 19100 37201? 39300 12 2770 577M Mean 20000 24' 20000 364000 - - 28700 12' 4799 445000 - - 50 3800 3800 39000 93 50 5666 115000 Medan 1'01 Calculaledas 1n2 mean Meals and $05 value: 30 Reference ID: 3399188 BEST AVAILABLE COPY BEST NDA 205-677 Melissa K. Banks-Muckenfuss, LE Table 22 Pharmacokinetic parameters derived from plasma 3113 concentrations in male rats administered tasimelteon by oral garage over 29 days at nominal doses of 25, 100, 250 and 500 mg/kg/dny Noninal Alina] number Day (ll) (IEMIIL) (MI) (Ill/Illa) (MI) 25 1 50.4 0.5 BID 268 0.4655 1.5 53.9 2 BLQ 102 0.5651 1.2 25 2 278 0.5 BIJQ 869 184 0.5 BBQ 679 0.3597 1.9 25 3 113 0.5 BLQ 303 03430 2.0 47.7 2 BLQ 188 0.3050 2.3 Mean 150 0.5' - 480 0.4043 1.7" 95.2 2? - 356 0.4099 1.7" Sd 116 - 337 77.0 280 0.1371 1(1) 4 211 8 31.12 2480 134 0.5 BLQ 694 100 5 363 0.5 BLQ 2380 302 0.5 BLQ 884 0.4331 1.6 100 129 0.5 311) 1120 131 8 13112 917 Mean 234 0.5' - 1990 - - 189 - 832 - - 25.0 5520 ?192 6 BLQ 4010 250 8 206 0 5 107 3220 0.0308 22 5 358 4 BBQ 3240 250 9 401 0.5 9 46 3670 0.2088 3.3 867 8 BLQ 4820 Mm 321 0.5? 47 2 4140 0.1198 5.8? 572 6? - 4020 - - $6 102 52.4 1220 264 790 500 10 437 24 437 7460 1350 2 BLQ 12100 500 11 451 05 187 3550 478 8 727 7170 0.2799 475 6780 720 0 5 BBQ 5060 Mean 600 0 5' 366 5930 - - 849 2' - 8110 - - Sd 271 156 2090 450 - 3610 Median for Calculated as Inl?meank Could not be xtliably cstim?cd ?om the data Means and calculated {ran rounded values Table 23 Pharmacokinetic parameters derived from plasma 3113 concentrations in female rats administered tasimelteon by oral garage over 29 days at nominal doses of 25. 100. 250 and 500 mg/ngday . on? umber Day c.ll 1., Cu (ll/ll) (?sh/IL) (ll/IL) (Ill/m1) (MIBLQ 208 03749 1.8 27 7 3 BIJQ 5860 12 25 15 154 05 BLQ 203 0.7536 05524 3 Mean 134 0 5I - 263 0 5643 15692 1.2270 770 0 5 BBQ 2450 100 17 214 0.5 5.77 1250 0.1701 4.1 241 0.5 BLQ 1520 01772 3 9 100 18 165 0.5 BLQ 905 161 0.5 BLQ 823 0.2931 24 Mom 396 0.5' 17.3 1480 - - 391 0.5- - 1600 0.352 291520 471 2 BLQ 6370 2.50 20 256 0.5 92.5 1900 227 12 BIQ 3140 250 21 53 0.5 92 6 2650 189 8 BLQ 3810 ?can 359 0.5' 73 3 3020 - - 296 8' - 4110 1970 500 22 380 0.5 99.7 1920 5 1080 0.5 307 9910 500 23 741 0.5 184 3220 1040 0.5 24.0 9960 500 24 536 0.5 130 2760 1280 0.5 56.0 15300 Wan 552 0.5' 138 2630 - - 1130 0 5' 129 11700 Sd 181 43 660 130 155 3100 Median for I'm cm?: as 1112mm 1: Mum and calcumed noun Minded values 31 Reference ID: 3399188 NDA 205-677 Reference ID: 3399188 Melissa K. Banks-Muckenfuss, Table 34 RIetabolite ratios of lletabolite 319 after daily oral administration of tasimelteon to rats Dose lerel Males Females Rat JD Metabolite ratioa Rat Metabolite ratiol numb" Day 1 Day 29 "limb0.15 0.23 13 0.15 0.26 2 0.11 0.23 14 0.16 0.Mean? 0.13 0.25 Mean? 0.17 0.28 100 4 0.06 0.45 16 0.08 0.10 :i 0.06 0.21 17 0.07 0.10 6 0.14 0.36 18 0.09 0.17 Meanb 0.03 0.32 Mean" 0.03 0.12 250 7 0.07 0.34 19 0.09 0.12 8 0.08 0.20 20 0.09 0.16 9 0.05 0.26 21 0.07 0.16 Mean" 0.06 0.26 Mean" 0.03 0.15 500 10 0.09 0.45 22 0.09 0.23 11 0.06 0.23 23 0.07 0.18 12 0.05 0.28 24 0.07 0.13 Mean 0.07 0.31 Mean" 0.03 0.17 Calculated from {tasimelteonj and corrected for relative molecular weight Geometric mean Table 35 LIetabolite ratios of Metabolite )Ill after daily oral administration of tasimelteon to rats Dose lerel Males Females (mgl'kgjday) Rat D) Metabolite ratioa Rat Metabolite ratio' numb1.18 1.55 13 2.45 5.78 2 1.11 2.46 14 3.42 3.60 3 1.26 2.58 15 2.78 3.60 Mean 1 13 2 14 Mean 2 86 4 21 100 4 1.23 3.03 16 2.98 2.47 5 1.32 2.25 17 2.04 2.01 6 1.40 2.31 18 1.69 2.95 Mean? 1.31 2.51 Mean? 2. 7 2.1.33 2.28 20 2.65 2.04 9 1.20 1.92 21 1.45 1.75 Mean" 1.24 2.03 Mean 2.15 1.75 500 10 1.45 2.95 22 2 23 2.1.21 1.78 24 1.79 1.53 Mean" 1.36 1.96 Mean" 2.17 1.74 Calculated from {tasimelteon} and corrected for relative molecular we1ght Geometric mean Table 36 RIetabolite ratios of Metabolite after daily oral administration of tasimelteon to rats Dose lerel Males Females (mgl'kgfday) Rat Metabolite ratioa Rat Metabolite ratio' numb" Day 1 Day 29 "mm0.01 0.01 13 0.01 0.01 2 0.03 0.06 14 0.01 0.01 3 0.02 0.02 15 0.01 0.01 Mean 0.02 0.03 Mean" 0.01 0.01 100 4 0.02 0.05 16 0.02 0.0.01 0.02 13 0.01 0.02 Mean" 0.02 0.03 Meanb 0.01 0.02 250 7 0.03 0.09 19 0.01 0.03 3 0.01 0.03 20 0.01 0.02 9 0.02 0.06 21 0.01 0.02 Mean" 0.02 0.06 Mean" 0.01 0.02 500 10 0.04 0.15 22 0.01 0.05 11 0.02 0.03 23 0.02 0.04 12 0.03 0.04 24 0.01 0.04 Mean 0.03 0.06 Mean" 0.02 0.05 Calculated from {tastmelteon} and corrected for relat1t-?e molecular u-?e1ght Geometric mean 32 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Although human metabolite M11 was not detected in rat, it was observed in mouse, pregnant rabbit, and monkey at low levels. Following a single oral gavage dose of 300 (b) (4) mg/kg in mice (non-GLP study 09628), the peak area and % tasimelteon (VEC-162) peak areas of M11, M12, M13, and M14 for mouse are summarized in sponsor’s Table 2, below. The sponsor further investigated the steady-state exposures of tasimelteon and metabolite M11 in CD-1 mice (27/sex/gp) after daily oral gavage administration of 100, 300, and 600 mg/kg tasimelteon for 29 days (GLP study TAJ0018). Animals treated with 600 mg/kg tasimelteon showed mortality and adverse clinical signs, and were sacrificed early; adverse clinical signs were also observed at 300 mg/kg. Generally, non-linear, dose-dependent kinetics were observed. The sponsor noted that enterohepatic recirculation was suggested. M11 represented <1% of the total drugrelated exposure in mice. BEST AVAILABLE COPY Only a single dose mass balance study in Cynomolgus monkeys (study PHZ00012, and (b) (4) addendum study 07815) was provided. Following a single 25 mg/kg [14C]tasimelteon PO dose administered to male and female monkeys, the highest mean radioactivity in plasma was observed at 2 hr post-dose, and the mean concentration of radioactivity decreased to below the quantifiable limit within 24 hr. Urine and feces accounted for approximately 80% and 5% of elimination, respectively; approximately 90% of the dose was eliminated by 24 hours after dosing. All tasimelteon human metabolites (i.e., M1, M3, M8, M9, M11, M12, M13, and M14) were confirmed in monkey plasma. Unchanged [14C]-tasimelteon accounted for 8.91% and 17.7% of the total radioactivity in 0-12 hr male and female monkey plasma, respectively. Metabolite M9 (a phenol-carboxylic acid derivative of tasimelteon) was also the most prominent circulating metabolite in monkeys and accounted for 10.3% (male) and 6.72% (females) of the total radioactivity in 0-12 hr plasma. Metabolite M12 was a predominant metabolite in the female monkey, representing 10.0% of the total radioactivity, while it represented only 3.72% in males. Exposures to the remaining human metabolites were each <50% of the tasimelteon exposure, and less than 5% of the total AUC. See the sponsor’s summary Tables S-2, 2, S-3 and 3. 33 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Table Concentrations (WC?162mg EqJ'g) of and Its Metabolites in 0.5. 1, 2, 3, and. 13 Hour Male Monkey Plasma and Estimated AUCMH, Entity Concentration ?EC-l?E-ng qug) AUCMI 15: Metabolites 0hf?ngEqig 31: Total Radioactivio' 10910 15040 22550 2060 660 102231 100 VEC-162 331 231 2412 69.4 :03 9553 3.91 MB 263 504 902 139 50.0 4430 4.13 MB 410 594 32 100 120 4265 3.93 M9 1592 2035 2331 33.9 20.0 10993 10.3 M12 142 352 925 33.4 :03 3991 3.22 M13 334 266 1012 22.2 1333 4420 4.12 MM 116 242 654 52.2 :03 2296 2.61 Table 2 Concentrations Eq.2?g) and M11 in 0.5-, 1-, 2-, 8-, and 12-Hour Male Monkey Plasma and Estimated Concentration Eq.2'g} AUCalh, Metabolites 0hr*ng qug "20 TRA I09l0 15040 22550 2060 660 1010101 100 M1 147 211r 3'10 ND ND 1532 1.43 M11 39.5 212. 431 22.2 ND 1393 1.110 radioactivity in plasma samples 3 Concentrations ?Va of 111342-162 or metabolite in a radio-pro?le ('I'able i) TRA. 01th ADC oflhe total radioactivity in plasma samples. ND: Nen-dctecrahle. Table S-3 Concentrations of VEC-162 and Its Metabolites in 0.5. 1, 2, 8, and. 13 Hour Female Monkey Plasma and Estimated Entity Concentration Metabolites 0.5 131 1 111 3. 1.11 8 131' 12 111' h?ngEqig 9-1: Total Radioactivib' 14310 13920 19530 6500 2290 123031 100 VEC-162 2303 3616 4132 340 332 22219 12.2 MB 392 552 623 343 120 5040 3.94 MB 362 230 359 242 153 5229 4.12 M9 2339 2342 1291 150 63.1 3606 6.22 M12 633 1169 1334 1035 329 12313 10.0 M13 1009 319 519 45.5 22.3 3163 2.42 MM 332 552 330 419 160 5530 4.36 Table 3 Concentrations qug) ofMl and M11 in 0.5-, 2-, 3-, and lE-[iour Female Monkey Plasma and Estimated AUCaizhr Concentration Eng} AUCU.L3 Metabolites 0.5 111?10 TM: 14010 10920 19530 0500 21190 1211031 100 Ml 153 232 234 36 41 1540 1.1?31 1.35 ?Total radioactivity in plasma samples. "Concentrations ?34: ofVEC-lt? or metabolite in a radio-profile [Table l) *5 TEA. ot'the AUC ot'the Iota] radioactivity in plasma samples. ND: Nonvdeteetable. 34 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In pregnant New Zealand rabbits, the plasma exposures of tasimelteon and metabolite M11 only were assessed following administration of 200 mg/kg tasimelteon for 14 days (GD6- GD19; GLP study TAJ0016). See the sponsor’s summary PK data, Tables 1 and 2 below. Little to no accumulation was reported for tasimelteon and M11; see sponsor’s Table 5, below. Metabolite M11 was observed to be approximately 1% of tasimelteon exposure on GD6, and approximately 3% of tasimelteon exposure on GD19. 35 Reference ID: 3399188 NDA 205-677 6 General Toxicology 6.1 Single-Dose Toxicity Melissa K. Banks-Muckenfuss, Ph.D. Single doses of BMS-214778 were tested in rats, mice, and monkeys. Generally, single doses of up to 400 mg/kg in rats and mice and 200 mg/kg in monkeys were tolerated. Following a single dose of 1750 mg/kg, rats showed CNS-related signs (i.e., ataxia, ptosis, and hypoactivity on day 1, and loss of righting reflex and prostration on days 2 and 3), labored respiration, and reduction s in body weight. According to the 1/16/98 review by Dr. Atrakchi, the dose of 1750 mg/kg in rats was considered the maximum non-lethal dose; higher doses could not be tested because of the toxicity of PEG-400 and the drug’s solubility limit. In mice, a single dose of 1750 mg/kg resulted in mortality, tonic convulsions, “inappropriate locomotion”, and “disequilibrium,” in addition to the signs previously noted in rats at that dose. 6.2 Repeat-Dose Toxicity Subchronic studies were conducted in mice, rats and monkeys by the oral route. The vehicle used in all toxicity studies was PEG-400. Most of the subchronic studies were reviewed by Dr. Atrakchi (see review dated 1/16/98). Pertinent results of some of the studies are summarized below. Target organs of toxicity have consistently included the CNS and liver, and kidney in rats. Reproductive organs have also been demonstrated to be a target. In a 3-month study in mice, BMS-214778 caused mortality at 800 mg/kg, and increased liver weights, dose-related minimal to moderate centrilobular hepatocellular hypertrophy, prostration, labored breathing, and slightly increased serum albumin and total protein (M) at 400 to 600 mg/kg. Relative kidney and prostate weights were decreased in males at 600 mg/kg. The reported no-effect dose was 100 mg/kg. In a 2-week study, rats showed increased liver weight and size, with dose-dependent centrilobular to panlobular hepatocellular hypertrophy observed at ≥ 100 mg/kg in males and ≥ 200 mg/kg in females. Increased hepatocellular mitoses were observed at 400 mg/kg. Hyaline droplet nephropathy was observed in male rats. Clinical pathology changes were reported at ≥ 200 mg/kg, including increased ALT, globulin, total protein, cholesterol, glucose (F), and triglycerides (F). The reported no-effect dose for 2 weeks was 50 mg/kg. In a 1-month study in rats, increased liver weight and size, minimal to marked centrilobular to panlobular hepatocellular hypertrophy, increased kidney weight (only 400 mg/kg in M), dose-dependent hyaline droplet nephropathy (≥ 100 mg/kg in M), increased heart and adrenal weight (only 400 mg/kg in F), and several changes in clinical chemistry parameters (e.g., increased cholesterol, triglycerides (F), glucose (F), total protein, albumin, globulin, and ALT) were observed at ≥100 mg/kg; most changes were at least partially reversible. Decreased serum urea nitrogen and creatinine were observed. Increased neutrophils (400 mg/kg) and RBC polychromasia and anisocytosis (400 mg/kg in F) were also observed. According to Dr. Atrakchi’s review, the NOEL was given as 25 mg/kg. 36 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In a 1-week study in monkeys, 175 mg/kg resulted in post-dosing emesis and/or salivation, decreased body weight and food consumption, and mild changes in clinical pathology parameters (including increased serum triglycerides (M), glucose (F), ALT (≥100 mg/kg), and fibrinogen, and decreased sodium (M), chloride, and phosphorus). According to Dr. Atrakchi, the NOEL was 50 mg/kg. In a 1-month study, 125 mg/kg was associated with increased liver weights (≥ 45 mg/kg), decreased spleen weights (M), and decreased SV/prostate and testes weights. According to Dr. Atrakchi’s review, the reported no-effect dose was 15 mg/kg, and 45 mg/kg was a LOEL. Chronic toxicity studies were conducted in rats (6-month) and monkeys (12-month, with an interim assessment at 6 months). These studies were terminated after the in-life portion and summary and/or interim reports were issued; upon restarting development, the sponsor contracted CROs to complete the histopathology assessments for these studies. (The rat fertility study was also completed in this manner.) However, it appears that GLP and QA conditions for these studies has been assured, except for the lack of a separate, signed pathology report for the 6-month rat toxicity study (this has been requested). The chronic toxicity studies demonstrated CNS, hepatic, renal, endocrine, and reproductive effects. These effects generally reflected toxicities previously seen in acute and subchronic toxicity studies, including: CNS signs (e.g., convulsions, hypoactivity, ataxia, labored breathing), body weight reduction, altered hematology parameters (e.g., increased neutrophils, reticulocytes, fibrinogen, RBC polychromasia), altered clinical chemistry parameters (increased ALT, globulin, total proteins, total cholesterol, glucose, and triglycerides), liver enlargement and histopathology (e.g., hepatocellular centrilobular to panlobular hypertrophy, increased hepatocellular mitoses), increased kidney weight and histopathology (hyaline droplet nephropathy), decreased spleen weight, decreased male reproductive organ weights (seminal vesicles, prostate, testes), and increased uterine weight. Also of note, from Dr. Atrakchi's review: a previous finding of eyelid edema and mild dyspnea after tasimelteon (in 50% PEG) dosing in monkeys was dismissed after it was demonstrated to not be reproducible. 37 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Study Title: BMS-214778: SIX-MONTH ORAL TOXICITY STUDY IN RATS Study no.: TAJ0007 /064164; BMS 98348 Study report location: EDR Conducting laboratory and location: In life BMS Pharmaceutical Research Institute, Dept. of Toxicology and Pathology, Mt Vernon, Indiana TK BMS Dept of Metabolism and PK Princeton, New Jersey Histonatholoav (slide readinal Date of study initiation: 10/27/98 GLP compliance: Yes (BMS- FDA) QA statement: Yes (BMS- FDA) Drug, lot and purity: BMS-214778, batch N031C-214778-01, 99.3% The in-Iife portion of this study was completed by BMS. A decision was made by the previous sponsor (BMS) to terminate the project as of 10/27/99; however, the current sponsor asked (12/18/06) to complete the study (histopathology evaluation and study report). Audited formulation analyses and TK reports were supplied by BMS on 3/9/07. The study was completed 7/3/07. Note: there is no separate, signed pathology report. The TK report (signed in 1999; Annex 5) was amended in 2007 ("Amendment to specify that it was audited for US GLP compliance. The NOAEL is 5 mg/kg/day, based on ?ndings in the liver, kidney, and spleen. Methods (see summary tables from the sponsor, below) Frequency of dosing: QD Route of administration: PO, by gavage FormulationNehicle: PEG-400 Species/Strain: Hsd:Sprague Dawley? rats (W) Age: ~7 weeks Weight: M: 198-275 g; F: 157-209 Dosed fasted or fed: Fed (ad libitum) Unique study design: After Week 3, animals were housed in wire- bottomed cages due to pica in all treated groups. Deviation from study protocol: None reported 38 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results Dosing Solution Analysis [Weeks 1, 6, &12] All formulations were homogenous and stable (at ambient temperature for two days and stored at approximately 4°C for 15 days). The mean concentrations of BMS-214778 in the formulations during Weeks 1, 6 and 12 were ±5% of nominal. Mortality [Twice daily] There was only one drug-related death (HDF) on D3. HDF (#2416) was sacrificed moribund on D3. Clinical signs included tonic convulsions, lacrimation, prolonged extension of limbs, hypoactivity, labored respiration, recumbency, ataxia, loss of righting reflex, hunched posture, and coolness to touch. No clinical pathology findings were reported (although the data were not provided). The sponsor reported no drug-related macroscopic or microscopic findings; however, the following findings were reported in this animal: glandular stomach (minimal, focal mucosal erosion), liver (slight centrilobular hepatocyte hypertrophy, with a prominent increase in mitotic activity), kidney (minimal cortical tubular basophilia), spleen (slight decreased cellularity), and reproductive organ (epithelial mucinification) changes. The animal was replaced with HDF #2421. Clinical Signs [Daily; detailed examination weekly] No signs were reported at LD or MD. At HD, clinical signs reflecting effects on the central nervous system occurred throughout the treatment period but were most prevalent during the first two weeks. A single HDF was sacrificed moribund because of the severity of the clinical signs (see 39 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Mortality). HDF were more often affected than HDM, particularly during the first four days of treatment; signs included (number affected; F/M): hypoactivity (19/12), labored respiration (16/6), ataxia (17/8), loss of righting reflex (11/0), extension of the limbs (14/1), recumbency (16/4), tremors (4/1), and convulsions (5/0). In HDF, tonic convulsions were observed on D3 (#2416; early mortality), D6 (#2407), D36 (#2408 and #2413), and on D3, 76, 113, and 120 (#2415). In animal #2408, clonic convulsions were observed on D4 and D5. There was evidence of dose-related pica in the treated animals; increased incidence of bedding material in the feces was observed on D12 (0, 3, 4, 10 in control, LD, MD, and HD males; 1, 0, 6, 15 in control, LD, MD, and HD females). The caging was changed to wire-bottomed cages from D12 on to prevent ingestion of bedding. A few other signs were noted at lower incidence. Salivation was observed in 3 HDM and 2 HDF. Red discoloration of the muzzle was observed in 4 HDM and 1 HDF. Chromodacryorrhea was observed in 1 conM, 1 MDF, 2 HDM, and 2 HDF. Lacrimation was observed in 2 HDF. Although of questionable relationship to drug, alopecia was observed in 1 female of each treated group. Body Weights [Weekly] Throughout the treatment period, average body weights were reduced 6-8% [ss] in treated males, compared to controls. A slight reduction in body weight gain was observed at all doses but was not consistently dose-related (Weeks 1-13: 10%, 11% and 13%; Weeks 1-26: 18, 12, and 13% for LD, MD, and HD males, respectively). In females, slight effects on average body weight gains appeared biphasic. The average body weight gains from Week 1-13 were -11%, -16% and +6%, and were -11%, -6%, and +10% in Weeks 1-26 ([nss] at LD, MD and HD, respectively). There was no significant effect on body weight at Week 26 in females. See the sponsor's Figure 1. APPEARS THIS WAY ON ORIGINAL 40 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, FIGURE 1 Bodyweight - group moan values vorsus period of treatment: males Group 2 3 4 Compound Control EMS-2 14TH EMS-2 1471's EMS-214T73 Dose 5 5 0 500 500.1} 450.0 - 400.0 - 350.0 - 300.0 - 250.0 14; [5010 - his] Bodwrergh 10070 - Period oftreau'nont [noel-us} FIGURE 1 -continucd Bodjnvoight - group mean values versus period of treatment: females Group 2 3 4 Compound Com! EMS-214773 EMS-2 I47T3 EMS-2147B Dose (may) 0 5 so son 500.0- 450!) - 400.0 - 350.0 - 300.0 - Badmight -Period oflrean'nent (weeks) 41 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Food and Water Consumption [Weekly] Food consumption was reduced 14% at week 1 in HDF; however, beginning week 4, HDF generally showed increased food consumption (~10%). No clear effect was observed in LDF, MDF, or treated males. HD animals consumed more water (3-4x in males, ~3x in females) in weeks 13 and 27. Ophthalmoscopy [Prior to study, Weeks 13 and 26] All animals were examined by means of a Keeler all pupil indirect ophthalmoscope. No drug-related findings were reported. One MDM showed anterior synechia of the iris. Hematology [Weeks 13 & 25, sample taken before dosing after an overnight fast] & Coagulation [Week 27] At Week 13, slight decreases (6-7%, [ss]) in RBC parameters were observed in HDF, but no clear effect was observed in males. Increases in reticulocyte (+9%, +12%) and platelet (+27%, +9%) counts were observed in HDF and HDM, respectively. At week 25, females showed a very slight, dose-related reduction (≤2-4%, [ss]) in RBC parameters; similar effects were not observed in males. Reticulocyte and platelet counts showed a slight, dose-related increase in females (up to ~20%), and HDM showed a 10% increase in platelets. At Week 13, WBC counts showed changes, predominantly in females. Increases in neutrophils (~2x in MDF and HDF, however, the MDF average resulted from 2 outliers at 4-7x the control average whereas nearly all HDF animals were 2x the control average), monocytes (30-60%, MDF and HDF), and LUC (~2x in HDF) were observed. Reductions in lymphocyte (15-35%, treated females, d-r, [ss] at HD) and basophil (0 vs 0.1, HDF vs conF) counts were observed. In HDM, increases in absolute lymphocyte (11%), monocyte (33%), and LUC (22%) counts were also observed. At week 25, MDF and HDF showed increases in WBC counts (15-44%). Neutrophils (2-2.5x, MDF and HDF), monocytes (2x, HDF), LUC (1.5-2.3x, MDF and HDF), and lymphocytes (+15%, HDF) were increased in females. In HDM, increases in neutrophil (1.7x), basophil (1.5x), and LUC (18%) counts were observed (total WBC count +9%). Coagulation parameters were dose-dependently affected in males at Week 27 (maximum increases of 19% in PT time and 15% for fibrinogen; maximum decrease of 4% in APTT time, all [ss]). In HDF, PT time and fibrinogen were increased (13% [ss] and 30% [ss], respectively), and APPT time was slightly decreased (-7%, [ss]). Clinical Chemistry [Weeks 13 & 25, sample taken before dosing after an overnight fast] Several clinical chemistry alterations were observed at both time points. At Week 13, increased ALT (d-r, up to 1.8x in HDF; also 24% in HDM) was observed. However, dose-related reductions in ALP (≤36%) and AST (≤40%) were observed in females; and, AST was decreased in HDM by 23%. Total protein was increased (MDF, 42 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. HDF and HDM, up to 19% in F and 4% in M), as were albumin (MDF and HDF up to 17%, HDM 5%) and globulin (MDF and HDF up to 23%). Triglycerides (d-r, up to 4.1x [ss] in HDF) and cholesterol (d-r, up to 2.7x [ss] in HDF) were also increased; in HDM, cholesterol was slightly increased (23%). Blood glucose was slightly increased in HDF (12%). BUN (-17%) and creatinine (-63%) were reduced in HDF. Generally, blood sodium and chloride were very slightly reduced (<5%), and calcium was slightly increased (5% in HDM, 17% in HDF). At Week 25, most of the changes observed at Week 13 continued. ALT was increased (42% in HDF, 20% in HDM, [ss]). ALP (d-r, ≤24% in HDF) and AST (d-r, up to 43% in HDF and 34% in HDM) were reduced. Although total protein remained only very slightly increased in HDM (2%, [ss]), total protein (d-r, up to 12% in HDF [ss]), albumin (d-r, up to 6%, [ss]), and globulin (d-r, up to 26%, [ss]) were increased in females. The A/G ratio was slightly reduced by 15% in HDF [ss]. Cholesterol (d-r, as much as 2.3x in F [ss], and 26% in HDM [ss]), and triglycerides (as much as 3.1x in MDF and HDF [ss]) remained increased. BUN (-16% in HDF [ss]) and creatinine (as much as -29% in MDF and HDF, [ss]) continued to be reduced. Blood glucose was slightly increased (up to 10%) in MDF and HDF. Blood sodium and chloride remained very slightly reduced [ss], and calcium remained slightly increased (12% in HDF, and 3% in MDF and HDM [ss]). Urinalysis [Weeks 13 & 27 (sic, 25), during 18 hr food deprivation, 1st 10 animals/sex/gp] At Weeks 13 and 25, urine volume was increased in HD animals (3- 5x, [ss]). Nearly all HD animals showed increased volumes. Urine specific gravity was also slightly reduced (1-3%), reaching statistical significance in HDM in Week 25. No ketones were present in HDM, in comparison to other groups having at least 6 animals showing trace levels. In females, there was a dose-related increase in the presence of protein in the urine (see the sponsor's table, below). One HDF (#2410) showed RBC, large numbers of WBC, many bacteria, and a few crystals in the urine. 43 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Gross Pathology Animals were sacrificed by carbon dioxide asphyxiation and subsequent exsanguination. Few organs showed macroscopic alterations. See excerpts from the sponsor's summary Table 9, below. Enlarged liver was the single clear finding in all HD animals. Stomach (in HDM), uterus, and mammary gland also showed gross alterations. Masses were observed in 1 MDF (proximal area of vagina) and one HDF (inguinal region of skin). Organ Weights [See the sponsor's list, below] Several organs demonstrated changes in weight. See the sponsor's summary tables, below. Heart, liver, adrenal gland, and kidney weights were increased in both males 44 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. and females. In females, ovary and spleen weights were also increased. Pituitary gland in both sexes and prostate in males showed decreased organ weights. BEST AVAILABLE COPY Histopathology [See list at end of general tox section; 4 m, H&E stained] Adequate Battery Yes Peer Review Report states yes, but no documentation provided (b) (4) Study Pathologist NOTE: A separate, signed pathologist's report was not provided in the report; a signed pathology report was provided upon request (N 205-677, SDN11). 45 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Histological Findings All tissues from control and HD animals were examined, as well as all early mortalities. Tissues reported as grossly abnormal were examined for all animals. Also, the target tissues were examined in all animals, including: kidneys, liver, and spleen. An additional section of kidney from each male rat was stained by Mallory-Heidenhain method. Other special stains used on the liver and/or kidneys to further specify drugrelated changes included Oil Red O, Gomori modified iron, periodic acid-Schiff (PAS), and Hall's and Kinyoun carbol fuchsin. Liver, kidney, and spleen were identified as clear target organs. See the sponsor's summary table of selected liver findings with severity, below. According to the sponsor, the centrilobular hypertrophy observed in the liver was presumed to reflect "an adaptive response" related to induction of microsomal enzymes (and possibly contributing to the changes in drug exposure over time, as well as the appearance of increased organ weight and gross enlargement), but the focal necrosis clearly indicated a "toxic" insult. A complete list of the histological liver findings is excerpted from the sponsor's summary data, below. Many of the clinical chemistry alterations (e.g., enzymes, glucose, triglyceride, cholesterol, protein) may reflect the "adaptive" and/or "toxic" changes. 46 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In the kidneys, the sponsor related the majority of the alterations to exacerbated chronic progressive nephropathy (CPN). See the sponsor's summary tables of kidney findings and selected findings with severity, below. It is of importance that HDF showed a higher incidence of more severe CPN. This is not the usual pattern for the finding; males are typically more affected. Kidneys showed increased organ weights. Clinical chemistry (e.g., BUN, creatinine, and electrolytes), hematological (e.g., WBC), and urinalysis (e.g., increased volume, decreased specific gravity, and proteinuria in HDF) alterations were believed to reflect this change. 47 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Hemosiderosis and extramedullary hematopoiesis were observed in spleen. The hemosiderosis was thought to reflect an increased rate of removal of RBCs from peripheral blood. The sponsor considered the splenic extramedullary hematopoiesis (and reticulocytosis) a "compensatory response." The sponsor further posited that the female prevalence reflected the higher exposures in females. Other organs also showed histologic changes. The stomach, lymph nodes (cervical, mesenteric), mammary gland, and female reproductive organs showed changes. See excerpts from the sponsor's summary Table 10, below. In addition to the noted changes, a few low incidence alterations of unclear toxicologic significance were observed. Focal pituitary hyperplasia of the pars distalis was observed in 1 HDM and 1 HDF. Changes in thyroid (called ”prominent ultimobranchial cysts” by the pathologist) 48 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. were observed in 3 HDM and 7 HDF (compared to 1 control of each sex). A recent paper may shed light on these structures, noted to change appearance as rats age (cf., Vezquez-Roman et al., 2013) The description suggests that these may represent forms that generally occur in older rats (predominantly female; “cystadenomata” forms at ≥ 18 months of age); the reason for and importance of the apparent early presence is unclear. Although ultimobranchial cysts are generally disregarded as embryological remnants without toxicologic import, there is a possibility that the observed pituitary and thyroid changes (cf. Chrisov et al., 1973; Conde et al., 1992; Martin-Lacave et al., 1992) reflect changes accompanying the observed centrilobular hypertrophy of the liver, exhibiting a known pattern of hormonal axis dysregulation in rodents. The gross observation of a vaginal mass was described histologically as a squamous epithelial cyst. Although heart weight was altered, there was no clear histopathological correlate. 49 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Toxicokinetics [See the sponsor's tables, below] Rpt MAP005/214778; 920002144 Blood samples were collected from the first 18 controls of each sex but were discarded without analysis. Systemic exposure to tasimelteon (BMS-214778) was dose-related. Cmax generally showed greater than dose-proportional increases on day 1 and less than dose-proportional increases at weeks 14 and 26 (see the sponsor's table below); AUC generally showed greater than dose-proportional increases on day 1 and approximately dose-proportional increases at weeks 14 and 26. Exposure was higher (2-5x) in females than in males. The sponsor indicated there was some accumulation at LD. The sponsor provided the following summary table for TK parameters. 50 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Interspecies Parent and Metabolite Comparison For reference, the sponsor also provided the following assessment of the estimated metabolite exposures in rats in the 6-month toxicity study (Table 8 from the Nonclinical Toxicology Written Summary, below). It is noted that the sponsor used 363 ng.hr/ml as the human exposure at the RHD (rather than the summary value of 411 ng.hr/ml across studies), and the Week 26 AUC exposures (i.e., the highest exposures after Day 1 AUC exposures) in male and females rats. 51 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Study Title: BMS-214778: ONE-YEAR ORAL TOXICITY STUDY IN MONKEYS Study no.: Study report location: Conducting laboratory and location: Date of study initiation: GLP compliance: QA statement: Drug, lot and purity: 057-005 EDR #97331 (BMS) (4) 11/24/98 Yes, except: 00A for the test article, Test article sample analysis, Bulk test article stability, missing QA statements for the TK data and report, and Stability of the formulated test article (previously determined in Bristol-Myers Squibb Study No. 97331) is not included in this report Yes Pathology QA BMS-214778-01, lot N031 C-214778-01, purity 99.3% Methods (see the sponsor's summary table, below) Frequency of dosing: Route of administration: Dose volume: FormulationNehicle: Species/Strain: Age: Weight: Dosed fasted or fed: Unique study design: Deviation from study protocol: Reference ID: 3399188 Daily PO, nasogastric intubation or oral gavage 1 Polyethylene Glycol-400 (PEG-400), lot M18621 and N01641 adult male and female Cynomolgus monkeys (19(4) Not provided M: 2.09 to 3.86 kg and F: 1.93 to 3.62 kg Not speci?ed Interim sacrifice: see sponsor's table below The study director stated that three deviations occurred, none of which affected the integrity of the study. 52 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results Dosing Solution Analysis Drug formulations were prepared one week prior to use. Duplicate samples were taken from the dosing formulations at Weeks 1,10, 27, 39, and 52. One sample of each formulation concentration was analyzed. Measured BMS-214778 concentration was within 6% of nominal in all samples. The sponsor stated that the stability of BMS-214778 formulations at the concentrations to be used in this study was previously established for seven days at room temperature in a previous study (i.e., Bristol-Myers Squibb Study No. 97331). Mortality [2x daily] No drug-related mortalities were reported; however, four animals died on study. Two males were found dead during the first week of the study and were replaced. MDM #169 and HDF #178 were found dead after approximately four and seven months of dosing, respectively. According to the sponsor, the three males each had acute fibrinous inflammation in the lungs and the female was remarkable for lung hemorrhage; these findings were consistent with accidental deaths. In addition to the findings related to the cause of death, a few other histopathologic findings were observed in these animals. Hemorrhage or vacuolation in the white matter of the brain and/or gray matter of the spinal cord was observed in the HDM and HDF. In the liver, mild congestion was observed in the MDM and HDM, and minimal chronic periportal inflammation was observed in all four early mortalities. In the kidney, minimal mineralization of the papilla was observed in the HDM and the HDF; chronic interstitial inflammation was also observed in the HDF. Mild atrophy of the thymus was observed in the HDM. In the HDF, changes were also observed in the aorta (minimal fibrous plaque), stomach (bacteria, ulceration, and lymphocyte accumulation), ovary (minimal mineralization), vagina (mild chronic inflammation), lachrymal gland (minimal chronic inflammation), and pineal gland (minimal mineralization). 53 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Clinical Signs [2x daily, prior to and after dosing; detailed physical exams pretest, Week 13, Week 26, Week 39, and Week 52] Convulsions were observed in 2 HD monkeys (M #183 on D34 and F #174 on D163). These animals also showed flaccidity (M only), salivation, gasping (M only), and labored breathing in association with the convulsions. (The HDM was sacrificed at the interim assessment.) According to the sponsor, drug-related “decreased appetite” was observed at ≥ MD (11/1, 10/2, 19/5, and190/6 in conM, LDM, MDM, and HDM, respectively, and 107/7, 68/6, 173/6, and 183/6 in conF, LDF, MDF, and HDF, respectively [# observations / # animals]). Occasional excessive salivation, emesis (F), and alopecia (head, limbs) was observed at ≥ MD. Decreased activity was demonstrated in most HD animals beginning after 6 weeks and continuing throughout the study. Of unclear relationship to drug (although the sponsor attributed to drug) are findings in one LDM (#143). This animal showed an episode of coldness, prostration, decreased motor activity, and ataxia on D177; the ataxia and decreased motor activity continued through D178 and D179, respectively. Veterinary care records indicated that this animal was "weak, unable to sit up" and showed "slight dehydration, pale mucous membranes" on D177; a 5% dextrose solution was administered SC. This animal's physical examination record in week 26 also showed thinness, paleness, a low body temperature (95.3 vs. 98-100o C in controls), and a prolonged capillary refill time of < 3s (vs. < 1s in most other animals). Body Weights [Predose on D1, weekly, last dosing day, and prior to necropsy] Slightly reduced (~5%) average body weights were observed for HD animals during the first 6 months of the study. During the last few months of the study, the LD animals tended to show increased average body weights (up to 15% in M and 10% in F). See the sponsor's Figures 1 and 2, below. APPEARS THIS WAY ON ORIGINAL 54 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAILABLE COPY Figure 1 - Mean Body Weights - Males I +G?oup 1 - JVehide Inmg 4.3 +G?oun 7 - 1 mgka +ts'oup 3 - 20 -O-G'oup 4 - ISO n'gkg KilogramStudy Day Figure 2 - Mean Body Weights - Females 3.9 3 7 3; I +004: 1 - O?Juhlcic Contro? myitg +Goup 2 - 3 lug/Kg Kilogtams +Goup 3 - 2C mg?ig +9on StudyDay 55 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Food Consumption [Daily, by visual assessment] Summary data were not presented. The sponsor indicated that ?any depression in appetite was noted in the study file?; this appears to have been reported under Clinical Signs, and qualitative measurements were not provided. Ophthalmoscopy [Pre-test, Week 13, Week 26, Week 39, and Week 52] An ophthalmological examination using a slit lamp was conducted (under ketamine anesthesia) on all available animals by a Board Certi?ed Veterinary Ophthalmologist, (5X4) No drug-related alterations were reported. ECG [Pre-test, Week 13, Week 26, Week 39, and Week 53] Electrocardiogram tracings were taken from all animals using six leads (I, II, and no anesthesia was used. The tracings were interpreted by and a written report was provided; some tracings were also sent to but no report was provided. The veterinary cardiologist did not report drug-related changes in heart rate, heart or ECG parameters (including at any dose. Altered blood pressure was not reported. However, blood pressures in HDF (systolic, diastolic, MAPR) were increased compared to controls (10-30%) on D269 and D365. In the LDM (#143) sacrificed early (at interim), the Week 26 HR and also appeared increased. Hematology [Pre-test, Week 13, Week 26, Week 39, and Week 52] Food and water were withheld overnight prior to collection, except for Weeks 13 and 52. The blood samples were evaluated for the following parameters: red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, red blood cell morphology, platelet count, white blood cell count, count, monocyte count, eosinophil count, basophil count, ?brinogen, activated partial thromboplastin time, prothrombin time, and other cells (as The clinical pathology data were interpreted by The sponsor reported reduced RBC parameters hematocrit, hemoglobin concentration, and RBC count) at the reduction was observed at Week 13 and throughout the study (to Week 39 in HDF). Eosinophils were increased (4x and 2.75x at weeks 39 and 52) in HDF. In the LDM (#143 sacrificed early (at interim), hematology assessment at W26 showed reductions in WBC RBC hemoglobin hematocrit and increased fibrinogen compared to conM. A few other changes of unclear toxicologic signi?cance were observed at individual measurement times. Some changes appeared most severe at Week 13 but appeared to resolve. Platelets were increased (28% in HDM only at Week 13; in HDF, 90% at Week 13, 30% at Week 26, and 40% at Week 39). Fibrinogen was reduced as much 56 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. as 28% in HDM (appeared dose-related) from Week 26 on; reductions were also seen in HDF from Week 13 on (10-35%), with some reduction in MDF as well. Prothrombin time was very slightly reduced at Week 13 (HDM, 8%) and Week 26 (MDM and HDM, 7%). APPT was increased ~26% in HDM only at Week 26. Clinical Chemistry [Pre-test, Week 13, Week 26, Week 39, and Week 52] Food and water were withheld overnight prior to blood collection, except for Weeks 13 and 52. The samples were processed and evaluated for the following parameters: total protein, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides, total bilirubin, urea nitrogen, creatinine, creatine kinase, alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), calcium, phosphorus, sodium, potassium, and chloride. The sponsor reported slightly increased serum ALT (2-3x that of ConM, highest at Week 13, with some increase in MDM; up to 2x that of ConF, from Week 26 on) and GGT (3050% in HDM, up to 70% in HDF); the sponsor indicated that these findings correlated with increased liver weight at necropsy. According to the sponsor, these increases were generally seen at Week 13 and, although they fluctuated, were usually seen throughout the study. Additionally, HD animals showed increased serum triglycerides (~2x ConM at Week 52 in HDM only) and reduced ALP (as much as 30-40%). In the LDM #143 sacrificed early (at interim), LFTs were increased 3-5x, and albumin and phosphorus were decreased (32% and 73%, respectively, compared to ConM) at Week 26. In addition to the changes noted by the sponsor, a few additional parameters showed changes of unclear toxicologic significance at individual measurement times. Some changes appeared greater at Week 13 than Week 26. Serum glucose was increased (20-25%) in HD animals at Week 13 only. Serum albumin was slightly increased (14%) in HD at Weeks 13 and 26. Serum total bilirubin was reduced as much as 75% beginning Week 13 in HDM; reductions (50-70%) occurred in HDF at Weeks 39 and 52. Creatine kinase values were extremely variable, but were reduced 40% in HDM at Week 52. Serum calcium was increased 6-10% in HDM. Serum potassium was increased 16% in HDF at Week 13 only. Urinalysis [Pre-test, Week 13, Week 26, Week 39, and Week 52] Food and water were withheld overnight prior to collection, except for weeks 13 and 52. The urine samples were evaluated for the following parameters: pH, protein, glucose, ketone, bilirubin, urobilinogen, leukocytes, specific gravity, color, clarity, occult blood, nitrite, and microscopic examination of urine sediment. No drug-related changes were reported. Ketones were occasionally increased at HD. Gross Pathology [D183/184 or D370/371] No summary table was provided. The sponsor reported enlarged liver in one HDM (#187) after one year of dosing, which correlated with increased liver weight. In addition to this, a few other findings noted in liver (cysts, foci, or pigment/mottled) were without 57 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. corresponding histology or were histologically observed as dilation of bile ducts (1 LDF), tension lipidosis (1 LDF), chronic inflammation (MDF), serosal/subserosal fibrosis (2HDF), or congestion (1 HDF). Although few organs showed a clear drug-related effect, there were a few changes noted in stomach, intestines, and/or lymph nodes (these appear to mostly be related to parasites), spleen, kidney, and ovary. White focus(i) in the spleen was observed in one LDF and one HDF; this correlated histologically to serosal fibrosis. One HDF showed a dark focus in the kidney that was histologically described as congestion. Gross observations in the ovary (i.e., cysts, focus, nodule) were not associated with microscopic changes. Organ Weights Organ weighs were assessed prior to fixation except for pituitary gland of #159. The sponsor reported increased liver weights at HD at 6 months and one year (~1.8x that of ConM [ss; d-r] and ~1.4x that of ConF); the increase was reported to correlate with reduced total systemic exposure and slightly increased serum ALT and GGT. Relative thyroid gland weight was increased up to 40% in MDM and HDM at 1 year (dose-related) but was slightly reduced in MDF and HDF (~20%). Histopathology Adequate Battery Yes, with a few omissions (see Appx. 2) Peer Review Yes, but limited (b) (4) See sponsor's summary table, below Separate, Signed Pathology Report Yes (revised narrative) (b) (4) 58 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Histopathology was performed on all tissues from all animals and all gross lesions. (b) (4) Fixed tissues were shipped to for trimming, embedding, and sectioning. Slides were stained with hematoxylin and eosin. Histological Findings The sponsor reported no drug-related histopathologic findings and noted that "parasites were observed frequently in tissue sections." The sponsor stated that the observed hypospermatogenesis in testis and oligospermia in epididymis (notably in LDM #143, showing the relatively severe clinical signs and sacrificed early at interim) were interpreted by the pathologist as sexual immaturity; other histopathological findings in this animal included animal chronic periportal inflammation in the liver, moderate, diffuse follicle dilatation in the thyroid, minimal chronic interstitial inflammation in the kidney, minimal chronic inflammation in the meninges of the forebrain, and minimal chromatolysis of the lumbar spinal cord. Noted in the report but not in the summary data tables was a "lack of corpora lutea" in the three remaining HDF (# 174, 176, and 178) at one year, which the pathologist stated "indicates a lack of recent estrus cycling." The sponsor stated that relationship to drug could not be excluded, but that it is "… not unusual to see monkey ovary sections that do not contain corpora lutea." Melatonin is known to have endocrine effects (and effects on estrus cycling were observed in rats); therefore, this finding is considered adverse by the reviewer. A few changes (increased incidence and/or increased severity) of unclear toxicologic importance were observed, at both interim and terminal sacrifice. Overall, minimal evidence for hepatic, cardiovascular, renal, and reproductive organ alterations was observed. In the liver, focal necrosis, pigmented macrophages, and minimal to mild fibrosis were observed. Chronic periportal inflammation was seen in most animals, but a slight increase in severity was suggested at HD. Inflammation of the heart and/or vessels was observed. In the kidney, chronic interstitial inflammation medullary tubule cyst, eosinophilic cast, and mild congestion were observed. In the spleen, mild serosal fibrosis was observed in treated females. Lactation of the mammary gland, ovum mineralization in the ovary, and minimal chronic inflammation of the cervix were observed. Although observed in all groups, increased severity of inflammation of the tongue and esophagus were observed in treated animals. 59 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, 6M Interim Males Females Organ/Finding Con (3) (3) (2) (3) (3) (3) (3) (3) Liver Periportal ?brosis Heart Chronic in?ammation, myocardium Aorta Chronic in?ammation, adven?a Vacuolation muscular wall Kidney Chronic interstitial in?ammation Med. tubule 0 1 0 Eosinophilic cast 0 0 0 0 0 1 Congestion 0 0 0 Mammary Gland Lactation 0 0 1* Ovary Ovum 1 3 2 2 mineralization Cervix Chronic 0 0 1 in?ammation *This HDF was found dead at 7 months, not interim sacri?ce 12M Terminal Males Females Organ/Finding Con (4) (4) (4) (4) (4) (4) (4) (3) Liver Focal necrosis 6M) Focal pigmented 0 0 1 0 0 0 macrophages Focal serosal ?brosis Focal subserosal 0 0 0 0 2 ?brosis Heart Chronic in?ammationmyocardium Spleen Serosal ?brosis Kidney Chronic interstitial in?ammation Medullary tubule 0 1 0 0 0 0 Pineal gland Focal necrosis Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Toxicokinetics Week 26, and Week 52] Plasma concentrations of EMS-214778 were determined after the ?rst dose and after a daily dose during Weeks 26 and 52 for those tasimelteon-treated animals (four/sex/group) designated for the twelve-month phase of the study. Blood samples were collected from the femoral vein (using K3 EDTA) at approximately hours after the first dose and after a daily dose during Weeks 26 and 52. GLP compliance for the toxicokinetic report was not provided. Dose-related increases in exposure were observed; however, the exposures were greater than dose-proportional between LD and HD. No clear sex difference was observed at LD (except on D1) and HD, but exposures were greater in males than in females at MD. There was some reduction in the systemic exposures with repeated once-daily dosing for one year. Dose" 5mm; [ng/mL] 1111101111? Day Male Female Male Female Male Female 5 1 1431701 24.11.1171 . 1.011.020) 1.0121110) 271114251 511512041 :82 2001;:25) 217(2201 1.0 (10,101 37111051 3431340) 12: 17.6) :22 (20:1 .0 (1 .0. 1.01 :01: 0. 1.01 3841061 352132.:1 20 572013317) 357513680) . 1011.01.01 1.0 (2.0.1.01 2393106182) 5750139521 554010021 315513.301) 1.0 (1.0. 2.01 1.0 r10. 1.01 137551102121 8430151361 153 28860100) 2619112418) 2.5 (1.0. 1.01 1.01;: 0, 2.01 1270214520) 6212: (22121 150 26889126621 27313185241 2512.05.01 3.5 12.0. 5.01 2135391639221 28702-1 1117961) 182 252201128117) 21144 (6366) 2.0 (1.0. 5.01 4.5110701 137156 (176321 1556151860821 563 1710419530) 2025814369)" 4.01.2.0. :21 3.011.050)" 115199 1519109) 139245109691)" as 'Median 1 minimum. maximum); ':Time 1 Ti ringed from '2 1130 '21 luhN 3 ?l CMAX ratio ratio Stud) Da) Male Female Male Female 1240:1853 1:15:11? 1:88:892 1:17:568 182 1:21:97 1:15:97 1:37:363 1:25:454 363 1:16:95 1:12:91 1:33:378 1:18:396 7 Genetic Toxicology Previously (see Dr. Atrakchi's review dated 1/16/98), it was determined that tasimelteon was neither mutagenic nor clastogenic. It is noted that the rat micronucleus assay was conducted as part of the 1-month toxicity study with tasimelteon and did not use a positive control; the assay showed no increase in micronuclei in PCE at up to 400 mg/kg. A chromosomal aberration assay was not available at the time; it is reviewed below. Tasimelteon demonstrated clastogenic effects in an in vitro chromosomal aberration assay in HPBL. Additionally, the sponsor has investigated the genotoxicity of metabolite M11 (which has poor coverage overall in the toxicology species and is not formed in vivo in rat). Metabolite M11 was not mutagenic in an Ames assay but was clastogenic in an in vitro chromosomal aberration assay in CHO cells. Reference ID: 3399188 61 BEST AVAILABLE COPY NDA 205-677 7.2 Melissa K. Banks-Muckenfuss, Ph.D. In Vitro Assays in Mammalian Cells Study title: CYTOGENETICS STUDY IN PRIMARY HUMAN LYMPHOCYTES Study no.: 97680 Study report location: EDR Conducting laboratory and location: BMS (Syracuse, NY) Date of study initiation: 12/1/97 GLP compliance: Yes QA statement: Yes Drug, lot #, and % purity: BMS-214778 (BMS-214778-01), Batch # C014A, purity 99.2% Methods Cell line: HPBL Concentrations in definitive study: 5 hr (+S9): 0, 100, 200, 400, and 800 g/mL 24 hr (-S9): 0, 25, 50, 100, and 200 g/mL Basis of concentration selection: A range-finding assay was performed, testing 1 - 2400 g/mL (the upper limit was based on drug solubility). Concentrationrelated mitotic index reductions were seen, 37% at 100 g /mL and 64% at 300 g /mL (24 hr [-S9]) and 30% at 600 g /mL and 100% at 1200 g /mL (5 hr [+S9]). (b) (4) Negative control: DMSO ( Lot #01735JQ) Positive control: 0.1 g /mL mitomycin C (Lot No. 25H0619, (b) (4) 4 g /mL cyclophosphamide (Lot No. (b) (4) 43H0269, Both prepared in sterile water (b) (4) Formulation/Vehicle: DMSO Incubation & sampling time: 37oC for 5 or 24 hr Study Validity The metabolic activation system used was Aroclor 1254-induced rat liver S9 fraction, (b) (4) purchased commercially from . (Note that rats in vivo do not produce metabolite M11.) The 24 hr (-S9) exposures were conducted in tissue culture flasks. The 5 hr (+S9) exposures were conducted in 15 mL centrifuge tubes, followed by two washes, then resuspended and transferred tissue culture flasks for an additional day of culture. Colcemid was used to arrest the mitotic cells in metaphase, and the flasks were incubated for an additional 3 hr. The sponsor's criteria for a positive result were based on statistical significance. 62 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Results The sponsor stated that BMS-214778 was not clastogenic when tested at the maximum cytotoxicity levels "recommended by ICH guideline"; however, the mean number of cells with aberrations and the aberrations per cell suggested an increase in the 24-hr cultures (without metabolic activation) and were more than double those of the vehicle control in the 5-hr cultures (with metabolic activation). The increase in chromosome aberrations occurred only at the highest concentration, was seen in the presence of cytotoxicity (mitotic indices of 45% in 24-hr and 24% in 5-hr cultures, compared to vehicle control), and did not reach statistical significance. The observed cytotoxicity in the 24-hr cultures (without metabolic activation) did not greatly exceed the recommended maximum of 50%). The cytotoxicity in the 5-hr cultures in the presence of metabolic activation was excessive (much greater than a 50% reduction) at 800 µg/mL, but did not show a reduction in mitotic index at the next lower dose; doses between 400 and 800 µg/mL should have been tested. While not reaching statistical significance, the results should be considered equivocal/positive (increased aberrations in the presence cytotoxicity). BEST AVAILABLE COPY 63 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, 7.4 Other Genetic Toxicity Studies In Vitro Reverse Mutation Assay in Bacterial Cells (Ames) Study Title: BACTERIAL REVERSE MUTATION TEST Study no.: Study report location: Conducting laboratory and location: Date of study initiation: GLP compliance: QA statement: Drug, lot and purity: UN0003 (4) 4/2/09 experimental start Yes 00(4) Yes, but the protocol was not provided. Metabolite M1 1, Lot WH-83-34-28, 98.5% pure Methods (see below, from the sponsor) Concentrations in definitive study: Basis of concentration selection: FormulationNehicle: Incubation sampling time: Reference ID: 3399188 Plate incorporation 8, 40, 200, 1000, or 5000 pg/plate Pre-incubation 8, 40, 200, 1000, or 5000 rig/plate, except TA1537 at 1.6, 8, 40, 200, 1000, or 5000 pg/plate An initial range-finding experiment was conducted by the plate incorporation method using TA98 and WP2 uvrA in the presence and absence of 89 mix. Duplicate plates were used. M11 concentrations of 1.6, 8, 40, 200, 1000, or 5000 mg/plate were tested. After approximately 24 hr incubation at :t the plates were examined for the presence of a complete bacterial lawn. (DMSO) 66 hr at :t 89 fraction (from Aroclor 1254-treated rats) from (low batch 2375 64 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Bacterial Strains: Positive Controls: Results The cytotoxicity range-finding assay suggested that the maximum concentration was adequate (see sponsor's table, below). There was one issue with the validity of the assay, as 2-AA was the sole indicator of the activity of the S9 mix; however, the study used a commercially prepared S9. In the plate incorporation and pre-incubation experiments, no drug-related increases in revertant numbers were reported. See the sponsor's summary tables, following. M11 was not a bacterial mutagen, under the conditions of the test. 65 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Table - Mean Number of Revertants Per Plate - Experiment 1 - Plato Incorporation Concentration of test article (uglplate? 5-9 Strain mix 0 3 40 200 1000 5000 PC WV TA1535 0 14.? 16.3 13.0 51'] 10.0 19.? 19.3 "146.? 0 9.3 St] 8.0 5r] 241.0 TA98 0 27.? 24.3 20.? 22.0 27.0 30.0 94.? TA10-0 0 10?.3 105.31 102.0 98.3 90.3 802.3 WPZ WM 0 33.0 29.0 30.3 21.3 34.3 20.? Concentration of test article 5-9 Strain mix 0 8 40 200 1000 5000 PC WV TA153S 10 9.3 13.3 15.3 9.3 11.3 144.? TA1537 10 11.3 8.0 8.3 11.0 11.? 9.3 121.? TA98 10 32.0 28.3 30.0 27.? 33.0 31.3 103.3 TA100 10 115.0 94.3 95.0 83.0 89.0 25.3 505.3 WPZ nvrA 1.0 3?.3 44.0 37.0 40.0 38.0 21.0 100.? PC: positive control, srl; reduced background bacteria] lawn. Table 2 - Mean Number of Revertants Per Plate - Experiment 2 - Pro-Incubation Concentration of test article 8?9 Strain mix 0 1.6 8 40 200 1000 5000 PC Viv TA1535 0 117 NT 19.0 16.0 16.? 13.3 19.0 T863 TA1537 0 6.3 . 9.0 8.3 11.? 15.? 86.0 TA98 0 26.3 NT 21.? 22.7 25.3 28.0 25 .0 10?.0 TA100 0 103.0 NT 98.3 102.?r 100.0 100.0 112.? ?140.? . WM 0 25.7 NT 25.0 26.3 29.0 31.7 27.? 1422.3 I Concentration of test article (ng/plate) S-9 Strain . mix 0 1.6 8 40 200 1000 5000 PC WV TA1535 10 12.0 NT 12.3 11.3 12.7 13.7 11.0 229.3 10 13.3 NT 17.0 13.? 10.3 10.3 11.0 122.3 TA98 10 29.? NT 32.3 33.0 30.3 31.3 25.7 723.? TA100 10 133.0 NT 132.0 113.0 118.7 128.3 108.0 2324.0 WPZ uvrA 10 40.? NT 40.0 35.3 38.0 42.0 31.3 283.3 PC: positive control. NT: not tesled. 66 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. In Vitro Assays in Mammalian Cells Study Title: IN VITRO MAMMALIAN CELL CYTOGENETIC TEST: CHINESE HAMSTER OVARY CELLS Study no.: UN0004 Study report location: EDR (b) (4) Conducting laboratory and location: Slide scoring: Date of study initiation: GLP compliance: QA statement: Drug, lot #, and % purity: 4/7/09 experimental start (b) (4) Yes, pg 5 Yes (except slide scoring, (b) (4) QA), but the protocol was not provided. Metabolite M11, lot WH-83-34-28, 98.5% pure 67 Reference ID: 3399188 (b) (4) NDA 205-677 Melissa K. Banks-Muckenfuss, Methods (see the sponsor's tables, below) Cell line: Basis of concentration selection: FormulationNehicle: Incubation sampling time: Positive Controls CHO cells Maximum concentration by solubility limit A range-finder tested M11 concentrations of 1.6, 8, 40, 200, or 1000 mg/mL. The 5000 mg/mL concentration was not tested due to excessive precipitation. (DMSO) Approximately 3 or 15 hr treatments Harvested at 15-39 hr 89 fraction (from Aroclor 1254-treated rats) from batch 2375 Metabolic Activation Positive Control Final Concentration(pg/ug/mL None MMC 0.2 and 0.25 rig/ml. Treatments (3 experiments) Experiment 1 Treatment Duration Metabolic Activation Serum Content 3 hours 10 v/v S-9 mix added at 10 None 3 hours None None 1.5 cell cycles None 10 v/v BS Experiment 2 time Harvest time Metabolic Activation mihofii?s) (hours after treatment began) con en Harvest 1 Harvest 2 IO v/v S-9 mix None 3 15 39 None 10 WV 15 15 39 Experiment 3 Tr tment time Harvest time Metabolic Activation erfmt 6111mm) (hours after treatment began) con en Harvest 1 Harvest 2 10 v/v S-9 mix None 3 15 39 68 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study Validity The mitotic index (MI) for each culture was determined from a minimum of 1000 cells; the doses used for scoring were based on cytotoxicity of 50-75%. Cytotoxicity was based on colony forming capacity (CFC). One hundred cells were counted from each culture for scoring. Results M11 was clastogenic in the presence of metabolic activation. Although the clastogenic response was not consistent throughout experiments, it was reproducible and dosedependent. M11 was negative in experiment 1, but was positive in experiments 2 and 3 (particularly 2nd harvest). The sponsor described the clastogenic response as "biologically relevant and reproducible" at the second harvest in the presence of S9 mix, and indicated that "M11 has the potential to delay cell cycling, which is confirmed by the potential increases in polyploidy, endoreduplication and hyperdiploidy (also observed in the absence of S-9 mix following 1.5 cell cycle exposure)". Generally, M11 was not clastogenic in the absence of metabolic activation. BEST AVAILABLE COPY 69 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, a, Che-n- Muted $6 ?In It. and- all all ?;Lm-p Mimi-ml- mlulw Mpg-[6.6.5.6.3&0 36.0 A 0 II 7.6.60 2 0 0 I 0 2.0 1.0 21!) 100 6 5.6.0 0.0 1000 100 I 5.5.36.0 36.0 mm 0 100 6.5.6.6.0 4.0 I000 I00 6 5.3.40.0 36 0 - Band-Wen: Table 8 - Experiment 2 Colony Forming Capacity Relative CFC Relative CFC Presence of metabolic activation Absence of metabolic activation Dose pg/mL 3 munch, 1.500 Irettmem 1000 130 10 134 1500 97 Y. 200 I21 1700 58 1000 7 1750 55 I 800 50 1500 48 96 1850 41 . I900 24 2000 0 A 70 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, mun h! I 3 $3 Cha-o-o mm swan-In nun-0- tun-I Haul-- hull-aunt ?ml-bun200 200 3.I0 0 0 5 0 0 0.0 0.0 I000 200 I500 zoo 0.25 50 I0 2.3&0 363.I000 I00 I500 100 400 400 939?3 0 200 3 4.4.0.0 0.0 I000 100 no 0 0 2 0 00 0.0 I500 I00 0 1.3.30 I 5 0 I 0 32.0 32.0 - *8 Gun-0- Nun-Hint:- $00.Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, m? Midli- I?m dual-anu- ?clan-om nan-1mg- D. 0 400 6 4.4.I500 200 7 3.14.0 14.0 (IA 10 125 27 2.21.6 214.0.0 0.0 15m 11!) 3.3.15.0 15.0 CPA 10 100 19 2.19.0 19.0 (Mu. II 0 200 4 4.4.1.0 1.0 1500 100 5 4.5.0 5.0 1000 100 13 2.13.0 13.0 CM 10 25 8 2.32.0 32.0 CPA - ?I?cInl Ala-rut mm ?at-hm wan-[mun- Cached Dan 0 400 5 5.1.3 1.3 1000 200 28 3.14.0 14.0 Oahu 18.0 103.10.0 10.0 72 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAILABLE Table ll} - Experiment 3 Celeny Ferming Capacity COPY Relative CFC ?2?0 D0 gme Presence ef metabelie activation SE 11 3 beer treatment 1400 03 '50 1500 34 '50 115W ll '30 Wilt} 9 Va 1500 5 Experiment 3 Aberratjen Data Presence 01f Metabolic 1"1 Harvest Dim Tm] Aberrant 151mm: curt-020412022 M??plc ?imu'iml aberra'liun: eel]: ih?'ra?nm Gaps ulle tEll? Ilnd? deletlan exchange deletl-nn I exchange Pal} I Emil: I Hyper will: gaps 11214110103505 Data 0 400 5 5.2.0.5 0.5 15?? 200 5 2.1.20.5 . 20.5 501111215291. 9 200 2 5.1.0 1.0 14?? 100 1 5.1.0 1.0 150'? 100 I 2.1.52.0 52.0 0010.154 0 '3 200 5 4.5.0 2.0 14?3? 100 0 2.0.0 0.0 15?? 100 2 5.1.15.0 15.0 CPA Cyclephusphamide Experiment 3 Aberration Data Presence 0fMetal501ie Aetivatien Harvest 1105: Total Aberrant Mitotic Gap: Chmmalid Chremseme mug]; 37410112214201 aberrations cells 21r_i_tl2 abern?em perm 55115 52118 Ind? 0511:5155 exchange 051201511 mhmge aberration: 1501;.- En?n H5159 with gape I ?51th gaps 4351-1100554 Data 0 400 15 4.5.5 1.5 1131] I5 3.212.5 I500 125 20 2.15.0 15.0 1:515:15 .4 0 200 5 4.5.0 1.0 1400 100 5 2.150 5.0 5.0 1530 ICICI 12. 1.91LED l?l? 002555: 101] 9 4.55 fr 3 CI 4.5 1.5 1400 100 3 3.512} 2 2 - 1 3.0 Ill 1500 25 2 5.52.0 15.0 73 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Appudlx'l -Hluorthonu'olDau(2003m2007) Raul: 130mammo-m unsung. mean 193240.9 men?25D 145.3 0.3 1.5 .1.3 0.6 0.3 0.5 0.7 0.0 0.7 0.1 0.3 0.3 -0.6 191 m+2s13 242.3 5.6 5.9 3.9 15 0.4 0.7 1.9 0.0 1.6 0.1 1.6 5.6 2.9 mi?mum 692002.5 7.0 4.0 men 34.7 41.5 2.1 5.1 21.4 262 1.6 2.6 0.5 03 0.0 0.4 415 39.1 :1 SD 262 12.2 1.1 6.4 11.9 13.0 2.9 3.6 1.3 1.0 0.0 1.3 122 12.1 mun-28D .17.7 17.0 0.0 .7.7 .2.4 0.1 4.1 .4.7 -3.2 -1.7 0.0 -2.1 17.0 14.3 We '25 mm+2so 37.1 66.0 4.3 17.9 45.2 52.3 7.3 9.9 4.1 2.2 0.0 3.0 66.0 63.4 minimum 60 3.6 02 0.0 0.0 3.6 0.0 0.0 0.0 0.0 0.0 0.0 3.6 3.6 mximnm 100.0 72.0 6.1 32.0 52.0 63.0 12.0 20.0 16.7 5.0 0.0 30 72.0 63.0 11.11- mun-m hum-I a. H396 1942 20.7 1.7 0.9 179 men-230 147.3 0.3 1.4 -1.7 0.3 .014 0man-+230 2406 6.0 6.0 4.5 1.3 05 0.7 1.3 0.0 1.3 0.2 1.3 6.0 3.1 1000.0 0.0 2010.0 3.0 0.5 3.0 10.0 4.5 mm 51.9 33.3 2.0 2.3 11.0 2733.5 31.5 SD 363 14.3 1.1 4.4 9.0 13.9 2.7 33 3.6 1.4 0.0 1.1 14.3 15.0 man-230 .307 3.3 0.3 .59 .71 .101 .44 .42 0.6 -2.2 0.0 .17 3.3 1.5 70 means!) 124.6 632 4.2 115 29.0 653 6.5 110 7.9 3.4 0.0 2.5 632 61.6 uni-1mm 1 1.0 6.0 0.1 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 6.0 3.0 100.0 76.0 5.0 24.0 52.0 96.0 20.0 120 29.4 7.3 0.0 4.0 76.0 63.0 men 35.7 39.0 1.9 5.7 192 22.5 3.3 22 05 05 0.0 0.1 39.0 35.9 so 27.1 13.7 1.0 6.1 11.1 11.9 4.0 3.4 1.1 1.3 0.0 0.4 13.7 13.5 man-230 -13.5 11.6 02 -6.4 0.0 .13 4.3 4.7 .1.9 .30 0.0 0.3 11.6 3.9 ?7 mozso 90.0 665 4.0 17.3 415 46.3 1116100.0 64.0 4.4 24.0 43.0 44.0 150 14.3 5.0 105 0.0 2.0 64.0 625 74 Reference ID: 3399188 NDA 205-677 8 Melissa K. Banks-Muckenfuss, Ph.D. Carcinogenicity Study title: VEC-162: CARCINOGENICITY STUDY BY ORAL GAVAGE ADMINISTRATION TO CD RATS FOR 104 WEEKS Study no.: TAJ0001 Study report location: EDR (b) (4) Conducting laboratory and location: Date of study initiation: 2/9/06 (b) (4) GLP compliance: Yes QA statement: Yes Drug, lot #, and % purity: VEC-162, batches 800156800, 800156790, 800167150, and 800167140, purity 99.1- 99.7% CAC concurrence: Yes (see ExecCAC minutes dated 8/23/99). Recommended doses were 20, 100, and 250 mg/kg. Key Study Findings FDA Statistically Significant Tumors: Uterus (F, 250 mg/kg): endometrial AC Sponsor’s Drug-related and Significant Tumors: Liver (M & F, ≥ 100 mg/kg): adenoma (+ carcinoma in M) Uterus (F, 250 mg/kg): endometrial AC and SCC Uterine Cervix (F, 250 mg/kg): SCC Adequacy of Carcinogenicity Study Overall, the study was adequate. The study used the doses recommended by the ExecCAC (see minutes dated 8/23/99). Mortality was increased in HDF at week 102; therefore, dosing was suspended in this group. All male groups were terminated at week 103 because of low survival in the control group. Appropriateness of Test Models The rat is a standard species used for 2-year carcinogenicity bioassays. Evaluation of Tumor Findings The only tumor reaching statistical significance was the uterine endometrial adenocarcinoma in HDF (see FDA Biostatistics review by S. Thomson). However, the sponsor reported drug-related tumors in liver (hepatocellular adenoma [and carcinoma in M], ≥ MD, M and F), uterus (endometrial adenocarcinoma & squamous cell carcinoma, HD, and uterine cervix (squamous cell carcinoma, HD). According to the sponsor’s statistical analysis, these tumors were statistically significant for trend, and for pairwise comparison at HD (liver adenoma in M, uterine endometrial adenocarcinoma). 75 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Methods Frequency of dosing: Dose volume: Route of administration: Formulation/Vehicle: Basis of dose selection: QD 2.5 mL/kg PO, gavage 100% PEG-400 MTD. At 100 and 400 mg/kg in a 1-month toxicity study, hyaline droplet nephropathy was present that did not resolve in males following a 1-month recovery period. In a 6-month toxicity study, one female given 500 mg/kg was sacrificed in moribund condition after 3 doses due to severe clinical signs, including seizures and labored respiration. The incidence and severity of progressive nephropathy were increased at 500 mg/kg. Therefore, 250 mg/kg was selected as the HD, with the LD and MD selected at appropriate intervals. Species/Strain: Crl:CD® (SD)IGS BR rats (b) (4) Age: 22 to 28 days of age Weights: M: 70-100 g, F: 60-90 g Animal housing: 5/cage Paradigm for dietary restriction: n/a, ad libitum food and water Interim sacrifice: All males were sacrificed at week 103 (treated for 102 weeks) based on mortality in the control group. Deviation from study protocol: The sponsor identified a total of 10 protocol deviations; the sponsor indicated that none of them were considered to have affected the integrity of the study. 76 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results Mortality [2x/daily] There was no statistically significant treatment-related effect on mortality, although survival was slightly lower in treated (particularly HD) females. Dosing was suspended for HDF during Week 101/102 because of low survival numbers; all female groups were terminated at 104 weeks. Survival in the control male group was reduced during Week 102, and all male groups were terminated at Week 102/103. The summary data are presented below (from the sponsor). There was an increased incidence of renal disease as cause of death (COD) in VEC-162-treated males. In HDF, there were increased incidences of “poor clinical condition,” endometrial carcinoma, and uterine squamous cell carcinoma as COD (deaths between Weeks 79 and 95; 4 uterine adenocarcinoma, 2 uterine SCC, 1 uterine cervix SCC). 77 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Clinical Signs [2x/daily, with detailed physical exams weekly, and detailed observations. In addition, detailed observations were recorded daily during Week 1, twice weekly during Weeks 2-4, once weekly during Weeks 5-13, biweekly during Weeks 14-52, and once every 4 weeks thereafter at the following times: immediately before dosing, immediately after dosing, on completion of dosing of each group, between one and two hours after completion of dosing of all groups, and as late as possible in the working day.] The sponsor reported "underactivity" in MDF, HDF, and HDM, beginning in Week 10/11 at approximately 30 minutes post-dose. This sign was occasionally reported at LD and in MDM. Chin/snout rubbing and increased salivation were also increased in tasimelteon-treated animals. Irritable, aggressive, vocalization, rales, irregular breathing, eyelid(s) partially closed, post salivation staining, hair loss, and piloerection were also occasionally reported. Observations of swollen, reddening and/or black spot(s) on tail were slightly increased in HDM. Few signs were recorded as “associated with dosing”, which is unusual for a study of such long duration. Although not reported by the sponsor, convulsions were observed in a few animals (1 ConM, 3 LDM, 2 MDM, 1 LDF, and 3 HDF); the number of occurrences was not doserelated in males (1, 9, 2, and 0 in ConM, LDM, MDM, and HDM) but was increased in HDF (0, 1, 0, and 6 in ConF, LDF, MDF, and HDF). The severity of the sign was notable in some animals (leading to death/euthanasia); some of the events were noted 78 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. to be of prolonged occurrence or frequency. Notably, there was some internal inconsistency in how this sign was managed by the laboratory. For example, MDM #199, LDF #386, and HDF #577 were sacrificed due to “prolonged/frequent convulsions,” with comments indicating the animals experienced convulsions lasting 30 to 35 seconds, while LDM #104 and HDF #559 experienced multiple convulsions of durations ranging from 30 seconds to 3 minutes. The reason for the discrepancy is uncertain. The sponsor reported that the group distribution, multiplicity, and mean onset time of palpable swellings were unaffected by treatment (see sponsor's table below). Body Weights [Pre-dose, D1, weekly for 12 weeks, and then every four weeks] Generally, the body weight gains of treated males and HDF were slightly lower than those of their respective controls (see the sponsor's summary data table, below). Overall, terminal body weights did not reflect a large difference. Average body weights at Week 102 were 96%, 96%, and 93% those of control males in the LD, MD, and HD groups, respectively. In females, average body weights at Week 104 were 108%, 97%, and 95% those of controls in the LD, MD, and HD groups, respectively. However, during Weeks 76 through 88, the average body weight in HDF fell to approximately 87% that of control females. For group mean body weights over the study, see sponsor's Figure 2 below. 79 Reference ID: 3399188 NDA 205-677 Group mean bodyweight gain elissa K. Banks-Muckenfuss, Group and sex 41" Dosage (Why100 250 Week 0-28 468 440? 439? 456? 204 205 199 188? As 3 of Group Week 28?80 150 154 141 102Group Week 0-102/104 572 545 S45 513Group Signi?cant when composed to Group 1: p<0.05; p<0.01 FIGURE 2 Bodywelgm - gimp mn values venus pemd of u'eatmem - Group 2 3 4 Compound Control VEC- 162 VEC-IGZ VEC-162 Dose (mg/kg/day) 20 100 250 m) .. Period of autumn (wedd 80 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Food Consumption [Weekly for 12 weeks, and then every four weeks] The sponsor reported that food consumption was unaffected by treatment. Food consumption values over 100 weeks of treatment were 101-103% and 105-106% of their controls in treated males and females, respectively. Generally, food consumption appeared slightly increased in treated females towards the end of the dosing period (~15%; approximately Week 92 onward). Ophthalmology [Weeks 52 and 100] The eyes of 20 animals/sex from the control and HD groups were examined by (b) (4) binocular indirect ophthalmoscope No drug-related effects were reported. Clinical Pathology [Week 52 (hematology only), Week 100 (M) or Week 101 (F)] Blood samples were taken from all animals. Limited hematological parameters were measured, including: RBC count, wbc count with differential, PT, and APTT. Clinical chemistry were evaluated (20/sex/gp), including: ALP, ALT, AST, Bili, Urea, Creat, Gluc, Chol, Trig, Na, K, CL, Ca, Phos, Total Prot, Alb, globulins (a1, a2, Beta, Gamma), and A/G ratio. The sponsor's summary data tables for animals dying during the treatment period state that the results are "… for information only, samples analyzed outside storage stability limits." 81 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. The sponsor reported slight reductions (≤ 5%) in RBC parameters in HD animals (low RBC count only in F) during Week 52, but not at Week 100/101. MCHC was very slightly reduced in treated males at Week 52, and was reduced (~3%) in HDF at Week 101. In MD and HD females in Week 52, neutrophil counts and PT were increased very slightly (~2%). At Week 100, platelets were increased slightly (~20%) in HDM. At Week 100/101, increased total cholesterol (as much as 35-50%) was reported at MD and HD, and was associated with increased triglycerides (as much as 65-80%) in MDM, HDM, and HDF. Alkaline phosphatase and AST were dose-dependently reduced (as much as 33%) in treated males; MD and HD females showed only a 10-25% reductions. Total protein was increased slightly (4-7%) at MD and HD; a1-globulin (13-14%, MDF, HDF, HDM) and beta-globulin (10%; MDM and HDM) showed slight increases. Changes in electrolyte levels were reported at MD and/or HD; reduced sodium (1%) and phosphorus (as much as 12%) were reported for MD and HD males, and reduced sodium and chloride (~1-2%) and increased phosphorus (~12%) were reported for HDF. Creatinine and urea were reduced 10-20% in HDF. Urinalysis [Week 100 (M) or Week 101 (F)] Urine samples were collected from 20 animals/sex/gp (16:00 hr until 08:30 hr the next day). Appearance, volume, pH, specific gravity, protein, U-Na, U-K, U-Cl, Gluc, Ket, Bili, UBld, and microscopy were recorded. The sponsor reported a few urinalysis changes in MD and/or HD males. A dose-related increase in urine protein (~50-75%) was observed in tasimelteon-treated males; specific gravity was slightly increased (~2%) in HDM. Generally dose-related increase in urine sodium (~30-90%) and chloride (~50-90%) were observed in tasimelteon-treated males; increased urine sodium was also observed in HDF (~25%). Although not noted by the sponsor, average urine volume was slightly increased in females (~20%); urine protein was reduced ~20% in HDF. The incidence of trace amounts of urine ketones was increased at HD (M > F). Organ Weights Adjusted liver weights were increased in tasimelteon-treated males (as much as 30%), MDF, and HDF (as much as 16%). Adjusted kidney weights showed increases up to 13-14% in treated animals (dose-related in males). Adrenal weights were increased in tasimelteon-treated males, although the sponsor indicated that these increases were driven by individuals in each group. Thymus weights were slightly increased in MDM and HDM. Gross Pathology Macroscopic findings were reported in the liver and uterus. Enlarged liver, liver masses, and pale areas on the liver (M) were observed at MD and HD. Uterine masses were reported in HDF. See the sponsor’s summary table, below. 82 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Group distribution of macroscopic findings related to treatment Group/sex 41" Dose (mg/Radar100 250 Enlargement Masses Pale areas Utems Masses Unscheduled; - Terminal kill; A - All animals In addition to the ?ndings reported by the sponsor, there were ?ndings of low or increased incidence of uncertain toxicological importance in several tissues, including adrenal, brain, eye, heart, kidney, liver, lung, spleen, mammary gland, pituitary, thyroid/parathyroid, reproductive organs and associated glands; these are listed in the reviewer?s table, below. A few general ?ndings, such as hair loss, thinness, and others, also appeared increased. A number of nodes showed low but increased incidences of changes such as enlarged, congested, and/or cystic in tasimelteon- treated animals. Organ/ Finding ConM LDM MDM HDM ConF LDF MDF HDF Adrenal Enlarged Brain Depression from Pituitary mass Dark areas Eyes Opaque Heart Enlarged Thin ventricle walls 83 Reference ID: 3399188 Reference ID: 3399188 NDA 205-677 Melissa K. Banks?Muckenfuss, Organ/ Finding ConM LDM MDM HDM ConF LDF MDF HDF Kidneys Enlarged Granular Pale Pale areas Dark Pelvic Dilatation Liver Dark 0 1 0 4 4 16 Lobes Enlarged Firm Lobular pattern accentuated Median cleft pale areas Lung Congested Dark Dark areas Spleen Enlarged Swollen Mammary Pituitary Mass Parathyroids Enlarged 0 1 2 0 0 0 Thyroids 1 3 3 2 2 2 Masses Preputial _glands Mass 0 0 0 1 Dark areas 2 7 2 3 Prostate Mass 1 1 2 3 84 NDA 205-677 Melissa K. Banks?Muckenfuss, Organ/ Finding ConM LDM MDM HDM ConF LDF MDF HDF Seminal Vesicles Oedematous 0 1 0 3 Testes Masses 2 1 3 6 Clitoral Glands Masses 3 13 13 9 Uterus Abnormal 3 1 6 Contents Fluid 2 3 3 5 distension Ovaries Cystic 3 2 2 5 Masses 0 1 3 Periov. sac 1 2 4 5 distension Vagina Abnormal 3 2 4 5 contents Stomach Corpus thickened Thick Limiting Ridge Antrum white nodules Skin, Hair loss General(moderate) hair loss Generalanimal thin Thorax Contained ?uid 0 0 1 2 2 4 Abdomen Masses Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Histopathology Adequate Battery Separate Signed pathology report Peer Review Yes (see Appendix 1) Yes (but limited version) (b) (4) Yes, from and a consultant, not identified Neoplastic The sponsor reported drug-related neoplasms in the liver (M & F), uterus, and uterine cervix. Increased neoplasm incidences were also suggested in ovary and mammary gland in HDF. In males, increased incidences of neoplasms were also observed in pituitary, testes, and skin. Liver The incidence of hepatocellular adenomas was increased at MD and HD in both sexes (9.2 and 12.3% for males and 9.2 and 10.8% for females respectively; carcinomas were also seen in MDM (but this incidence, 4%, was within the relevant historical control range (study range: 0-5% in males [mean= 1.88%, N=744])). The incidence of hepatocellular adenomas was above the historical background range from contemporaneous studies at these laboratories (study range: 0-5% in males [mean= 2.15%, N=744] and 0-4.6% in females [mean= 1.34%, N=744]). See the sponsor’s summary table, below. Also see the biostatistical evaluation by FDA reviewer S. Thomson (Table 3 excerpt, below). The sponsor considered the development of these hepatic tumors to be either a secondary response to microsomal enzyme induction (which would not be relevant to humans) or “increased hepatocellular turnover.” 86 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh Male Rats LIVER # Evaluated 65 65 65 65 Adj. # at Risk 43.7 36.1 49.9 43.2 HEPATOCELLULAR ADENOMA 2 2 6 8 .0123 .0444 .1802 Adj. # at Risk 43.7 36.1 50.7 43.2 Hepatocellular Adenoma/Carcinoma 2 2 8 8 .0145 .0444 .0746 Female Rats LIVER # Evaluated 65 65 65 65 Adj. # at Risk 45.4 39.6 40.5 40.4 HEPATOCELLULAR ADENOMA 2 1 6 7 .0087 .0539 .0980 plow vsVeh .6222 .6222 .8511 Female (and Male) Reproductive Organs Neoplasms and/or other proliferative effects were seen in uterus and uterine cervix, and possibly ovary and mammary gland in females. The sponsor stated that similar effects were not observed in mice, and posited the effects were due to a “species-specific effect of unknown etiology on aging female rats.” The sponsor attributed the uterine tumors to a rat-specific effect on prolactin, which they suggested would lack human relevance. There was limited evidence of hyperplasia and neoplastic effects in male reproductive organs (i.e., see hyperplasia and interstitial cell adenoma in testes, following). Uterus The incidences of endometrial adenocarcinoma and uterine squamous cell carcinoma were increased in HDF; these tumors were identified as drug-related causes of death by the pathologist. See the sponsor’s summary table, below. The sponsor stated that the incidences of endometrial adenocarcinoma and squamous cell carcinoma (17.2 and 4.7%, respectively) were above the relevant historical control range (study range for adenocarcinoma 0-3.1% [mean= 0.67%] and for squamous cell carcinoma 0-0% [N=744]). Also see the statistical evaluation by FDA biostatistical reviewer S. Thomson (Table 3 excerpt, below). The sponsor indicated that uterine tumors are common in rats treated with dopamine agonists, which reduce prolactin levels; this mechanism is not believed to be relevant to humans. The sponsor further argued that chronic activation of melatonin receptors in the pars tuberalis (cf. Morgan and Williams, 1996) may similarly lower prolactin levels in rats; however, the sponsor provided no data (i.e., serum prolactin levels) to support this assertion. 87 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh UTERUS # Evaluated 64 65 64 64 Adj. # at Risk 45.3 39.7 39.3 40.6 ENDOMETRIAL ADENOCARCINOMA 2 2 2 11 .0002 .0035 .6360 Adj. # at Risk 45.3 39.7 39.3 40.6 Endo. Adenoma/-Adenocarcinoma 3 2 2 11 .0005 .0102 .7722 [Adj. # at Risk 45.5 39.6 39.5 39.9 SQUAMOUS CELL CARCINOMA 1 0 1 3 .0543 .2558 .7160 plow vsVeh .6360 .7722 1 ] Uterine Cervix Two HDF showed squamous cell carcinoma of the uterine cervix. The incidence (3.1%) was above the historical background range from contemporaneous studies (range 0-0% [N=744]). There was a statistically significant positive trend. See excerpt from biostatistical review by S. Thomson (below). 88 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh plow vsVeh UTERINE CERVIX # Evaluated Adj. # at Risk SQUAMOUS CELL CARCINOMA . 64 65 64 64 45.2 39.6 39.3 39.3 0 0 0 2 .0568 .2126 . Additionally, the observed squamous cell carcinomas in uterus and in uterine cervix should be combined for evaluation, yielding an incidence of 1, 0, 1, and 5; this incidence was considered positive considering that the tumors would be considered rare (the sponsor’s provided historical control incidences are zero in both tissues). See excerpt from the biostatistical review by S. Thomson, below. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh Uterus(w/cervix) # Evaluated Adj. # at Risk SQUAMOUS CELL CARCINOMA 64 65 64 64 45.5 39.6 39.5 40.8 1 0 1 5 .0059 .0765 plow vsVeh .7160 1 Ovaries Two HDF females showed a benign sertoliform tubular adenoma. The sponsor indicated that the incidence (3.0%) was within the background range for this tumor (study range 0-8.3% [mean= 1.08%, N=744]). The combination of ovarian stromal tumors was assessed. See the FDA statistical reviewer’s assessment below (Table 3 excerpt). Although the trend test was close to reaching statistical significance, no pairwise comparison was found significant. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh OVARIES # Evaluated 64 65 64 63 Adj. # at Risk 45.2 39.6 39.3 38.2 SERTOLIFORM TUBULAR ADENOMA 0 0 0 2 .0546 .2066 . Adj. # at Risk 45.2 39.6 39.4 38.2 Thecal Cell Tmr/Serto.Tub.Adenoma 0 0 1 2 .0525 .2066 .4643 89 Reference ID: 3399188 plow vsVeh . . NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Mammary Gland Although considered an incidental finding by the sponsor, the incidence of mammary adenocarcinoma was increased in HDF (and there was a slightly increased incidence of mammary adenoma in HDF). The sponsor noted that the HDF incidence (27.7%) was, however, within the historical background range from contemporaneous studies (study range 9.4-38.3% [mean 20.6%, N=742]). See the FDA statistical reviewer’s assessment below (Table 3 excerpt). The sponsor also stated that there were no treatment-related increases in associated hyperplasia or other non-neoplastic findings to suggest a proliferative response in the mammary gland. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh Female Rats MAMMARY # Evaluated 64 65 64 65 Adj. # at Risk 47.6 42.4 43.5 44.8 MAMMARY ADENOCARCINOMA 10 10 12 18 .0146 .0356 .3134 plow vsVeh .4864 Of uncertain toxicological importance, a few neoplasias were observed at low incidence and/or with unclear dose-relatedness for a few tissues in males (see excerpts from the sponsor’s summary table, below). Where appropriate, hyperplastic changes in the same tissue were also noted. The FDA statistical reviewer’s evaluation is excerpted from Table 3, below. 90 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Table 3. Potentially Statistically Significant Neoplasms in Rats Sex/ Incidence Significance Levels organ/ Veh Low Med High ptrend phigh pmed ____tumor_________________________________________________________vsVeh vsVeh Male Rats PITUITARY # Evaluated 63 65 65 64 Adj. # at Risk 47.6 45.6 55.0 46.9 ADENOMA, PARS DISTALIS 19 28 30 23 .4822 .2361 .1104 [TESTES # Evaluated 65 65 65 65 Adj. # at Risk 43.3 35.7 49.2 43.2 INTERSTITIAL (LEYDIG) CELL ADENOMA 3 1 4 6 .0589 .2417 .5735 SKIN # Evaluated 65 65 65 65 [Adj. # at Risk 43.1 35.7 50.3 43.4 FIBROMA 1 0 5 3 .0920 .3080 .1404 Adj. # at Risk 43.1 35.7 50.3 43.9 Fibroma/Sarcoma NOS/Fibrosarcoma 1 0 5 4 .0407 .1800 .1404 plow vsVeh .0296 .9135] 1] 1 (Note: The FDA statistical reviewer did not include testes and skin fibroma in his assessment of potentially statistically significant neoplasms; these data were taken from Table A.2.1 in the body of his review to parallel this reviewer’s discussion.) Non-neoplastic The sponsor reported non-neoplastic histopathology in the liver (both sexes), kidney (F), ovary, and uterine cervix; other organs showing non-neoplastic alterations include kidney (M), spleen, uterus, mammary, testes, and pituitary. A few other low incidence alterations or alterations with slightly increased incidence were observed in a number of other organs. Liver Several alterations were noted in the livers of tasimelteon-treated males and females. See the sponsor’s summary table, below. Dose-related increases in the incidence and severity of centrilobular hepatocyte hypertrophy were reported in both sexes. The sponsor attributed the hypertrophy to “adaptive” drug-induced induction of hepatic enzymes. Increased incidences of bile duct hyperplasia were also reported in MD and 91 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. HD animals; the sponsor considered this change a response to biliary excretion of the drug and/or its metabolites. In MD and HD males, increased incidences of regenerative hyperplasia, cystic degeneration, and centrilobular hepatocyte vacuolation were reported. In MD and HD females, pigment in the hepatocytes (identified as lipofuscin) was reported, as well as a low incidence of cystic degeneration in tasimelteon-treated females. Additionally, a few other findings of generally low incidence were observed, including: prominent increase in mitotic activity, focal fibrosis and portal bridging fibrosis, portal inflammation, and pigment in macrophages (see excerpts from the sponsor’s summary table, below). The sponsor noted that reduced incidences of basophilic foci were reported for MD and HD females. 92 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Kidney Dose-related increased incidences of cortical tubular pigment (identified as lipofuscin) were reported in tasimelteon-treated females. The incidence of papillary/pelvic epithelial mineralization was reduced in treated females, compared to controls. See the sponsor’s summary table, below. Although no findings were reported in males, a few low incidence alterations were observed (see excerpts from the sponsor’s table, below). A slightly increased incidence of cortical cysts was observed in tasimelteon-treated males. In MD and HD males, cortical tubular basophilia and an increased incidence of tubular hypertrophy were observed. In HDM, increased incidences of pelvic dilatation (also in HDF) and mineralization (cortex, papilla, and/or pelvic/papillary epithelium) were also observed. 93 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, ConM LDM MDM HDM ConF LDF MDF HDF Cortical Cortical tubular basophilia Tubular hypertrophy Pelvic dilatation Mineralization, 2 2 2 4 cortex Mineralization, Papi/Ia 0 1 1 2 Spleen Hemosiderosis was observed with increased incidence in tasimelteon-treated males and HDF. See the summary table below. ConM LDM MDM HDM ConF LDF MDF HDF Hemosiderosis Ovaries Increased incidences of were reported in MD and HD females. See the sponsor?s summary table, below. A few stromal tumors were observed in MDF and interstitial cell hyperplasia and Sertoli cell hyperplasia were increased in incidence. In addition, absence of corpora lutea and focal ?brosis were observed (see excerpts from the sponsor?s summary table, below). Summan? of treatment related findings in the ovaries for all rats Groupi?sex 1F 21" 3F 41" Dose (mg .rkg/day) 0 20 00 250 Cyst(s) Slight 2 5 6 Moderate 2 4 4 5 Marked 0 0 1 0 Total 4 5 10 1 Number of animals examined 64 6? 64 63 94 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Uterus In addition to uterine tumors, glandular dilatation and hyperplasia were observed in MD and HD females (see excerpt from the sponsor’s summary table, below). Uterine Cervix Increased incidences of epithelial hyperplasia and epithelial keratinization were reported in HDF. Also, the incidence of mural/stromal hypertrophy/hyperplasia was slightly increased in HDF (see excerpt from the sponsor’s summary table, below). 95 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Mammary Gland Although some neoplastic changes were observed in females (adenomas [HD], adenocarcinomas [MD and HD]), drug-related increases in the incidences of nonneoplastic alterations were not clearly observed in females; ductular hyperplasia was detected only in one HDF. However, the incidences of ductular dilatation and galactoceles were slightly increased in tasimelteon-treated males and in MD and HD males, respectively. See excerpts from the sponsor’s table, below. Testes There was only a suggestion that interstitial Leydig cell adenomas were increased (HDM and possibly MDM); however, the incidence of interstitial cell hyperplasia was doubled at MD and HD. There was a very slight increase in arteritis/periarteritis. See excerpt from the sponsor’s table, below. Pituitary Although there was only a suggestion of an increase above the spontaneous incidence of adenomas in the pituitary pars distalis in males, the incidence of hyperplasia was clearly increased at HD. Hypertrophy and angiectasis was slightly increased in 96 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, tasimelteon-treated males. Vacuolated cells in the pars distalis were increased in HDM. Drug-related pituitary changes were not observed in females. Pituitary Angloclasls llvperplasla - Pars Dlstalls. Focal Hypertrophy - Pars Dislalls. [50ml Vacuolated Cells - Pars l)lsla ls. l-?ocal Other organs Number Examinedfew tissues showed limited, low incidence changes of unclear toxicological importance (see table below for details). Changes were suggested for the eyes, lung, heart, urinary bladder, stomach, pancreas, thyroid, adrenal, bone marrow, Peyer?s patches, nodes, thymus, skin and integumentary tissues, prostateepididymides. Organ/Finding ConM LDM MDM HDM ConF LDF MDF HDF Eyes Retinal atrophy Lung Alveolar epithelial hyperplasia Pigmented alveolar - - - - 0 0 1 3 macrophages Peribronchiolar aggregates Heart Vascular Mineralization Urinary Bladder Lumina/ Dilatation Stomach Dilated glands Submucosal inflammationglandular Pancreas Acinar cell vacuolation Islet cell hyperplasia ISLET CELL ADENOMA ISLET CELL CARCINOMA Thyroid Follicular Cell hypertrophy Follicular cell atrophy - - - - 0 0 3 3 Follicular cell - - - - 0 0 2 3 97 Reference ID: 3399188 NDA 205-677 Melissa K. Banks?Muckenfuss, Organ/Finding ConM LDM MDM HDM ConF LDF MDF HDF Adrenal Cortical/Cystic/Hemorrhagic Degeneration Sinusoidal Dilatation/Congestion Sternum and Marrow Myeloid hyperplasiamarrow Femur and joint Myeloid hyperplasiamarrow lleum/Peyer?s patches GALTincreased cellularity 4 2 1 0 0 2 Mesenteric LN Dilated/Cystic Sinuses Mandibular LN Dilated/Cystic Sinuses Paracortex, increased cellularity Thymus Epithelial hyperplasia Skin Areas of Prominent Collagen Epidermal ulceration 0 0 3 1 2 2 Pinnae Epidermal hyperplasia Epidermal ulceration Tail Epidermal hyperplasia Lacrimal gland Acinar atrophy Harderian gland Acinar atrophy concretions Clitoral Gland aggregates 14 26 22 22 Prostate Acinar hyperplasia, focal 3 1 Inflammation 4 4 2 7 Epididymides In?ammation 0 3 1 4 98 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Toxicokinetics [Week 4 and Week 26, as well as during Week 96 from 3/sex/gp from main study; see the sponsor's summary table below] The blood samples from Week 96 were not analyzed but were stored frozen for potential future analysis. Throughout the study, plasma samples taken from control animals demonstrated tasimelteon exposures above the LLOQ (0.2 to 10.6 ng/mL); while suggesting that there was some contamination, these levels are well below exposures achieved in the tasimelteon-treated animals (at least 100-fold). Generally, systemic exposure to tasimelteon increased over the dose range, in a less than doseproportional fashion (with the exception of week 4 AUCs). Week 26 exposures were similar to Week 4 exposures (except the AUC increased between Week 4 and Week 26 in LDM). A sex difference in exposure was demonstrated (females > male). See the sponsor's summary exposure data, below. 99 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. (* Note: Livers from the satellite TK animals were harvested and snap frozen in liquid (b) (4) nitrogen. The tissues were then shipped on dry ice to The results of any analyses performed on these tissues were not reported here.) Dosing Solution Analysis [Weeks 1, 13, 26, 39, 52, 65, 78, 91 and 103] Dosing formulations were prepared each week and stored refrigerated (2-8oC) and protected from light. Previously, homogeneity and stability were demonstrated at 7.5 and 100 mg/mL for 24 hours at ambient temperature (nominally 21°C) and for 15 days refrigerated (nominally 2-8°C). The dosing formulations prepared for Weeks 1, 13, 26, 39, 52, 65, 78, 91 and 103 of treatment were analyze. The mean concentrations were within ± 10% of the nominal values, with individual results within 3% of the mean value. 100 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, lnterspecies Parent and Metabolite Comparison For reference, the sponsor provided the following summary table of estimated metabolite exposures in the rat carcinogenicity study (see sponsor?s Table 18 from the Nonclinical Toxicology Written Summary, following.) Table 18: Tasimelteon metabolite levels in rats estimated from tasimelteon plasma BEST levels measured at Week 26 in Study using the AVAILABLE metabolite/tasimelteon AUC ratios determined in Study TAJ0014 COPY (ugh-?mL) Rat Tasimelteon ratioa ratioa ratioa ratioa 20b 6.520 0.25 1.630 2.14 13.953 0.03 196 0.29 1.891 9.900 0.28 2.772 4.21 41.679 0.01 99 0.25 2.475 100 38.800 0.32 12.416 2.51 97.388 0.03 1.164 0.58 22 504 2.000 0.12 5.040 2.45 102.900 0.02 840 0.26 10.920 250 52.500 0.26 13.650 2.08 109.200 0.06 3.150 0.73 38.325 101.000 0.15 15.150 1.75 176.750 0.02 2.020 0.43 43.430 Humanc 363 -- 423 -- 619 -- 353 -- 192 20 mg QD Metabolite/tasimelteon AUC ratios from Study at comparable doses (corrected for molecular weights). Metabolite/tasimelteon AUC ratios for 20 mg/kg were those determined for 25 doses from Study 0014. Data for tasimelteon and M9, M12. and M13 are from Study 10: An Open-Label, Single Sequence Study to Assess the Effect of Multiple Doses of Tasimelteon on the Cytochrome P450 3A4 and 2C8 Enzymes using Midazolam and Rosiglitazone as Substrates. Data for M3 are ?om group 2 in Study VP-VEC-162-1107: An open-label. single-dose. parallel group study to assess the effects of smoking status. age and body size on the pharmacokinetics, safety, and tolerability of tasimelteon in healthy volunteers. 1 01 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study title: VEC-162: CARCINOGENICITY STUDY BY ORAL GAVAGE ADMINISTRATION TO CD-1 MICE FOR 104 WEEKS Study no.: TAJ0002 Study report location: EDR (b) (4) Conducting laboratory and location: Date of study initiation: GLP compliance: QA statement: Drug, lot #, and % purity: CAC concurrence: 3/14/06 (b) (4) Yes Yes, VEC-162, batches 800156790, 800156800, and 800167150, 99.7100.7% pure Yes (see ExecCAC minutes dated 8/23/99). Recommended doses were 30, 100, and 300 mg/kg. Key Study Findings No neoplasms reported by the sponsor. Adequacy of Carcinogenicity Study Overall, the study was adequate. The study used the doses recommended by the ExecCAC (see minutes dated 8/23/99). Appropriateness of Test Models The mouse is a standard species used for 2-year carcinogenicity bioassays. Evaluation of Tumor Findings None. 102 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Methods (See summary design table from the sponsor’s submission, below.) Frequency of dosing: QD Dose volume: 4 mL/kg Route of administration: PO, oral gavage Formulation/Vehicle: 100% polyethylene glycol-400 (PEG400) Basis of dose selection: MTD. In a 3-month dose-ranging study, a single dose of 800 mg/kg caused mortality following severe clinical signs including labored breathing; the dose was lowered to 600 mg/kg without further mortality. At 600 and 400 mg/kg, clinical signs of hypoactivity, prostration, and labored breathing were observed in addition to centrilobular hypertrophy of the liver. Based on lethality at 800 mg/kg and similar signs at 600 mg/kg, a HD of 300 mg/kg was recommended by the ExecCAC. The remaining doses were placed at appropriate intervals, with the LD expected to provide a plasma AUC equal to that anticipated in humans at a dose of 50 mg. Species/Strain: Crl: CD-1™ (ICR) BR mice (b) (4) Age: 37 to 43 days at initiation Weight: M: 22 to 42 g; F: 20 to 33 g Animal housing: 3/sex/cage, unless reduced by mortality or isolation Satellite groups: TK (see Toxicokinetics for details) Deviation from study protocol: The sponsor noted 4 deviations that were not considered to have had an impact on the integrity or validity of the study. 103 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results Mortality [2x/day] There was no clear drug-related effect on mortality. See the sponsor’s summary table and Figure 1, below. Histopathological findings reported as factors contributing to death included: bronchioalveolar carcinoma (tasimelteon-treated females) and lung lesions, laryngeal/tracheal lesions (HD animals), sarcoma NOS (tasimelteon-treated females), and skin/ear lesions (HDM). 104 Reference ID: 3399188 NDA 205-677 FIGURE 1 Percentage survival versus period of treatment Group 1 2 3 Compound ontrol EEC-162 1WEE-16 2 Dose 30 100 4M Survival [itsPeriod of treatment {weeks} 1 continued Percentage survival Tversus period of treatment Group 1 '3 Compound ontrol - 162 Dose {mgt?kgr?dayj CI 30 3 100 4F Survival 20Period of treatment (weeks) 105 Reference ID: 3399188 Melissa K. Banks-Muckenfuss, 4 TEES-162 300 T8 91 104 .1 TEE-1452 300 91 104 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Clinical Signs [2x/daily, with detailed physical exams weekly, and detailed observations recorded daily during Week 1, twice weekly during Weeks 2-4, once weekly during Weeks 5-13, biweekly during Weeks 14-52, and once every 4 weeks thereafter] Observations were performed with following timing on the specified days: immediately before dosing, immediately after dosing on return of the animal to its cage, on completion of dosing of each group, between one and two hours after completion of dosing of all groups, and as late as possible in the working day. The sponsor reported few clinical signs. “Underactive” was reported in most HD animals after dosing, at 30 to 120 minutes after dosing; this occasionally lasted until the end of the workday. MD (beginning week 50) and LD (beginning Week 52) animals were also reported as “underactive” occasionally. Partial closure of the eyelids was reported intermittently in MD and HD animals beginning in Week 26 and was most consistent during Weeks 50 to 52; it was observed at 1 or 2 hours after dosing and sometimes lasted until the end of the workday. Dark eye(s) were observed with slightly increased incidence in tasimelteon-treated females. No drug-related effects on palpable swellings were reported. See the sponsor’s summary Table 3 below for details. Body Weights [Week-1, Day 1, weekly for 12 weeks, every 4 weeks thereafter] Body weight gain was reduced in HD animals (males [ss] > females [nss]) compared to controls. This was most apparent between Weeks 36 and 80; see the sponsor’s summary table and Figure 2, below. Average body weight was reduced approximately 106 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfussthe study. Overall, LDF and MDF tended to gain more weight than the respective control. Group mean bodyweight gain Group and soDose (un?t/day100 300 Week 0-36 13.8 14-3 14.2 12.3 13.0 13.2 13.5 12-5 As a omeup Week 36-80 2.8 3.0 2.7 1.1? 4.9 5.7 5.0 3.5 As a%omeup1 - 107 96 39 - 116 102 71 Week 80-104 -3.3 -2.1 -4.4 -2.2 -4.0 -3.6 -4.6 -2Week 0-104 14.9 13.9 12.7 11.1? 13.7 14.6 15.2 14.0 As a ofGroup 1 - 93 85 74 - 107 111 102 Signi?cant when compared to Group 1: p<0.05; p<0-01 FIGUREZ Bodyweight - group mean values versus petiod of treatment - males Group 1 2 3 4 Compound Control VEC-162 VEC-162 VEC-162 Dose (mg/kg/dayPendofuummedn) 1 07 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Food Consumption [Weekly for 12 weeks, every 4 weeks thereafter] Food consumption was reduced in HD animals beginning approximately Week 16 (slightly earlier in females) compared to controls. See the sponsor’s summary table, below. 108 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Hematology [Week 52, Week 104] Blood samples were obtained from the orbital sinus of 20 main study animals/sex/group at Week 52, and the remaining animals (not sampled at Week 52 were sampled at Week 104. A limited list of parameters were evaluated, including: hemoglobin, hematocrit, erythrocyte count (RBC), total leucocyte count (WBC), neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC), and morphology. The sponsor reported slight dose-related reductions (~4-6%) in red cell parameters in MDF, HDF and/or HDM; hemoglobin and hematocrit (Week 52 only), MCH (both times, HDF), MCHC (both times, MDF and HDF), and MCV (Week 104, HDF and HDM). Platelet counts were increased (~5-15%) in HDF at Week 52 and in MDM, HDM and HDF at Week 104. Organ Weights Terminal body weights were reduced approximately 9% in HDM; average terminal body weights of all other groups were within 5% of their respective controls. The sponsor identified alterations in liver, kidney, and heart, and stated that apparent alterations in brain and adrenal were considered incidental. Average relative liver weights were increased 14% in HDM. Relative kidney and heart weights were reduced 10-17% and 7-10%, respectively, in MDM, HDM, and HDF. Brain weight was slightly reduced in HD animals (~4%). Absolute adrenal weight was reduced in HDM (16%). The relative adrenal weight reduction would be approximately 7% (if adjusted by the 9% reduced body weight); also, if adjusted for the slight increase in average body weight (3%) in HDF relative adrenal weight would be similarly reduced (~8%). A few other noted differences were of unclear toxicologic concern. Average spleen weight was increased (~20%) in HDM. Average relative thymus weight was reduced approximately 10 to 20% in HDF. Average uterus weights were reduced approximately 10 to 15% at MD and HD. Average ovary weights were highly variable, but an increase of 81% was observed at HD (and LD). Gross Pathology The sponsor reported no drug-related macroscopic changes; however, there were a few changes of low or increased incidence. Changes were observed in liver, kidneys, ureters, heart, brain, thymus, reproductive organs, trachea, lung, eye, GI tract, and adipose tissue. In addition to these changes, a number of lymph nodes were noted to be enlarged in tasimelteon-treated animals. 109 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, (301p 1 2 3 4 Como! VEC-162 Dose 0 30 100 300 Camp/sex 1M 2M 3M 4M 11" 2F Tissue and ?nding Numhet Examinett 66 66 66 66 66 66 Liver In?egular DepressionbUneven Thickeoed 2 4 2 8 Thymus Dark Dark muUtuus Abnormal contents - - - - Elongated - - - - 6 8 8 8 Testes DadMama?accid 20 14 - - - - Small Seminal veucles Small Pxepun'al glands Cyst(Cystic 0 0 - - - - Cvsuc' enla?zemmt 0 - - - - Tasha Mammal Lungs blonchi 2 4 I 4 1 4 Optic nerve; Thin Stomach Coupns dark andsMasksPale nubGenenl comments Ahoonml contents :1 tract Pallet of heme] tissueMine: tissue Dark Datk aea(MasksPin-1:: Thickened 1 3 1 10 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Histopathology Adequate Battery Separate, Signed Report Peer Review Yes (see Appendix 1) Yes (limited version) (b) (4) Yes, from and a consultant, not identified Neoplastic The sponsor reported no drug-related neoplasias. The FDA statistical reviewer did not report any statistically significant incidences of neoplasms; his assessment is included the following excerpts from Table A.2.2, below. There were a few low incidence findings of unclear toxicological importance. Lymphoma, leukemia, and sarcoma metastases appeared in numerous tissues in tasimelteon-treated animals. Skeletal muscle and skin (all sarcoma NOS occurred in early decedents), GI tract (stomach and intestines), mammary gland (most acanthomas occurred in early decedents), and reproductive organs showed a few neoplasms. APPEARS THIS WAY ON ORIGINAL 111 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. [Intestines B-adenoma M-adenocarcinoma Combined 0 2 2 112 Reference ID: 3399188 0 0 0 0 0 0 4 2 6 0 0 0 0 0 0 0 0 0 1 0 1] NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Non Neoplastic The sponsor reported drug-related histopathological changes in the liver at ≥MD in males and at HD in both sexes. See the sponsor’s summary table for the incidence and severity of hepatocyte hypertrophy observed, below. Also, a slightly increased incidence of necrosis was observed (see excerpt from the sponsor’s table). In addition to the liver change noted by the sponsor, there were a few low or slightly increased incidence observations of unclear toxicological importance. There was minimal evidence of changes in kidney, urinary bladder, GI tract, immune and hematologic tissues, respiratory tract, integument, Harderian gland, optic nerve, and reproductive tissues. 113 Reference ID: 3399188 Reference ID: 33991 88 114 Colon Epithelial Hypetplasia - Glandular Region Adenonntom Hypaplasia - Region Laxynx ln?matory?xudaehLI-nen Mucosal Hypuplasia Spleen Trachea Tismeandl-?indinl Opdcnervcs Alarm? Acinx Necrosis/WW with In?ammation Gliods Atrophy Seminal Vesicles Mucosal [?oatation/In?ammation Foreign and In?znlnatotv Emulate NDA 205-677 Grow Coupound Dose (mafia/day) 3. Oedana Scab(s) Tail Epidermal Ulnention Hank-?an Glands Acinx Hypapluia Epithelial Hypexplas'n In?amation/Pibtosis Epithelial Hypuplasia In?mation/Ulcm Alveolar Haemorrhage White Pulp - Increased Bronchioloalveolnt Hypelplada In?ammation-in bronchial Lumen In?anmawty Cells acrosis 2 30 3 IN Nnmba? '66 1 IF Nnmba?xamined: MW: 66 Nnmbet?nmined: 61 65 .660? 66 4 8 22 62 0150.791?0 C. Lfow 05 fl??'?000.690[00 001.9067.? 2'glugws 26 5 2 7 24 000 09919 65 61 NumbetExaminedz666666666666 2 59 5 64 3 65 00009 97v0.79 3 63 3 z? 9.7? 1 65 66 24 0 OEZOSIIW 6666 OIISZ 66 0 2 65 0 2F3P4F 66 66 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Toxicokinetics [Week 4 and Week 26] TK was assessed in a satellite group of animals. The sampling schedule was provided by the sponsor (below). Satellite animals were discarded without necropsy following scheduled sampling. APPEARS THIS WAY ON ORIGINAL 115 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Some control samples were found to contain tasimelteon, and were confirmed upon reexamination. A total of three animals (out of 36) were affected, and plasma levels were low (4-20 ng/mL). Absorption was relatively rapid, and plasma exposures increased (generally less than or approximately dose-proportionately, with the exception of LD::MD in Week 4) with increasing dose. See the sponsor’s summary PK table, next page. There were no clear, consistent sex differences in plasma exposure (HDF at Week 4 excluded). There was some evidence of accumulation at LD and at HD between Weeks 4 and 26 (see the sponsor’s table, below). The terminal rate constants and corresponding half-lives could not be calculated for all groups; the sponsor believes that tasimelteon may show time-dependent kinetics. 116 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Dosing Solution Analysis The drug substance was generally stored refrigerated (2-8oC; although batch 800156790 was stored at ambient temperature, 21oC) and protected from light. All formulations were prepared weekly and stored refrigerated (2 to 8°C) and protected from light. The sponsor stated that homogeneity and stability were demonstrated in the rat carcinogenicity study (TAJ0001) at 7.5 and 100 mg/mL for 24 hr at ambient temperature (nominally 21oC) and for 15 days refrigerated (nominally 2-8oC). Formulations prepared for Weeks 1, 13, 26, 39, 52, 65, 78, 91 and 103 were analyzed for drug concentration. Four samples (nominally 1 mL accurately weighed) from all formulations were obtained, from which two samples were analyzed (the remaining two samples were retained frozen). The formulation concentrations were within ±7% of nominal, and individual results were within 3% of the mean. 117 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Interspecies Parent and Metabolite Comparison For reference, the sponsor provided the following summary estimates of M11 exposure (see sponsor’s Table 14 from the Nonclinical Toxicology Written Summary, following). The focus was solely on metabolite M11 (not a major human circulating metabolite) because rats do not produce M11 in vivo. 9 Reproductive and Developmental Toxicology 9.1 Fertility and Early Embryonic Development As suggested by some effects in the toxicity studies, several effects were demonstrated in the reproductive toxicology studies. The fertility study in rats tested 0, 5, 50, and 500 mg/kg tasimelteon (BMS-214778) in males and females, and then females only (separately) after a reduction in fertility was observed. At HD, 3 HDF were euthanized moribund. Treatment with tasimelteon caused a clear reduction in body weight in males, but only a transient effect in females. Two HDM showed small male reproductive organs (i.e., testes and/or epididymides); however, matings with these males resulted in pregnancies within 1-2 days. Irregular estrus cycles were observed in MD and HD females. Although >90% of the animals mated during the first receptive period, increased infertile matings and a reduction in fertility parameters were observed at MD and HD. A re-evaluation was conducted pairing the treated males with untreated females; no effect on fertility was observed. 118 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Study Title: BMS-214778: ORAL STUDY OF FERTILITY AND EARLY EMBRYONIC DEVELOPMENT IN RATS Study no.: Study report location: Conducting laboratory and location: Date of study initiation: GLP compliance: QA statement: Drug, lot and purity: Reference ID: 3399188 Bristol-Myers Squibb Study No.99009 (4) Reference EDR (Live phase- all raw data, dated 1999) BMS, Pharmaceutical Research Institute, Department of Pathology, New Brunswick, NJ (Test article and final report. dated 2919;) (4) 2/11/99 Yes BMS) Yes "We; BMS) BMS-214778, NO31C-214778-01, 99.3% 119 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Methods (see the sponsor's summary of the design, below) Frequency of dosing: M: QD for 4 weeks before pairing until termination F: QD for 2 weeks before pairing until D7 after mating Dose volume: 3 mL/kg Route of administration: PO, oral gavage Formulation/Vehicle: Polyethylene glycol-400, PEG-400 Lot number M18621 Species/Strain: Crl:CD®(SD)IGS BR rats (b) (4) 8-9 weeks of age at initiation M: 292-331 g, F: 208-288 g Amendments/Deviations Amendment 1: Fertility rates were decreased at to/from study protocol: MD and HD in treated female rats. The sponsor added a group of untreated females to be bred with the treated males, in order to determine "whether male or female reproductive function was affected by drug treatment." Amendment 2: BMS decided to discontinue development of BMS-214778; a final report was not issued. Amendment 3: Documents the change in "ownership" of the study and study report. Extension (Only Males Treated): 120 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results Dosing Solution Analysis [1st and last weeks] The concentrations of the tasimelteon (BMS-214778; batch N031C-214778-01) formulations were within ±5% of nominal. Mortality [Twice daily] The sponsor reported no drug-related deaths in males, LDF, and MDF. The sponsor reported that three HDF were euthanized in moribund condition after 11 daily doses; in these animals, clinical signs of perioral, perinasal, and periocular substance, urine-stained and ungroomed coat, lethargy, and dehydrated skin were observed. Two of these animals also showed reduced, soft, and/or loose feces and/or fecal stained coat, fluid in the large intestines, and/or dark red discoloration of the liver. Necropsy did not identify a clear cause of death for any of these animals. In addition to these animals, 1 conM and 1 LDM were found dead after 42 and 87 daily doses, respectively. One LDM was euthanatized due to excessive aggression following 25 daily doses. No drug-related changes were reported in any of these animals at necropsy. Four males (2 LDM, 1 MDM, and 1 HDM) and two females (1 LDF and 1 HDF) were found dead or euthanized in moribund condition from intubation accidents (after 5 to 70 daily doses); one or more findings consistent with intubation injuries were noted at necropsy). All other rats survived to scheduled necropsy. Clinical Signs [Daily, prior to dosing and 1 hr post-dose] In males, drug-related clinical signs occurred at all doses, consisting of dose-related increased incidences of perioral substance (red, brown, and/or clear), fecal-stained coat, and low incidences of periocular substance (red, brown and/or clear). Urinestained and ungroomed coat and low incidences of soft feces were observed at MD and HD. Decreased motor activity and reduced feces were observed in one HDM. In females, dose-related increased incidences of perinasal and perioral substance were observed. At HD, increased incidences of periocular substance, ungroomed coat, fecaland/or urine-stained coat, and localized alopecia were observed. Low incidences of reduced feces, decreased motor activity, and dehydrated skin were also observed at HD. Body Weight [M: Twice weekly, F: Twice weekly during treatment and post-mating D0, 4, 7, 10, 13, and 16] Average body weights were dose-dependently decreased in males. At HD, body weight gain was reduced in the first five days of treatment (~5%), compared to control. At the first pairing (D29), average body weights of treated males were 5 - 11% lower than 121 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, those of controls. At D92, treated males weighed on average 5 - 16% less than controls. See the sponsor's ?gure, below. . i i COPY Lorry-nun! tcmi um.? mu; 1: kuu?ulu Bodyweighlgain (9) treated males 300 250 -, :Conzroi 2m I xSmQIKglday I A ASOmg/kglday a oSOOmglko'day .a . ?53 2150 turmoil! There was no clear effect on body weight in females at LD and MD. HD females showed average body weight loss during the first week of treatment, and average body weight continued to be reduced compared to controls) through pairing. During gestation, average weight gain remained reduced compared to control through the treatment period (GD7, up to and then showed recovery from through GD16 2% reduction compared to controls). gain of before panng a Control . 5 mg'kgmy A 50 m/kolday 5(1) mokgld Bodywolghl mm (9) Day o'lrvamari 1 22 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY Food Consumption [Weekly during treatment, for F: twice weekly GD] There was no clear effect on food consumption at LD and MD. Food consumption was reduced (84 - 90% of control values) in HDM during the four weeks before first pairing, and remained lower than controls throughout the study. In HDF, food consumption was reduced throughout treatment (80-87% of controls, before pairing to GD7), then was similar to controls after treatment ceased. Toxicokinetics [N/A] Necropsy [M: D92 of treatment, F: GD16] The sponsor reported no gross lesions in animals not dying prior to scheduled termination. However, 2 HDM (animals 4M0180 and 4M0192) were observed to show small right testes and small and/or short right epididymides (mating with both of these males resulting in pregnancies within 1-2 days). Estrus cycles Before treatment, females showed estrus cycles of 4 or (rarely) 5 days duration. There was no clear effect of drug at LD. In MDF and HDF, drug was associated with an increased incidence of females showing irregular cycles. See the sponsor's summary data, below. 123 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Fertility Parameters (Mating/Fertility Index, Corpora Lutea, Preimplantation Loss, etc.) Animals were paired one-to-one for a period of up to 3 weeks. The day on which evidence of mating occurred was designated GD0, and the animals were separated. In the extension, untreated females were paired with the surviving males for a maximum of 14 days. At least 92% of animals mated during the first estrous period (see sponsor's Text Table 1). Infertile pairings were increased at MD and HD; the sponsor indicated that most of these resulted from mating on the first night at an infertile stage of the female's cycle. However, there was a slight reduction in fertility at MD and HD (see the sponsor's summary table, below). 124 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY Due to the slight reduction in fertility at MD and HD, the males were re-assessed after 10 weeks of treatment by pairing with untreated females (see sponsor's summary Tables 16 and 20, below). There was no evidence that treatment of the male rats had any adverse effect upon litter parameters when mated with untreated females. 125 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY 9.2 Embryonic Fetal Development The EFD study in rats tested 0, 5, 50, and 500 mg/kg BMS-214778. Maternal toxicity (morbidity, decreased body weight and food consumption) was observed at HD. Some embryofetal toxicity (i.e., slightly delayed development at MD and HD and slightly decreased fetal weight at HD) was observed. Delayed development was observed as an increase in skeletal variations (i.e., incomplete ossification). 126 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study Title: ORAL STUDY OF ENBRYO-FETAL DEVELOPMENT IN RATS Study no.: 99005 Study report location: EDR Conducting laboratory and location: Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, NJ Date of study initiation: Not given, protocol dated 1/28/99 GLP compliance: Yes QA statement: Yes Drug, lot #, and % purity: BMS-214778, batch N031C-214778-01, 99.8% Key Findings:  Maternal toxicity (mortality, decreased body weight and food consumption) at 500 mg/kg  Some embryofetal toxicity, including slightly delayed development at 50 and 500 mg/kg and slightly decreased fetal weight at 500 mg/kg Methods Doses: Frequency of dosing: Dose volume: Route of administration: Formulation/Vehicle: Species/Strain: 0, 5, 50, & 500 mg/kg QD, GD6-15 3 ml/kg PO, oral gavage PEG 400 Crl:CD®(SD)IGS BR Sprague Dawley F rats 203-225 g 8-10 weeks of age (b) (4) Number/Sex/Group: 22 F Study design: Dose selection based on BMS-214778: Ten-Day Oral Range-Finding Study in Pregnant Rats (Study No. 98046). Evaluations on GD20. Dam evaluations: survival, clinical observations, body weight, food consumption & uterine contents Fetal evaluations: viability, gender, body weight, and gross external, visceral, and/or skeletal alterations. Important Deviations from Tissues (i.e., uterus, ovaries and uterine study protocol: contents) from the one HD dam sacrificed moribund on GD11 were discarded. Observations and Results Dosing Solution Analysis Solutions were prepared as needed and used within 7 days; stability had been previously shown. The solutions used were within ±10 % of nominal. 127 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Mortality [2x daily] There were no drug-related LD and MD maternal deaths. One drug-related death occurred at HD. The HD dam was euthanized in moribund condition on GD11. This rat had lost weight beginning on GD8 and had reduced food consumption starting GD9. Clinical signs included perioral, perinasal and/or periocular substance (clear, red or brown), reduced and/or soft feces, urine-stained and/or ungroomed coat on GD8-11. Necropsy did not reveal gross lesions. There were 13 embryos present in utero, which were reported as normal for gestational age. Clinical Signs [up to 2x daily; pre-dose and 1 hr post-dose] There were no drug-related signs at LD or MD. At HD, perioral and perinasal substance (12/22; clear, red or brown), reduced feces (10/22), localized alopecia (6/22; hindlimbs, dorsal surface or ventral surface), urine- and/or fecal-stained coat (4/22), and decreased motor activity (3/22; including ataxia in 1; over 1-2 days) were observed. Body Weight [daily] The numbers of dams in control, LD, MD, and HD groups were 15, 18, 20, and 22/21; non-pregnant animals were not included. Also, two dams were excluded from the control mean due to having litters with a single fetus. No changes occurred at LD or MD. Significant decreases in body weight (3-9%) and body weight gains (including losses) occurred during the dosing period (GD6-15) in HD dams. Statistical significance was reached by GD9, and remained through GD16. Increased body weight gains were noted at HD over GD16-20. Food Consumption [daily] No changes occurred at LD. Food consumption was transiently slightly decreased at MD on GD6 to GD7. Significant decreases in food consumption occurred at HD during the dosing period (GD6-16; averaging ~25%), with increases noted during GD16-20. Toxicokinetics [N/A] Necropsy [GD20] All dams were reported as normal. Cesarean Section Data (Implantation Sites, Pre- and Post-Implantation Loss) No drug-related changes in maternal or litter parameters at cesarean-sectioning were observed. Numbers of corpora lutea and implantations, litter sizes, numbers of live and·dead fetuses, resorptions, and fetal sex ratios were comparable among groups. Fetal weight was very slightly reduced (~3% [nss]) at HD. See the sponsor's summary tables, provided below. 128 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAILABLE annual u-aum noun" COPY mt can tug/Inlay11:11.1) nun." nun.? ?(100.01 mam-m I u. a a canon bum 13.; 1312.. n.9 so a 1.. 3.: 2.1 man. man a a 6.0 1.. 11.9 13mun an: u.c 11.. 310.0: - 0.01 0.0: at 0.0) man-1m. a - mm. a. In.? and 1mm all: In single nu." ma mm In mm- 1003:. ?sunny WM: than-Qu- uncut mum-11v. local. W?nu- n- um um - Wilt-jun may a no. Who-mun an (?.mmuWI It?. 1 29 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY Offspring (Malformations, Variations, etc.) All fetuses were evaluated for gross external alterations; half were examined for visceral alterations and half were examined for skeletal alterations (KOH-alizarin red-S technique). Notably, no malformations were reported in any group, which is unusual. There were no clearly drug-related effects. See the sponsor’s summary table of alterations, below. A slight increase in skeletal variations was observed but there was not a clear dose relationship; incomplete ossification suggested a very slight delay in development (e.g., dumb-bell shaped thoracic vertebrae) at MD and HD. 130 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, I I 0 can: Inc-urns mam-u mm non (QM/day) 0 I so 000 uranmunnun W) mom an: mama-u am at mt n.u' am with Mun can by nun-um n: 08.1.) 70.? II ?u mu: 1.0 110.0) at 11.0! at am ?n hum rich any mm In) 01.0) 00.8) 10.0) 00.1! um: 1.0 1.0 1.1.0 0.0 with IO 0.0 10Ian. ..I ?an0.0 0.0 0.0 0.0 mum: m?mumum?m. non-mnmmum?mwumu m. 1 31 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Excerpts from the sponsor's summary table for skeletal observations: BEST AVAILABLE COPY The EFD study in rabbits tested 0, 5, 30, and 200 mg/kg. Maternal toxicity (i.e., clinical signs, reductions in body weight and food consumption) was observed at HD. Black ovarian cysts were observed in treated does, and there was an increase in abortions at HD. Embryofetal effects at HD included increased post-implantation losses (late resorption in MD and HD) and slightly reduced fetal weights, with some suggestion that male fetuses were slightly more affected. Also at HD, an increase in the incidence of incompletely ossified pubes (indicating a developmental delay) and a slight increase in total variations and malformations were observed (generally not due to any specific alteration). The appearance of a general slight increase in variations and malformations may have resulted from the large number of abortions at HD (resulting in a reduced number of fetuses). 132 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study Title: ORAL STUDY OF EMBRYO-FETAL DEVELOPMENT IN RABBITS Study no.: 99001 Study report location: EDR Conducting laboratory and location: Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, NJ Date of study initiation: Not given, protocol dated 1/15/99 GLP compliance: Yes QA statement: Yes Drug, lot #, and % purity: BMS-214778, (Batch No. N031C214778-01, 99.3% Key Findings:  Maternal toxicity at 200 mg/kg (clinical signs, decreased bodyweight, & food consumption)  Embryo-fetal toxicity (aborted litters, slightly decreased fetal weight, some evidence for increases in malformation/variation rates, and slightly delayed ossification, esp. pelvis) at 200 mg/kg. Methods Doses: Frequency of dosing: Dose volume: Route of administration: Formulation/Vehicle: Species/Strain: 0, 5, 30, & 200 mg/kg QD, GD 7- 19 1 ml/kg PO, oral gavage polyethylene glycol-400 (PEG-400) (b) (4) Hra:(NZW)SPF rabbits nulliparous, timed-mated, 5-6 mo of age, 2.85 to 4.24 kg Number/Sex/Group: 20 F/group Study design: Dose selection based on BMS-214778: Thirteen-Day Oral Range-Finding Study in Pregnant Rabbits (Study No. 98052). Evaluations on GD29: Dam evaluations: survival, clinical observations, body weight, food consumption & uterine contents Fetal evaluations: viability, gender, body weight, and gross external, visceral, and/or skeletal alterations. Observations and Results Dosing Solution Analysis Solutions of tasimelteon (BMS-214778) in 100% PEG-400 were prepared as needed and used within 5 (200 mg/ml) or 7 days (5 and 30 mg/ml). The formulations were within ±10% of nominal concentration. 133 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Mortality [2x/daily] There were no drug-related deaths during the study. Two LD does did not survive to scheduled cesarean-sectioning on GD29. One (#31) was euthanatized in moribund condition on GD14 as the result of an intubation accident (verified by necropsy); this animal had 10 embryos present in utero, all of which appeared normal for their developmental age. The second LD doe (#30) was euthanatized in moribund condition on GD21 as the result of persistent bodyweight losses and anorexia; at necropsy, numerous gross lesions of the stomach, liver, gallbladder, intestines, kidneys, and ovaries were noted. The uterus contained 7 early resorptions as well as 5 fetuses described as "unremarkable." No cause of morbidity was determined for this doe. Additionally, a number of does were euthanized following abortions. Increased incidence of abortion was observed at HD, although abortion of 1 or more fetuses occurred in all groups (see sponsor's Table 1, below) on GD19-GD29 prior to scheduled cesarean delivery. Necropsy observations included black and/or hardened stomach contents, ingested conceptuses and placentae, fluid-filled intestine(s), hardened gallbladder, thinned and/or pitted stomach lining, mottled liver, and ovarian cyst(s). All fetuses in the aborted litters were reported as "normal for their developmental age." APPEARS THIS WAY ON ORIGINAL 134 Reference ID: 3399188 NDA 205- 677 Melissa K. Banks-Muckenfuss, em no. 99001 new 1 BIS-214778: our. STUDY 0? amino-mu. 0mm mans or mum MD CLINICAL 08.00! 1 2 3 mum Control HIS-211118 Ins-214713 Its-214718 mm nose (mike/day) 5 10 200 men! asst-.(Total) 1 2 1 5 I?ound Deed 0 mm-mmusu 0 2 0 Aborted I 2 2 1 5 CLINICAL oesnwmousx mans 20 20 20 20 feces, colt ll 3(2) 6(1) 11(5) Feces. reduced end/or 1) 5(2) 10(4) 9(1) 11(3) ebeent Pettugml enbetence. 3 1(1) 2(2) 1(1) 5(5) red led euhetence ?/02 1(1) 2(2) 1(1) 5(5) tissue 1n cue pen Coet. tecel-etelned )l 11(2) 11(3) 11(1) 16(5) Pertom subetence. 0 2(2) 0 ye11cv. red uncle: brown 9.an unbalance. red 0 1(1) wheezing. ?bored )l 0 2(2) 0 0 breathing, end/or mes Localized dopecte. It 0 M2) 2 2(1) (oxen-Me). Mont-Me). tail. ventral mtece end/or done]. eeriece mauled ekin leelcn. I 1 0 1(1) end/or ventral surface Decrees?) actor acti?ty a 1?1) 0 0 021m. none I 0 1(1) 0 1 Cent. (wine-stew I 3(2) 2 1(1) 2 lunbe: o! rebut: out): the ebeervetlon Inch died or were euthenetleed 9:10: to scheduled cesarean sectioning. - Value- c1ted refer to the e! m1. with the obemeuon mum o: pregnancy etetu. Clinical Signs [1-2x daily, predose 1 hr post-dose] Drug-related clinical signs at HD included increased incidences of soft, reduced, and absent feces, and observations associated with abortion (red peri-vaginal substance, red substance and/or tissue in cage pan). See sponsor's summary Table 1, above, for details. Reference ID: 3399188 135 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Body Weight [Daily, GD7-29] Average body weight loss was observed at HD (maximum of 2%); the GD7 average body weight was not regained until GD13. Decreased maternal body weights (5%) and body weight gain were observed at HD throughout the dosing period and remained at GD29. No clear drug-related maternal body weight differences were observed at LD or MD. Food Consumption [Daily, GD7-29] The sponsor reported decreased food consumption at HD during the dosing period. Decreased food consumption was observed in a few individuals, with the greatest number observed at HD (10 HD, 3 MD, 4 LD, and 1 control). Animals with continually very low food consumption often, but not always (1 LD, 3 MD, and 2 HD), aborted. Necropsy [GD29] No drug-related findings were reported in does surviving to GD29. Pale kidneys were observed in one MD doe. Small ovaries were observed in 2 LD does and 1 MD doe. Black ovarian cysts were noted in 2 LD (early mortalities), 1 MD, and 2 HD (early mortalities) does. Observations in other organs (e.g., stomach, intestines, liver, gall bladder, and kidneys) were only observed in early mortalities (see Mortality for details). Cesarean Section Data Intact gravid uteri (including ovaries) were weighed. Corpora lutea and implantation sites were counted and the placement of implantation sites, early and late resorptions, and live and dead fetuses were noted. Each placenta was examined for grossly for alterations. The sponsor reported minimal drug-related decreases in fetal body weight at HD (≤ 5%; see sponsor's Table 7, below); no other drug-related findings were reported for maternal or litter parameters. However, post-implantation losses (due to resorptions) were increased in treated animals (although this was not dose-related). The increase appeared related to early resorptions at LD, but both early and late resorptions were increased (compared to controls) at MD and HD. See the sponsor's summary Table 6, below. Additionally, the sponsor excluded data from the following does in the summary data presented: 1 LD doe with complete post-implantation loss (8 early resorptions), 1 LD doe with a single implantation leading to a live birth, and 1 MD doe with complete post-implantation loss (2 implantations, 2 early resorptions). Male fetuses were very slightly more affected than females (see % live male fetuses per litter and mean fetal body weight, male). 136 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, 8100! 110. $9001 I BEST AVAILABLE COPY nus-aura: our. sum or mun-mu. mm at mm or cumulus snoop 1 2 3 4 9mm:- Contxo1 Ill-210718 nus-21m; nus-214113 any can mam/m) 30 zoo nun-nuns pm um zouoom zouoom somemam? I 18 10? 18 15 comm 0.2 0.6 0.0 0.2 SD 1.8 1.5 1.7 2.0 WINS 8.2 8.8 8.1 80 2.0 1.6 1.0 2.1 manna?!? we? mm 11.1 7.4 1.3 3.7 SD 10.0 9.5 5.8 5.6 murmur mesh mm 0.3 3.1 2.1 SD 2.8 172.0 2.2 2.1 man2.0 2.2 2.1 1.9 DEAD P890838 1.82!) I 1 10 8 0 0.Ill! 0.1 0.7 0.2 0.2 8D 0.2 1.5 0.0 0.0 1288 manual: I 0 0 1 cans um an uncommon: um 5.5; 23.5) s: 21.? 25.1) 0088 no van was um om 0.0) oz 9.0: at 0.0) you In: ?(100.0! 18(100.0) 181100.01 15(100.? statisticu music: Analysis of variance with Mott's procedure vu nod to: continuum data. mum-mm. with Dunn's procedure and for mutation data. Hahn's Duct test was and to: proportion date. a - rumination lou calculat-d u: ?Carport but? - mlutaumucozpon Int-a1 100. - Dani-plantation lou calculated u: (mud ruonbod mmtucsllilomutiml 8 100- - Dachau do. "0030 that had a single-com litter consisting o! on. live tutu. 1 37 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAILABLE COPY 5mm: :40. 99001 nan: 7 nus-214770: 0m 5100! or mam-mu. 0mm nun-s sum: or man m-mxmso 1.1mControl ans-214770 Bus-214770 Bus-214770 0011.? 0062 (mike/day) 0 30 200 LINERS 018 OR MORE LIVE ON on 29 07 Gammon a 10 14a 10 15 um muses 2.0 2.2 2.1 1.9 LIVE was a 73 46 7s 62 0 LIVE ms causes mm 51.3 40.3 46.0 47.1 030. 0mm so 19.2 16.7 21.6 10.4 mm mm. 0001! warn 40.95 40.33 40.15 33.11 term) 0314 turn so 5.45 5.15 4.50 4.05 0411.: mm 41.45 41.93 40.455 39.49'3 so 5.68 6.91 4.25 0.01 PM: masts um 39.05d 39.24 39.77 30.10 so 4.49 4.79 4.04 6.71 4 pm on ?000330 m0 0.6 0.1 4.4 2.7 domain-asks mm mm so 2.4 17.0 7.9 4.0 Statistical Analyeia: Analysis of variance with Dunnatt'a procedure was used to: continuous data. Kruekal-lallie with Dunn's procedure used for enumeration data. a - Excludea doe 290036 that had single-conceptus litter consisting of one live lotus. - excludes litte: 3!0045 which had no sale fetuses. ?uid: excludes litter 490076 which had no aale fetuses. - N817: excludes littez 1?0014 which had no female fetuses. All fetuses were weighed and examined for gross external, visceral (fresh dissection) and skeletal (stained using KOH-alizarin red-S technique) alterations. The only reported drug-related change was a slight increase in the incidence of fetuses with incompletely ossi?ed pubes at HD (see sponsor's Table 11, below). The sponsor indicated that this delayed ossification was concomitant with reduced fetal bodyweight. Additionally, a slight increase variations was noted overall at HD. Externally, arched palate occurred in a HD fetus born to doe #61, and a number of skeletal variations occurred at HD (see sponsor's Table 11). However, in addition to the increased incidence of variations, there was a slight increase in malformations observed at HD (and also see sponsor's Table 8). Single 138 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, HD fetuses showed gross malformations or variations (see sponsor's Table 9). Spina bifida occurred in a fetus born to doe #69. Malrotated paws occurred in doe #75's litter, and discolored/misshapen hindlimbs and paws occurred in doe #76's (this was coded as a variation rather than a malformation). No clear drug-related visceral alterations or differences in ossifications sites were observed. BEST AVAILABLE COPY 8700! NO. 99001 TABLE 0 nus-214770: ORAL may 01? mam-mm warm RABBITS my 0? sum. muons? GROUP 1 2 3 4 serum Control nus-214173 ans-214778 sets-nun DAILY boss ("Ito/dear) 30 200 P370825 SVALBARD 147 115 157 129 Live fetuses 147 115 157 129 Deed retusee (hellornetions Verietions) Petuses with Any Mt) 11( 7.5) at 7.0) 10( 6.4) 12( 9.3) Alterations Litters with tetues am 0( 44.4) 26.7) 0( 44.4) 8( 53.3) with Any Alterations Percent Petuses per HEAR 9.0 7.7 7.2 10.1 bitter with Alteretions so 13.9 15.2 9.9 12.4 !etuses with My NH) 9( 6.1) St 5.2) 10( 6.4) 10( 7.8) Verietions Litters with fetuses 8(0) 7( 30.9) 2( 13.3) 44.4) 7( 46.7) with Any Variations Percent Petuses per mu 7.8 4.6 7.2 0.9 Litter with Verietions SD 13.7 13.4 9.9 12.3 fetuses with Any mm 2( 1.4) 2! 1.7) 0( 0.0) 3! 2.3) Malformation Litters with NM) 2( 11.1) 2t 13.3) 0( 0.0) 3? 20.0) with Any mtomtions Percent retuses per new 1.1 3.2 0.0 2.3 bitter with Meltornetions SD 3.3 9.1 0.0 5.1 Stetisticel. Analysis: Analysis of Verience with Dunnett's procedure see used tor continuous data. l'isher's Mot rest ves used for proportion date. - A11 percentages were ce1cu1eted on the besis o! the number at live tetnses in eech group. - Col-on observetions in this species end strain end reversible deieys or eoceleretions in deve1opnzent. - Irreversible chenges occurring et 1w incidences in this species end strain. 1 39 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAEABLE COPY INDY l0. 9,001 9 "-214118: mu. 0' autonew; AD mmous? my I. a 3 01:32:01 ?-21.77. ?-3141?! ?-2167?! land will tau/boldlyrota? 14nu]. Incidence 0 0 0 1( 0.8) ?the: Incidence 0 1( 0.7) Paul Incidence 1( Line: Incidence um 6.1) (I) teal mid-ac. 0.8! mum: Incidence 3.1) (I) foul Incidence am 0 1! 0 0 at?: Incidence um 6.1) 0 Poul lucid-1c. It.) 0 1( nine: Incidence 0 1( 6.7) (I) automaton (V) I mum a - All mama an enemas? the but. at 11v. fame. in ?calm. van no nus-labia pummel automatons. 140 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, BEST AVAILABLE COPY new no. ?001 mm 11 nus-211711: outmum Control nus-214111 nus-211171 3113-21.17. mm nos: (mike/day) 30 zoo mu mm )1 111 115 151 129 Live umum: ammo I 11) 15 10 15 hm add-ace um I) 3.1). when Incidence It.) 0 0 31 20.0) (I) he? mum:- 21 1.0 0 1C 1.11m lacunae. 21 11.1) 0 3! 11.1) 1( 6.7) van mm. m) 3,7, 5) 5.2) 1.9) 1) 0..) 1.11m Incidence 11m 2) 11.1) 2) 13.3) a) 16.1) 1: 6.7) run mew-nee am 2) 1.4) 2.5) 3: 2.1) 1.111.: helm 11m 2( 11.1) 22.2) 20.0) mm run :ncidmc um 1) 0.7) ?no: Incidence 1t 5..) 0 0 0 hm hem-nu am 0 1) 0.9) 0 0 ?an: lucid-11cc 6.1) 0 Nu! he?d-OI 11 0..) 1? 0")b ucu: mun:- 1? W: 70:11 Incidence um 1) 1.11m chidcun 0 1) 1) run Incidence nm 9 11 ?b 1.111.: Inca-m 2m 0 0 1) 5:7) (I) - Mallet-nation W) - mutton sun-11:11 Alums: thing-a not not. ?walkway ?than: he. ovum: ?0.05. b-mulemm?l-u. 141 Reference ID: 3399188 NDA 205-677 9.3 Melissa K. Banks-Muckenfuss, Ph.D. Prenatal and Postnatal Development TAJ0015: Tasimelteon: PRELIMINARY STUDY FOR EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT STUDY IN THE CD RAT BY ORAL GAVAGE ADMINISTRATION (b) (4) Conducted by non-GLP, initiated 6/20/11 Final report issued 7/12/12, Amendment dated 10/19/12 Species: Female Crl:CD(SD) rats (b) (4) Drug: Tasimelteon (VEC-162), , batch 800167140, 99.8% pure Dosing/Formulation: in 100% PEG-400, PO (gavage), QD In Study TAJ0001, tasimelteon (VEC-162) in PEG-400 formulations (7.5 and 100 mg/mL) were found to be homogeneous and stable for 24 hours at ambient temperature, and for up to 15 days when refrigerated. The homogeneity and stability of tasimelteon in PEG 400 formulations at 200 mg/mL was performed as part of the (b) (4) ongoing pivotal pre- and post-natal study ( Study TAJ0017). (b) (4) 142 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. One HD dam (#19) was killed on GD25 after failing to deliver a litter. This dam appeared to have aborted (blood in the cage on GD22; no pups apparent); one placenta was observed in the uterus at necropsy. Drug-related clinical signs after dosing the dams included: chin rubbing, salivation, excessive chewing, underactivity, and piloerection, as well as pica (eating feces; MD and HD), “muscle reaction- unsteady” (HD), partially closed eyelids (HD), and flat posture (HD). One HD dam (#23) was observed to show pale feces on GD12. Body weight gains were reduced 36% at HD, compared to controls, from GD6-14; afterward, gains were similar or increased throughout gestation and lactation (e.g. tasimelteon-treated dams gains increased approximately 20% compared to controls LD1-11). Food consumption was reduced up to 12 and 35% in MD and HD dams, respectively, at the beginning of dosing (GD6 to GD10 or GD14, respectively); afterward, consumption was similar throughout gestation and lactation. All dams delivered after a normal gestation period (22-23 days), but HD dams showed a very slightly increased gestation length. The sponsor reported no drugrelated macroscopic changes. The sponsor reported no tasimelteon-related adverse effect on group mean number of implantations, litter size, fetal survival, fetal clinical signs, or fetal necropsy; however, the calculated values exclude data from the HD dam that did not deliver (presumed abortion). For this reason, an effect on fetal survival cannot be excluded. Additionally, a few pups from treated dams did not survive. One male LD pup was missing on D4. One LDF pup, one MDM pup, and one HDF pup were found dead between birth and LD1. Both the LDF and HDF pups had no milk present in the stomach (the LDF pup also showed an unexpanded lung). One HDF pup was pale on LD1. According to the sponsor, sex ratios (i.e., % males) at birth were slightly high in the Con and LD groups and slightly reduced in the MD and HD groups. Mean fetal body weights were reduced (approximately 5 to10%) at MD and HD, both at LD1 and LD11 (difference greater at LD11). The sponsor indicated that tasimelteon treatment was believed to have “delayed fetal development in utero and also reduced the rate of growth postpartum” at HD; a similar fetal effect was observed at MD. Based on the results of this study, doses of 0, 50, 150, and 400 mg/kg were selected for the pivotal pre-/post-natal development study. 143 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study title: TASIMELTEON: STUDY FOR EFFECTS ON PRE- AND POSTNATAL DEVELOPMENT IN THE CD RAT BY ORAL GAVAGE ADMINISTRATION Study no.: TAJ0017 Study report location: EDR (b) (4) Conducting laboratory and location: Date of study initiation: 6/24/11 (b) (4) GLP compliance: Yes QA statement: Yes (b) (4) Drug, lot #, and % purity: tasimelteon (VEC-162), batch 800167140, 100.4% pure (w/w) Methods Frequency of dosing: Dose volume: Route of administration: Formulation/Vehicle: Species/Strain: Study design: Deviation from study protocol: QD 2.5 mL/kg PO (oral gavage) 100% polyethylene glycol-400 (PEG-400) Crl:CD(SD) rats, females See sponsor’s table, below None identified. 144 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Observations and Results F0 Dams Survival: There were no F0 mortalities. Clinical signs: Few dose-related clinical signs were observed. After dosing, chin rubbing and/or salivation was observed in most VEC-162-treated dams. Convulsion (1 HD; see description below), partially closed eyelids (6 HD), unresponsive (3 HD), flat posture (2 HD), breathing fast (1 HD), limited use of limbs (1 HD), and underactive (5 MD and 22 HD, LD5 to end), and/or unsteady gait (2 MD and 20 HD, for 1-3 days on average; see sponsor’s definition below) were observed after dosing. During the detailed physical examinations, hair loss, piloerection, pale feces, irritable behavior, and /or vocalization were observed with increased incidence during the gestation or lactation periods in a few VEC-treated animals. One HD dam was observed to have a 30 second convulsion on LD15; this dam was previously observed to be underactive, unresponsive, and have flat posture shortly after dosing on LD8, underactive on LD12 and LD19, and underactive with unsteady muscle reaction (“lack of muscular coordination giving rise to staggering or otherwise abnormal gait”) on LD15. 145 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Body weight: During gestation, average body weights were very slightly reduced in MD and HD dams (see sponsor’s Figure 1, below; ~3-5%). Overall average body weight was reduced (3 to 5%) in HD dams throughout gestation. Average body weight gain at HD was decreased 83% from GD6 to GD10 and was reduced 15% overall (GD6-GD20). At MD, average body weight gain was reduced approximately 30% from GD6 to GD10, but was similar to controls (-5%) during the remainder of the gestation period. There were no clear differences in LD dams. During lactation, average body weights were generally slightly lower than those of controls (<5%); HD dams did not show the loss exhibited by the other groups at the end of the lactation period (LD18-LD21), which generated a net increase in body weight gain over the lactation period at HD (~30%). BEST AVAILABLE COPY 146 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Food Food consumption was reduced 15% on GD6 to GD9 in MD dams and consumption: throughout the gestation period in HD dams (35% GD6-GD9, average reduction 16% GD6-GD19). Food consumption was also reduced (7-13%) during LD7 to LD17 at MD and HD. Uterine Gestation length was within the expected length (22-33.5 days) for all content: groups, but the mean gestation length was slightly increased in tasimelteon-treated groups (dose-related, [ss]). Two LD dams were found not pregnant, and one LD dam was sacrificed on GD23 after showing total litter loss “shortly after the commencement of parturition.” However, a relationship to drug cannot be established since similar events did not occur at MD or HD. There were 22, 19, 22, and 22 litters for examination in Control, LD, MD, and HD groups. The sponsor reported no drug-related effects on the number of implantations or litter size (but note that data from the LD#32 with total litter loss were removed). LD#32 had 14 implantation sites; this dam was coded as “no live litter” and the sponsor stated that “most offspring were dead or presumed eaten at parturition.” Necropsy The sponsor reported no drug-related macroscopic changes. Increased observation: hair loss was reported in tasimelteon-treated dams. Two HD dams showed kidney changes (punctate depressions, pelvic dilatation). One HD dam showed multiple punctate dark areas on the thymus. A few changes of unclear toxicological significance observed in ovaries (cysts), mammary gland (pale, inactive), and spleen (enlarged) were observed only at LD. Toxicokinetics: Plasma exposures to tasimelteon (VEC-162), and metabolites M9, M12, M13, and M14 were evaluated at GD6, GD17 and LD4. Repeat analyses were necessary for a number of samples for M12 and M13; one sample for M14 was repeated. Control animals showed some exposure to tasimelteon and/or its metabolites, but were “generally close to or below the limit of quantitation” (i.e., < 5.438 ng/mL for M14 and < 5 ng/mL for all others). Low level exposures were also observed in samples from offspring animals (5.07 to 15.5 ng/mL). After an investigation, the sponsor attributed these observations to contamination during sample handling. See sponsor’s Tables 68-72 for summary plasma exposure data in dams and Tables 73-76 for summary plasma exposure data in the offspring. 147 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Plasma exposures to tasimewlteon were generally less than doseproportional (see sponsor’s Tables 68 and 11), and little to no accumulation was observed in the dams (see sponsor’s Table 12). There was a >100fold margin from the plasma tasimelteon AUC at the LD to the AUC following a 20 mg dose in humans (~411 ng.hr/mL). 148 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Plasma exposures to major metabolite M9 were generally less than doseproportional (see sponsor’s Tables 69 and 14); however accumulation was observed at HD (see sponsor’s Table 15). 149 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Plasma exposures of major metabolite M12 were generally less than doseproportional (see sponsor’s Tables 70 and 18), and accumulation was not observed (see sponsor’s Table 19). Metabolite M12 plasma exposures were up to 3 times that of parent at LD, 2 times that of parent at MD and 12 times that of parent at HD. 150 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Plasma exposures of major metabolite M13 were generally less than doseproportional (see sponsor’s Tables 71 and 22), and some accumulation was observed only at HD (see sponsor’s Table 23). 151 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Plasma exposures of major metabolite M14 were generally less than doseproportional (see sponsor’s Tables 71 and 26), and some accumulation was observed only at HD (see sponsor’s Table 27). 152 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Dosing Solution Formulations were prepared weekly and stored refrigerated (2-8oC) before Analysis use. Homogeneity and stability at 7.5 and 100 mg/mL were established in Study TAJ0001; homogeneity and stability at 200 mg/mL was confirmed in this study. Suspensions were homogenous and stable for 8 days at ambient temperature and for up to 15 days refrigerated. The mean concentrations were within 3% of nominal. F1 Generation Survival: The sponsor reported no drug-related effects on litter size, survival indices, or sex ratios; however, there was a slight reduction in the live birth index at HD (see sponsor’s Table 49, below). No milk was present in the stomach of 4, 1, 2, and 11 fetuses in the control, LD, MD, and HD groups (presumed stillborn; from 4, 1, 2, and 5 litters, respectively). One deceased HD fetus was noted to be of “small build” with bruising to the nares. Also, the live birth index should be adjusted for the sponsor’s removal of data from LD#32 (14 implantations, “no live litter” although the individual line data show 1 male and 3 female pups at delivery; est. adjusted live birth index ~94.6%). Clinical signs: For early observations of the F1 pups, the sponsor reported a low incidence of clinical signs, including: cold to touch, little milk in stomach, minor cuts, and bruising. In tasimelteon-treated groups, underactive, swollen area on the abdomen, small build were also observed in a few offspring. Although the protocol defined how selection of the F1 mature animals would be performed (i.e., one/sex/litter), it is unclear how this selection was actually done. For example, there were 22 litters for the control and MD groups (i.e., 2 litters in each group were not represented) and, although there were 19 LD 153 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. and 22 HD litters, the sponsor appears to have selected the representative F1 generation from 18 and 19 litters, respectively. The F1 mature animals were then numbered in such a way that one cannot determine from which litter each animal was born. At maturation, few clinical signs were reported, including: broken teeth (2 LDM), vocalization (2 MDF), abnormal staining on head (1 MDF), swollen area in the ventral abdomen (1 MDM, 1 HDM), and kinked tail (1 HDM). During early gestation for the F1 dams, few clinical signs were reported, including: vocalization (2 LDF), irritable (1 LDF, 1 HDF), and abnormal staining on head (1 MDF). APPEARS THIS WAY ON ORIGINAL 154 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Body weight: Generally, average body weights of the F1 pups were reduced during the entire lactation period. However, LD and MD pups were slightly heavier than control pups at LD1, which the sponsor attributed to the slightly longer gestation lengths; although gestation length was increased 0.7 days for the HD pups, their average body weight on LD1 was 0.3 g less than that of control pups. The sponsor noted that the birth weight of the HD group was 0.8 g less than that of controls of similar gestation length, and stated that “there may have been a delay in pup growth in utero contributing to the extended gestation length.” There was an incremental body weight gain reduction in the HD pups between LD4 and LD18 (-12% at weaning, compared to controls). Body weight gains over LD1 to LD21 were reduced 15% in the HD pups, and they weighed 14% less than controls at 25 days of BEST AVAILABLE COPY age. 155 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. After weaning on LD21 (although the sponsor nominally defined the “start” of the F1 generation as 28 days of age), average body weights in the F1 animals continued to be reduced in the tasimelteon-treated groups, especially the HD group (see sponsor’s Table 39, and Figures 8 and 9). Generally, average body weights in the LDM and MD groups were reduced 5 to 10 percent, and average body weights in the HD groups were reduced 10 to 20 percent. In the F1 females, this average body weight reduction continued during gestation (~5% at MD and ~10% at HD; see sponsor’s figure 9). 156 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Figure 7: Bodywelght for males - you) mean nines formalcs (Fl) Grow 2 3 4 Compound Control Tasimelteon Tasimeheon Tasimelneon Dose (mg/kg/dayFigure 8: Bodyweight for females before pairing Bodyweight goup meanvalues forfemalesbefoue pairinga?l) 2 3 4 Compound Conan] Tasimclacon Tuimchoon Taxmdwon Dose (mg/kg?dayBodyweigl! (5) 135 115 75 Day before poking 1 57 Reference ID: 33991 88 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY Food consumption: Data not provided Necropsy Of the pups dying prior to scheduled termination, there was an increase in no Observations milk present in the stomach at HD (4/4, 1/1, 2/2, and 11/5 [pups/litter] in the control, LD, MD, and HD groups). A few observations were noted in the F1 pups at scheduled termination. Small build was reported for 1/1 and 45/6 (pup/litter) at MD and HD, respectively. Dilated renal pelvis was observed in 4/4, 4/4, and 1/1 (pup/litter) in the LD, MD and HD groups, respectively. (Of unclear importance were 3 LD pups [1 litter] observed to show fluid-filled dilated ventricles in the brain.) At maturation, few alterations were observed. In tasimelteon-treated males, alterations in testes (flaccid, blue, and/or small; 1 LDM, 2 HDM), epididymides (small; 1 LDM, 2 HDM), seminal vesicles (abnormal shape; 1 HDM), and tail (kinked; 1 HDM) were observed. Of unclear toxicological importance is 1 LDM that showed dilated ventricles in the brain. In tasimelteon-treated females, alterations in kidney (pelvic dilatation, enlarged, and/or dark; 1 LDF, 1 MDF), ureters (distended, thickened; 1 MDF), and urinary bladder (thickened, distended, dark, and calculus present; 1 MDF) were observed. 158 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Physical Sexual maturation was assessed beginning at 28 days of age for females development: and 38 days of age for males. Mean age at balano-preputial separation for males was not clearly affected, but it was achieved at a lower average body weight at HD. Mean age at vaginal opening for females was increased at HD (although body weight was similar to that of controls); the sponsor stated that “sexual maturation was occurring when the females reached a critical body weight.” See sponsor’s summary Table 56 for details. BEST AVAILABLE COPY Neurological The HD pups demonstrated an increased age to achieve surface righting assessment: (assessed from LD2 until achieved) and air righting (assessed from LD14 until achieved), compared to controls; see the sponsor’s summary Table 55, below. Additionally, an increased number of HD pups failed to achieve surface and/or air righting within the allotted time period (4, 1, 2, and 11 in the control, LD, MD and HD groups, respectively); data for these pups were not accounted for by the sponsor’s method of reporting the summary data. The sponsor reported no drug-related effect on pupil reflex and startle response on LD20 (as assessed by % of animals passed). Two LD pups showed no or partial pupil dilation; this was not reported in other groups. 159 Reference ID: 3399188 NDA 205-677 Reference ID: 3399188 Melissa K. Banks-Muckenfuss, Table 55: Pre-weaning examinations BEST AVAILABLE Pre-weaning examinations - group mean values for (F 1) COPY Group 1 2 3 4 Compound . Control Tasimelreon Tasimelteou Tasimelteou Dose 0 50 150 450 Surface Group righting Air righting Pupil re?ex Startle response Dayof age Day of age pass) (?13 pass) Statistical test Wi Sh 1 Mean 3 .4 15.9 Number of animals tested 216 216 SD 0. 73 0.37 Number of animals failed 0 0 22 22 $3 of animals passed 100.0 100.0 2 Mean 3 .2 15.9 Number of animals tested 0.56 Number of animals failed 1 19 19 9.3 of animals passed 99.5 100.0 3 Mean 3 .3 15 .9 Number of animals tested 218 218 SD 0 7] 0 79 Number of animals failed 0 0 22 22 9.6 of animals passed 100.0 100.0 4 Idean 3.9? 16.5? Number of animals tested 220 220 SD 0.8] 073 Number of animals failed 0 0 22 22 ?36 of animals passed 100.0 100.0 Sh Treated groups compared to Control using Shirley?s test Wi Treated groups compared to Control using Williams? test. 3 p? 0.05 Motor activity testing was assessed at LD22, and showed no consistent drug-related effects (see sponsor?s Figure 5, below). While an apparent slight reduction in beam breaks in the MD and HD males started at 42-48 minutes, compared to controls, it is of questionable toxicological importance given that the SDs at these times were approximately equal to the averages. Likewise, tasimelteon-treated females were more active during the first 6-12 minutes, but averages were within 1 SD. APPEARS THIS WAY ON ORIGINAL 160 NDA 205-677 Reference ID: 3399188 Melissa K. Banks-Muckenfuss, Ph. D. Figure 5: Motor netin'ty BEST AVAILABLE Motor acuntv - group mean scores (beam breaks) for male lfl) COPY Group 3 4 Compound Control Dost (mg?kg?dajr) 50 150 450 II-ISM 50 - 200 - 300 Total scores 1 000 Iotnl 45 75 .r ., High beams '3 300 Lowbeams Tune (minutes) Time (minutes) FIGURE 5 - conunucd Motor - group mean scorcs (beam breaks) for female (Fl) Group 1 3 3 4 Compound Control Tasinicltron Dost (mg?kg?dayTotalacmes Ioral scores SW 1000 45 - a High beams 200 175 Low beams ?Time (minutes) Tum (mmutcs) At 31i1days of age (and again at 48? 1 days of age for HDM), Morris Water maze performance was assessed. F1 HDM demonstrated clear de?cits (see sponsor?s summary Table 58 and Figure 6). Trial times, sector entries, and failed trials were increased for HDM on all four testing days. While the day-to-day testing showed improvements similar to 161 NDA 205-677 BEST AVAILABLE COPY Melissa K. Banks-Muckenfuss, Ph.D. those of controls, the absolute values remained consistently higher than those of controls and many of the absolute values were outside of the historical control range. The MDM showed deficits in performance on the first day of testing, but subsequent performance was similar to that of controls. The performance of F1 females was not affected. 162 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. BEST AVAILABLE COPY Although not standard practice (and not encouraged due to known effects of repeated testing), the sponsor re-tested the F1 males at 47 to 49 days of age to determine whether the observed deficits would resolve. While some inter-group variation remained in the performance of F1 males, the sponsor reported no drug-related consistent differences (see sponsor’s continuation of Table 58, below). 163 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Reproduction: After at least 9 weeks of age, mating pairs were assigned within the same treatment groups (with care taken to avoid sibling pairings). Mating pairs (one-to-one) were left together for up to 2 weeks. Pre-coital interval and fertility parameters were not clearly affected (see sponsor’s Tables 65 and 66, below). Three LDF and one HDF were not pregnant; additionally, another HDF had been pregnant (#257; as indicated by a single resorption), but had no viable implants. The sponsor calculated litter data based on 20, 17, 20, and 18 litters in the control, LD, MD, and HD groups. The sponsor reported no drugrelated effects on F1 reproduction, although a slight reduction in the number of corpora lutea in the HDF was noted. The average numbers of corpora lutea were 17.0, 16.4, 16.8, and 15.7 in the control LD, MD, and HD groups (SDs were similar, 2.27 to 2.73). See sponsor’s Table 67, below. (Note that data from the HDF with a single resorption were excluded, which yields reductions in a number of the fertility parameters 164 Reference ID: 3399188 NDA 205-677 BEST AVAILABLE COPY Melissa K. Banks-Muckenfuss, Ph.D. for the HDF group. For reference, the reviewer-calculated means including that HDF are 15.0 for corpora lutea, 14.4 for implantations, 0.89 for early resorptions, 0.37 for late resorptions, 1.26 for total resorptions, 13.2 for live embryos, 7.0 for pre-implantation loss %, and 12.7 for postimplantation loss %.) 165 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Other (TK): Plasma exposures to tasimelteon (VEC-162) and metabolites M9, M12, and M14 were measured in the F1 offspring on LD 4 were as follows (see sponsor’s Tables 73 to 76). Metabolite M13 plasma exposures were below the limit of quantitation, with the exception of males from one litter (~8 ng/mL, 4 hr post dose of dam). Some sex differences were observed (cf. tasimelteon Cmax and AUC). The measured AUCs in the F1 pups at LD on LD4 exceed the AUC for a 20 mg dose in humans (411 ng.hr/mL). 166 Reference ID: 3399188 NDA 205-677 10 Melissa K. Banks-Muckenfuss, Ph.D. Special Toxicology Studies The sponsor conducted ocular irritancy and skin sensitization tests. Also, a photo safety assessment was undertaken; a tissue distribution result suggested that tasimelteon binds melanin. Study 99AB38.350: BOVINE CORNEAL OPACITY AND PERMEABILITY ASSAY (b) (4) Conducted by Dated 4/22/99 GLP (except test article characterization) Drug: Tasimelteon (BMS 214778-01), COA not provided 167 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Tasimelteon scored an average in vitro score of 1.5 (mild irritant) on the Bovine Corneal Opacity and Permeability Assay (BCOP). Five freshly obtained corneas were tested. (b) (4) Saline (0.9% USP injectable saline [ was added to solid BMS 214778-01 to achieve a 20% mixture (w/v) and the vial was vortexed for approximately one minute. A 20% (w/v) solution of imidazole was used as the positive control. See the sponsor’s excerpted explanation of scores and summary Table 1 of results, following. Of the five individual corneas, the in vitro score ranged from 0.3 to 5.3 (all mild irritant). Study BMY 378/993273/SS: BMS 214778-01 SKIN SENSITIZATION TO THE GUINEA-PIG (MAGNUSSON & KLIGMAN METHOD) Conducted by (b) (4) Dated 8/20/01 GLP, except for test article characterization Drug: Tasimelteon (BMS 214778-01), lot 032, purity 99.3% Ten healthy female guinea pigs (nulliparous, nonpregnant; 4-7 weeks of age; 349-400 g) were tested with tasimelteon; 5 animals were used as controls. Intradermal and 168 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. topical irritancy was tested at the following concentration (see the sponsor’s summary, below). Following initial exposure to tasimelteon (the 'induction' period comprising intradermal injections and topical application) the animals were subjected, approximately two weeks after the topical induction exposure, to a 'challenge' exposure of the test substance in order to establish if a hypersensitive state had been induced. During preliminary investigations, the “maximum practical concentration” of tasimelteon for topical application (50%) produced slight irritation in 2 of 4 animals. Dermal responses to the induction and challenge phases were scored as follows (see excerpt from the sponsor’s submission). Sponsor’s Tables 1, 2, and 3 below provide the results of the induction and two challenge phases. In this study tasimelteon did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. 169 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, TABLE 1 Dermal reactions ohsen'ed after each induction Gmup Animal Intredermel injections Topical number Site number applieetiun 2. Control 1595 1596 1597 1599 1599 Test 1666 1661 1662 1663 1664 1665 1666 166? 1663 1699 h1trederms1 injections Tepieal appliea?on Control animals: See 631m: 1 {previous Control morsels: Alembieol page) Test animals: EMS Test animals: See ?gure 1 {pmvious pegs} 5996 in Munhieel No enr?sema Necrosis No: in?ation irritation TABLE 1 Dermal reactions observed after the ?rst challenge applieat'len with BRIE Freuml's treated controls Soore 1 Guineanpig number ?edeme 24 Home 43 Home A. A 1595 I 1 1596 2 2 0 1 I 1591r 1 till 1 9 1 CI 0 l] 1596 I 1 0 Ell 9 1599 1 It} C- 6 9 A Anterior site. exposed tn EMS 313% w?i in Alembieel Pestes?iur site, imposed to: EMS 15% in Alembieel 170 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, 2 Dermal reactions nheerved after the ?rst challenge app?ealiun with EMS 114713-111 {een?nue-d} Test animals Scare i Guinea-Pie Emilia-m 11111an ID Dedema 24 Home 43 Hem A A 1601.1 1 [l (J ID {1 16111 1 1 ?1 CI 1 El 1611! 1 L1 1 Cl 1 1603 16414 1 1* 1 16115 {l 1} {1 I) 161116 1 Ii} {1 1611':r El '11 El C- 16113 1 ID El Cl {1 ID 16115Leealjeed dermal rue?ee (re?ned to a small area of the challenge sire] and 510qu efthe epidermis Anterior eite. to EMS 2141'7'8451. 30% in alemhieel Pesleder site, expesed to EMS In?ll?01. 15% new in Alemhieel '1:le TABLE 3 Dermal reae?ens ehserved after the challenge application with ELLIS 214173-111 Freund?e treated eemmIe Scare Gluten-pig - Earthen number 0 ?edeme 24 Home 43 Home A A 1595 l} 1596 [l [l 0 l} [l [l 155'?r l} 1593 0 1555Anemia: site, expueed to EMS INTER-DI, 15% MW in Mmhicnl Pusterier site, expueed to EMS 15% We in?lembieul 171 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Study No. VCR-TMI-121012: MOLAR EXTINCTION COEFFICIENT DETERMINATION FOR TASIMELTEON AND TASIMELTEON METABOLITES M3, M9, M11, M12, M13, and M14 Conducted by Non-GLP report dated 1/29/13 (b) (4) but reported by Vanda A Molar Extinction Coefficient (MEC) determination was performed for tasimelteon and its metabolites. The solutions for all compounds except M11 were prepared at a concentration of 0.03 mg/mL in deionized water; due to better solubility, the solution for M11 was prepared at a concentration of 0.1 mg/mL. All UV determinations were performed with a cell path length of 1 cm. Because the absorption maxima for tasimelteon and all metabolites occurred below 290 nm, which is the lower limit of the 290 nm to 700 nm wavelength range specified in the ICH guidance, the MEC values were calculated at both the absorption maxima and at 290 nm. There was no absorption in the 300 nm to 700 nm wavelength range for tasimelteon or its metabolites. The obtained MEC values for tasimelteon and its metabolites M3, M9, M11, M12, M13, and M14 are provided in sponsor’s Table 1, below. 172 Reference ID: 3399188 NDA 205-677 11 Melissa K. Banks-Muckenfuss, Ph.D. Integrated Summary and Safety Evaluation Pharmacology Tasimelteon and a number of its metabolites showed affinity at melatonin (MT1 and/or MT2) receptors, with slightly greater affinity at MT2 receptors. Tasimelteon and metabolite M13 showed nanomolar affinity at MT1 receptors; tasimelteon, M11, M12, M13, and M14 showed nanomolar affinity at MT2 receptors. In an early study, tasimelteon was shown to have affinity similar to melatonin at MT1 receptors, and greater than melatonin at MT2 receptors. Tasimelteon did not show affinity at other targets in a receptor binding screen. Toxicology With regard to this application, there are three general issues that warrant discussion as they potentially impact the adequacy of the toxicology development program: the use of PEG-400 as a vehicle for the drug, the termination (and subsequent reopening) of a number of the toxicology studies, and coverage of the exposures to the major human circulating metabolites (M9, M12, and M13) in the toxicological species. For the toxicity studies PEG-400 has been used as the vehicle. Although relatively widely used as an excipient, both toxic and some protective effects have been observed for PEG-400 itself. According to Dr. Atrakchi's review, the sponsor indicated that the high doses tested were limited by the toxicity of PEG-400 itself and the finding of cecal dilation in the 2-week rat toxicity study was attributed to PEG-400. GI and related effects (e.g., loose feces, reduced food consumption, reduced body weights) have been observed with PEG-400 (Hermansky et al., 1995), and renal toxicity has been suggested. More recent evidence demonstrated that when administered by gavage (5, 50, or 100% v/v% at 5 ml/kg/day), PEG-400 caused histopathological changes localized 173 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. to the stomach mucosa, but not in the intestine (Ueda et al., 2011). Conversely, it has been shown that PEG-400 protects against some compounds causing gastric mucosal damage (cf. Gutierrez-Cabano, 1995, Gutierrez-Cabano, 2000). It is unclear whether the use of PEG-400 as the vehicle had an effect on the apparent toxicity profile of tasimelteon. While body weights and food consumption were affected in a number of studies, there were no clearly adverse histopathological effects on stomach and/or forestomach. Secondly, it is notable that the chronic toxicity assays in rat and monkey were terminated after the in-life portion of the assays during development and summary and/or interim reports were issued; upon restarting development, the sponsor contracted CROs to complete the histopathology assessments for these studies. (The rat fertility study was also completed in this manner.) However, GLP and QA statements have been submitted for these studies (although a separate, signed pathology report for the chronic, 6-month rat toxicity study was not included in the report and was submitted in a later NDA submission only upon request). Although it is an unusual circumstance, these studies appear to have been completed in a reasonably appropriate manner. According to the Clinical Biopharmaceutics staff, M9, M12, and M13 were identified as the major human circulating metabolites, with exposures 130%, 180%, and approximately equal to those of tasimelteon, respectively. With the exception of the pre-/post-natal development study in rats, exposures to the major human metabolites were not measured in the toxicology studies. The sponsor conducted a separate study to determine exposures to the major human metabolites at steady state in rats, and used the obtained ratios (metabolite/tasimelteon) to extrapolate the metabolite exposures achieved in the toxicology studies. Only single dose metabolite exposure data were obtained for monkey, and only exposure data for M11 (not a major human circulating metabolite) were obtained at steady state in mice and at the HD tested in the embryofetal study in rabbits. Estimated exposures to M9, M12, and M13 at the high doses in the rat toxicology studies, using the steady state metabolite exposure ratios obtained, suggest that the major human circulating metabolites have been tested at exposures equal to or exceeding those anticipated in humans at the RHD. The one exception is offspring exposure (measured on LD4) to metabolite M13. Metabolite coverage was not always achieved at the observed NOAEL doses. General Toxicity The chronic toxicity studies generally reflected the toxicities demonstrated in subchronic studies, including slight effects on body weight and food consumption, clinical pathology, and a number of target organs (i.e., liver, kidney, and reproductive organs, as well as spleen and endocrine organs). Liver toxicity, in particular, was of increased severity and demonstrated at lower doses. Following the summaries of the chronic rat and monkey studies, an overview of the target organs/systems of tasimelteon toxicity will be discussed. 174 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. The 6-month chronic toxicity study in rats tested doses of 0, 5, 50, and 500 mg/kg. Convulsions (tonic and/or clonic), tremors, hypoactivity, labored respiration, ataxia, loss of righting reflex were observed in rats. In rats, pica was observed in both sexes at HD, and in females at MD. Over the course of the study, average body weights were slightly reduced in males, but a similar effect was not seen in females. Several clinical pathology changes were observed. Slight reductions in RBC parameters were observed in females, and reticulocytes and/or platelets were slightly increased in both sexes at HD. WBC increases were also observed in both sexes (especially, neutrophils, monocytes [F], LUC [F], and basophils [M]). PT time and fibrinogen showed slight increases. ALT, cholesterol, and triglycerides were increased in HDF (some increase in males); BUN and creatinine were slightly reduced. Serum electrolytes showed very slight but consistent changes over the treatment period (sodium and chloride slightly reduced, calcium slightly increased). Urine volume was increased at HD. Grossly, enlarged liver was observed in all HD animals. There was an increased incidence of dilatation of the uterus in treated females. Liver, heart, adrenal gland, kidney, ovary (F), and spleen (F) weights were increased; pituitary gland and prostate (M) weights were reduced. Histologically, liver, kidney, spleen, and endocrine/reproductive organs were identified as target organs. In the liver, minimal to moderate centrilobular hepatocyte hypertrophy (≥MD) and minimal to slight focal hepatocyte necrosis (4 HDM and 3 HDF) were observed. In the kidney, exacerbated (slight to moderate) chronic progressive nephropathy, minimal to slight cortical tubules with hyaline droplets, minimal to slight cortical tubular pigment, and interstitial pigmented macrophages were observed in both sexes in a roughly dose-related manner. In MD and/or HD females, slight cortical tubular dilation, interstitial inflammation, and simple tubular hyperplasia were also observed. In the spleen, minimal to slight hemosiderosis and minimal to slight increased extramedullary hematopoiesis were observed. The sponsor considered this reflective of an increased rate of RBC removal from peripheral blood leading to the "compensatory" response. Thyroid changes (called prominent ultimobranchial cysts by the pathologist) were observed in 3 HDM and 7 HDF (compared to 1 Con/ sex); focal pituitary hyperplasia of the pars distalis was also observed at HD (one/sex; see Zabka et al., 2011). The incidence of ovarian follicular cysts (4 HDF vs. 1 ConF) and luminal dilatation of the uterus (14 HDF vs. 6 ConF) were increased at HD. In the mammary gland, two HDF showed galactocele(s) and three HDF showed secretory activity. Plasma tasimelteon exposures were greater in females, some accumulation at LD, and slightly reduced exposures with repeated dosing for 6 months (at ≥MD in males, HD in females). The 1-year toxicity in monkey tested 0, 3, 20, and 150 mg/kg. Convulsions (2HD), flaccidity (HDM with convulsion), labored breathing, decreased activity (HD), salivation (≥ MD), emesis (F, ≥ MD), alopecia (≥ MD), and decreased appetite were observed. The sponsor noted that for a single two-day period, one LDM was noted to be coldness, prostrate, ataxic, weak, unable to sit up, slightly dehydrated, and had pale mucous membranes; this animal showed elevated LFTs and decreased wbc and RBC counts. Slightly reduced average body weights were observed at HD over the first 6 months of the study (which appeared to resolve), and slightly increased average body weights were observed over the last few months of the study at LD. Blood pressure was slightly 175 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. increased in HDF on D269 and D365. Reductions in RBC parameters (<15%) were observed at HD, and increased platelets and decreased fibrinogen were suggested. Serum ALT (2-3x) and GGT (50-70%) were increased at HD, and ALP was decreased (30-40%); triglycerides were increased (~2x) in HDM. Liver weights were increased (1.4-1.8x) at HD, as was thyroid weight (~40%) in MD and HD males. Few clear histologic changes were observed. Although noted only in the comment field but discussed by the sponsor, the three remaining HDF at terminal sacrifice showed a lack of corpora lutea; the sponsor indicated that this could indicate a "lack of recent estrus cycling." Mild focal necrosis, minimal to mild periportal fibrosis, mild focal serosal fibrosis, mild subserosal fibrosis, centrilobular fatty change, and/or minimal focal pigmented macrophages were observed in the livers of individual MDM, MDF, HDF and HDM. The incidence of minimal to mild chronic interstitial inflammation in the kidney was increased in treated males. Minimal chronic inflammation of the heart and/or aorta was observed in a few treated animals. Plasma tasimelteon exposures showed some reduction with repeated dosing for a year. Target Organs/Systems The primary target organs of tasimelteon toxicity include: the CNS, liver, kidney, and reproductive organs. Some alterations were also observed in the hematologic and endocrine systems (i.e., thyroid, pituitary, and adrenal glands), and related organs. Generally slight reductions in weight and sometimes food consumption were observed across the studies; these were usually not severe enough to hamper the studies significantly. Post-dosing emesis was observed in monkeys, and salivation was observed in many species. In the chronic study in rats, pica was observed in both sexes at HD, and in females at MD; average body weights were slightly reduced in males, but a similar effect was not seen in females. In the one-year monkey study, slightly reduced average body weights were observed at HD over the first 6 months of the study (which appeared to resolve), but slightly increased average body weights were observed over the last few months of the study at LD. CNS CNS signs including convulsions (clonic and/or tonic), tremors, loss of righting reflex, flaccidity, and/or ataxia were observed in single dose studies using higher doses and usually at relatively high doses tested for longer durations in rats, mice, and monkeys. Reduced activity, ataxia, and occasionally labored breathing were noted throughout many studies. Of unclear relationship to drug were observations in one LDM (3 mg/kg) in the 12-month monkey study; this animal was noted to be cold, prostrate, ataxic, weak, unable to sit up, and slightly dehydrated, and had pale mucous membranes, for a single two-day period; this animal showed elevated LFTs and decreased WBC and RBC counts. A few of these signs resembled those seen at higher doses (and could therefore be considered drug-related), but the overall severity of the findings were not replicated in a clearly dose-related manner in animals treated with higher doses. If the signs in this animal were, in fact, drug-related, it appears to be an outlier. 176 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Liver The liver was clearly a target organ in most of the toxicological species used. Rodents showed the effects on liver most clearly. Subchronic studies in rats showed increased liver weight and size, dose-dependent minimal to marked centrilobular to panlobular hepatocellular hypertrophy (generally at ≥ 100 mg/kg), and increased hepatocellular mitoses at high dose (400 mg/kg). In the chronic (6-month) study, minimal to slight focal hepatocyte necrosis (500 mg/kg, males and females) was also observed; tumors were observed in the rat carcinogenicity study at ≥ 100 mg/kg. These histopathologic changes were accompanied by clinical pathology changes such as increased: ALT, globulin, albumin, total protein, cholesterol, glucose (F), and/or triglycerides (F); these were generally at least partially reversible in subchronic studies. The NOAEL in rats generally decreased with increasing dose (e.g., 50 mg/kg for 1-week, 25 mg/kg for 1-month, 5 mg/kg for 6-months). Increased liver weights, dose-related minimal to moderate centrilobular hepatocellular hypertrophy, and slightly increased serum albumin and total protein (M) were also observed in mice. A number of the effects, particularly in rats, may be related to hepatic enzyme induction (demonstrated in a separate study using liver tissue samples from TK animals at 6 months in the rat carcinogenicity bioassay), but there is some evidence of damage in a few animals at higher doses (i.e., necrosis, regeneration, fibrosis). In monkeys, the effects in liver were more pronounced (and at lower doses) with longer duration of treatment. In a 1-week study, mild changes in clinical pathology parameters (including increased serum triglycerides (M), glucose (F), ALT (≥100 mg/kg), and fibrinogen, and decreased sodium (M), chloride, and phosphorus) were observed after 1-week (175 mg/kg). After a month of treatment, 125 mg/kg resulted in increased liver weights (≥ 45 mg/kg); the reported no-effect dose was 15 mg/kg. After 1-year of treatment, histopathological changes were observed in individuals (at ≥ 20 mg/kg; including mild focal necrosis, minimal to mild periportal fibrosis, mild focal serosal fibrosis, mild subserosal fibrosis, centrilobular fatty change, and/or minimal focal pigmented macrophages), liver weights were increased (at 150 mg/kg; 1.4-1.8x), and clinical pathology changes were observed (at 150 mg/kg; increased serum ALT (2-3x), GGT (50-70%), and triglycerides were increased (~2x in M)). Kidney Kidney toxicity was most clearly observed in rats (that demonstrate speciesspecific renal toxicity with aging, chronic progressive nephropathy [CPN]); however, the pattern did not strictly follow the usual pattern for CPN and some evidence was observed in other species. Kidney toxicity was observed with as little as 2-weeks of tasimelteon administration in males rats, demonstrated as hyaline droplet nephropathy. In a 1-month study in rats, increased kidney weight (only 400 mg/kg) and dose-dependent hyaline droplet nephropathy (≥ 100 mg/kg) were observed in males; however, decreased serum urea nitrogen and creatinine were observed. In the 6-month study in rats, both sexes showed 177 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. histopathological alterations with other concomitant alterations. At HD, BUN and serum creatinine continued to be slightly reduced, and serum electrolytes showed very slight but consistent changes over the treatment period (i.e., sodium and chloride slightly reduced, calcium slightly increased). Kidney weight was increased, as was urine volume. Histologically, exacerbated (slight to moderate) chronic progressive nephropathy, minimal to slight cortical tubules with hyaline droplets, minimal to slight cortical tubular pigment, and interstitial pigmented macrophages were observed in both sexes in a roughly dose-related manner. In MD and/or HD females, slight cortical tubular dilation, interstitial inflammation, and simple tubular hyperplasia were also observed. In this study, there was a suggestion of equal or slightly greater severity findings in females, which does not follow the generally male-predominant pattern accepted for CPN. In mice, kidney weights were increased in males at high doses (i.e., 600 mg/kg) in the 3month study, but were slightly reduced in the carcinogenicity bioassay and showed minimal histopathological changes (e.g., few animals with cortical tubules with hyaline droplets and/or pigment). Monkeys showed minimal evidence of renal toxicity (decreased sodium, chloride and phosphorus in the 1week study; minimal to mild chronic interstitial inflammation and medullary tubule cyst in treated males in the 1-year study). Reproductive organs A number of reproductive organ effects occurred in rodents, rabbits, and monkeys. In female rats, several effects were seen in ovary (i.e., increased ovary weight, of ovarian follicular cysts, absent corpora lutea, and interstitial or Sertoli cell hyperplasia),uterus (i.e., dilatation, luminal dilatation, glandular dilatation, glandular hyperplasia, adenocarcinomas, and squamous cell carcinomas), and uterine cervix (i.e., epithelial hyperplasia and keratinization, mural/stromal hypertrophy/hyperplasia, squamous cell carcinomas). In the mammary gland, galactocele(s), secretory activity, ductular dilatation, and a slightly increased incidence [nss] of adenocarcinoma (at 250 mg/kg) were observed. In male rats, effects were observed in the prostate gland (i.e., decreased weight, inflammation) and testis (i.e., interstitial cell hyperplasia and a slight increase in Leydig cell adenomas in the carcinogenicity bioassay). Mice showed fewer, but similar, effects, including: cysts in the ovary and uterus, decreased prostate weight (600 mg/kg), epithelial hyperplasia in the seminal vesicles, and cystic atrophy of the preputial gland. In the EFD study, rabbits demonstrated small ovary and black ovarian cysts. A few changes were also observed in monkey, including: decreased seminal vesicle and prostate and testes weights (1 –month), lactation of the mammary gland (1 HDF in the 1-year study), and a lack of corpora lutea in the ovary (HDF at terminal sacrifice in the 1year study); the sponsor indicated that the last finding could indicate a "lack of recent estrus cycling," which is consistent with the irregular estrus cycles demonstrated in rats. 178 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Hematologic System (Including Spleen) Effects on hematologic function were observed, mostly at high doses, in rodents and monkeys. Generally slight to mild (5 to 15%) reductions in red blood cell parameters were observed, with increases in reticulocytes and/or platelets. In an early (1-month) subchronic rat study, RBC polychromasia and anisocytosis were observed at 400 mg/kg. In the chronic rat study, slight increases in WBC were also observed in both sexes. Effects in the spleen (rat carcinogenicity and/or 6month rat study) reflected these alterations (i.e., increased spleen weight (F), minimal to slight hemosiderosis (M and F), and/or minimal to slight increased extramedullary hematopoiesis); the sponsor considered this reflective of an increased rate of RBC removal from peripheral blood leading to the "compensatory" response. Endocrine System Alterations in the endocrine system (i.e., adrenal, thyroid, and pituitary) were suggested. In rat, increased adrenal weights were observed but did not show clear histopathological changes; however, in the rat carcinogenicity bioassay, VEC-162-treated animals showed cortical/cystic/hemorrhagic degeneration and/or sinusoidal dilatation/congestion. In the In the 6-month study in rats, alterations were observed in endocrine organs at HD, but generally with low incidence: decreased pituitary weight, cysts and/or focal hyperplasia (and/or hypertrophy) of the pars distalis of the pituitary (6-month study, carcinogenicity bioassay), and thyroid changes (prominent ultimobranchial cysts in the 6-month study, hypertrophy, atrophy, and/or cyst in the carcinogenicity bioassay). Some of these findings may reflect the observed hepatic enzyme induction in rats (cf. Zabka et al., 2011), or may be a reflection of aging. In the 1-yr monkey study, increased thyroid weight (~40%) was observed males (≥ 20 mg/kg; but was slightly, ~20%, decreased in females); histologically, only a few instances of follicular cysts were observed in treated monkeys (not clearly dose-related). Heart Minimal evidence of alterations in heart was observed; primarily these were seen as changes in organ weight. Increased heart weights were observed in female rats, without clear histologic changes. However, heart weight was slightly reduced carcinogenicity bioassay in mice. In monkeys, minimal chronic inflammation of the heart and/or aorta was observed in a few treated animals; slightly increased blood pressure was observed in HDF late in the 1-year study. Genetic Toxicology Tasimelteon was neither mutagenic in an Ames assay nor clastogenic in an in vivo rat micronucleus assay (see review by Dr. Atrakchi, dated 1/16/98); however, an in vitro chromosomal aberration assay demonstrated clastogenic effects. It is noted that the rat micronucleus assay was conducted as part of the 1-month toxicity study with tasimelteon and had some flaws (i.e., it did not use a positive control and there is some question about the adequacy of the maximum dose tested). The sponsor also investigated the genotoxicity of M11 (a non-major human circulating metabolite that has 179 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. poor coverage in the animal species). Metabolite M11 was not mutagenic, but was clastogenic in an in vitro chromosomal aberration assay in CHO cells. Taken together, there is limited evidence that tasimelteon has genotoxic properties. It is notable that a similar compound, ramelteon, was also shown to be positive in a chromosomal aberration assay. Given the positive results in the carcinogenicity assay in rats, there would not seem to be a need to conduct additional genetic toxicology studies. Carcinogenicity Two-year carcinogenicity bioassays were conducted in rats and mice. Tasimelteon was administered orally (by gavage) at doses of 0 (vehicle: PEG-400), 20 (LD), 100 (MD), and 250 (HD) mg/kg male and female Sprague Dawley rats (65/sex/group) for up to 104 weeks. Dosing was suspended for HDF during week 101/102 when the number of survivors fell to 20, and this group was terminated at 104 weeks. All male groups were terminated at 103 weeks due to low survival in the male control group. Overall, there was no statistically significant drug-related effect on survival rates in males or females. Body weights at 102 weeks were 96%, 96%, and 93% those of control males in the LD, MD and HD groups, respectively. In females, body weights at 104 weeks were 108%, 97%, and 95% those of controls in the LD, MD, and HD groups, respectively; however, during weeks 76-88, body weight in HDF was approximately 87% that of control females. The sponsor indicated that tumors were observed in liver (MD & HD, both sexes), uterus, and uterine cervix. In addition to these tumors, there were non-significant increases in neoplasms in ovary, mammary gland, testes, and skin. FDA biostatistical evaluation indicated that only the incidence of uterine endometrial adenocarcinomas in HDF reached statistical significance. The FDA Executive CAC concluded that the observed neoplasms in liver, uterus and cervix were drug- related, based on statistical significance or exceeding the historical control (HC) range (see table below; cf. meeting minutes dated 10/16/13). Histopathologic alterations were observed in liver (e.g., centrilobular hepatocyte hypertrophy, centrilobular hepatocyte vacuolation, cystic degeneration, regenerative and bile duct hyperplasia, pigment in hepatocytes), kidney (e.g., cortical tubular pigment, tubular basophilia or hypertrophy, pelvic dilatation), ovary (e.g., cysts), uterus (glandular dilatation and hyperplasia), uterine cervix (epithelial hyperplasia and keratinization), mammary gland (duct dilatation, galactoceles), testes (interstitial cell hyperplasia), and pituitary (e.g., focal pars distalis hyperplasia and hypertrophy). 180 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. Tasimelteon was administered orally (by gavage) at doses of 0 (vehicle: PEG-400), 30 (LD), 100 (LMD), and 300 (HD) mg/kg male and female CD-1 mice (66/sex/group) for up to 104 weeks. There was no drug-related effect on survival rates in males or females. At week 104, body weight in HDM was reduced approximately 10% compared to control males. Body weight was not affected in females. No drug-related neoplasms were identified. Slight, but non-significant, increases were observed in intestines (male) and uterine cervix. Few histopathologic changes were observed in the study, most notably centrilobular hypertrophy in the liver. The sponsor postulated mechanisms underlying the observed increases in liver, uterine and uterine cervix tumors in rats that they believe to negate, or ameliorate, human relevance. The sponsor attributed the observed hepatic tumors to hepatic enzyme induction in rats. While there is some suggestive evidence (e.g., enzyme induction, hepatic hypertrophy with early evidence of increased hepatocellular mitoses), it is not clear whether the increased liver tumor incidences in rats is related to the observed hepatic enzyme induction (cf. Ennulat et al., 2010; Holsapple et al., 2006; IARC Monograph 79, 2002; Maronpot et al., 2010; Whysner et al., 1996; Williams et al.,2001; Williams, 1997; Wojcikowski & Daniel, 2009), particularly since some evidence of other potential mechanisms was observed (i.e., damage, clastogenicity). Additionally, it is notable that clear effects on thyroid were not observed. (As a side note, the observed increases in uterine and uterine cervix tumors may suggest an increase in exposures to estrogens, which is in itself a potential mechanism for hepatocarcinogenesis.) A marketed drug with a similar mechanism, ramelteon (Rozerem) also demonstrated increased hepatic tumors (although of greater incidence, and in both rats and mice) and clastogenicity; these effects were reported in the label and human relevance was not excluded. Without more objective and complete data, it is not clear that the relevance of the hepatic tumors to humans can be excluded. With regard to the reproductive organ tumors, the sponsor attributed the tumors in the uterus and uterine cervix (i.e., endometrial adenocarcinoma and squamous cell carcinoma) to reduced serum prolactin levels, possibly resulting from chronic activation 181 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. of melatonin receptors in the pars tuberalis (cf. Morgan and Williams, 1996). However, no data were provided to support this hypothesis. The sponsor also stated that similar effects were not observed in mice, so the effects may be due to a “species-specific effect of unknown aetiology on aging female rats.” Melatonin, and therefore melatonin receptors, are known to have roles in the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes (Chuff et al., 2011a; Chuff et al., 2011b; Diaz et al., 2000; Dubocovich & Markowska, 2005; Malpaux, 1999; Preslock, 1984; Slominski et al., 2012); these role(s) have not been fully elucidated. In general, melatonin is generally believed to result in a progesterone dominance over estrogens, and has been suggested to possess anticancer effects in estrogen-dependent cancers, especially breast cancer (cf., Cos et al., 2008; Dubocovich & Markowska, 2005). This does not appear to be the case with tasimelteon; although uterine endometrial tumors are generally believed to be an estrogen-dependent tumor (cf., Ito et al., 2007; Ito et al., 2011; Slominski et al., 2012), uterus and uterine cervix showed increased tumor incidences in rats. Additionally, and contrary to the sponsor’s hypothesis, mammary gland did not appear protected from carcinogenesis (as would be expected with a drug that reduced serum prolactin); in fact, a slight, but non-significant, increase in adenocarcinomas was observed at HD. In males, only a slight, non-significant increase in Leydig cell hyperplasia and adenomas was observed in testes following administration of tasimelteon. Notably, the label for marketed drug ramelteon (Rozerem) does not indicate increases in female reproductive organ or mammary gland tumors, but does indicate an increase in Leydig cell tumors of the testes; increased prolactin and decreased testosterone have been reported in humans (see Richardson et al., 2009; Richardson & Wang-Weigand, 2009). There is not sufficient objective evidence to exclude human relevance of the uterine and uterine cervix tumors. Reproductive Toxicology The reproductive effects of tasimelteon were tested in rats and rabbits. As previously suggested in the toxicity studies (e.g., effects on reproductive organs, such as the lack of corpora lutea in the ovaries of female monkeys in the chronic toxicity study), effects on female fertility and fetal development (into adulthood) were demonstrated. A slight increase in infertile pairings demonstrated in a fertility study dosing both males and females later clarified that the effect on fertility resulted from tasimelteon exposures in the females. Irregular estrus cycles, slightly increased infertile pairings, and slight reduction sin fertility parameters were observed at MD and HD in females. The embryofetal development (EFD) studies in rats and rabbits demonstrated some embryofetal toxicity. Drug-related slight delays in development and decreased fetal body weights were observed at MD and HD in rats and rabbits. Rabbit does also demonstrated an increase in abortions at HD, and slightly increased late resorptions at MD and HD (although early resorptions were increased at LD and MD, which puts the significance of this apparent change in question). Some maternal toxicity was observed at HD (i.e., clinical signs, and slight reductions in body weight and food consumption) in the dams and does. Clear toxicity in the F1 generation was demonstrated in the pre/post-natal study, particularly as developmental delays and a reduced growth rate postpartum and into 182 Reference ID: 3399188 NDA 205-677 Melissa K. Banks-Muckenfuss, Ph.D. adulthood. Limited F0 maternal toxicity was demonstrated (i.e., slightly [~3-5%] reduced body weights during gestation at MD and/or HD, and during lactation in all VEC-162-treated dams). The gestation period was very slightly increased in all VEC162-treated dams, and the live birth index was slightly reduced at HD. Pups from VEC162-treated dams showed an increased incidence of “underactive,” “swollen area on the abdomen,”and “small build.” Although average pup birth weights were similar (<5% difference; provided as the average of the litter averages for each sex), HD pups demonstrated reduced (~15%) body weights throughout lactation and into adulthood. Sexual maturation was achieved at a lower body weight in males (similar PND) but was delayed in females. Other developmental milestones (e.g., righting responses) were similarly delayed. Learning and memory testing (Morris Water maze) demonstrated clear deficits in males (i.e., trial times, sector entries, failed trials), but not females, when tested at PND 31/32; when re-tested at PND 47/49, no consistent differences were reported. A few effects were demonstrated on F1 generation reproduction. Reductions in body weight were accentuated in the adult female F1 generation during gestation, and there was some evidence for reductions in fertility parameters (e.g., corpora lutea, implantations, late resorptions, post-implantation loss %, and live births) at HD. The F1 generation was exposed to tasimelteon, M9, M12 and M14 (as measured on LD4), although to a much lesser extent than the dams. APPEARS THIS WAY ON ORIGINAL 183 Reference ID: 3399188 NDA 205-677 12 Melissa K. 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Chuffa LGA, Fábio Seiva FRF, Fávaro WJ, Teixeira GR, Amorim JPA, Mendes LO, Fioruci BA, Pinheiro PFF, Fernandes AAH, Franci JAA, Delella FK, Martinez M, & Martinez FE (2011b) Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation. Reproductive Biology and Endocrinology, 9: 108-116. Conde E, Moreno AM, Martin-Lacave I, Fernandez A, & Galera H. (1992) Immunohistochemical study of the ultimobranchial tubule in Wistar rats. Anat Histol Embryol, 21: 94-100. Derelanko MJ. “Section 10: Clinical Pathology.” Toxicologist’s Pocket Handbook, Boca Raton: CRC Press, 2000. Page 97. Diaz E, Pazo D, Esquifino AI, & Diaz B (2000) Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats. Journal of Reproduction and Fertility, 119: 151–156. Dubocovich ML & Markowska M (2005) Functional MT1 and MT2 Melatonin Receptors in Mammals. Endocrine, 27( 2), 101–110. 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Banks-Muckenfuss, Ph.D. Appendix 2: Histopathology Inventory Study Species Adrenals Aorta Bone Marrow smear Bone (femur) Brain Cecum Cervix Colon Duodenum Epididymis Esophagus Eye Fallopian tube Gall bladder Gross lesions Harderian gland Heart Ileum Injection site Jejunum Kidneys Lachrymal gland Larynx Liver Lungs Lymph nodes, cervical Lymph nodes mandibular Lymph nodes, mesenteric Mammary Gland Nasal cavity Optic nerves Ovaries Pancreas Parathyroid Peripheral nerve Pharynx Rat Monkey 6 month 6 mo or 1 yr X* X* X X X X (taken but not (rib) examined) X (incl X (rib) marrow) (also sternum, but not processed) X* X* X X X X* (with uterus) X X X X X X X X X (w/optic X (w/optic nerves) nerves) N/A X X* X X X X* X X X* X X* X X* X X X* X (w/ bronchi) X 2-Yr Bioassay Mouse X* X (thoracic) X (with joint) X (with joint) X* X X (w/uterus) X* X X (w/uterus) X X X* X X X X X* X X N/A X X X* X X X X X* X X X* X X X* X* X X* X X X* X* X X X X X X X (inguinal) X X (caudal) X (caudal) X (w/eyes) X* X X (w/thyroid) X (w/ eyes) X* X X (w/thyroid) X x* (w/oviducts) X X (w/thyroid) X X* (w/oviducts) X X (w/thyroid) 186 Reference ID: 3399188 2-yr Bioassay Rat X* X (thoracic) NDA 205-677 Pituitary Prostate Rectum Melissa K. Banks-Muckenfuss, Ph.D. X* X* X (not processed or examined) X X X* X X* X X X (not X X processed or examined) X (submandibular) Salivary gland X X (submandibular (submandibular) Sciatic nerve X X X X Seminal vesicles X* X X X Skeletal muscle X (biceps) X X (biceps X femoris, Diaphragm [not processed]) X Skin X (dorsal thorax X (dorsal X thorax) Spinal cord X X X (cervical, X lumbar, mid-thoracic) Spleen X* X* X* X* Sternum X X (w/bone marro X Stomach X X X (cardia, X fundus, pylorus) Testes X* X* X* X* Thymus X X* X X* Thyroid X* (w/ X* X* (w/ X parathyroids) /parathyroids) Tongue X X X X Trachea X X X X Urinary bladder X X X X Uterus X X* (w/cervix) X* (w/cervix) X* (w/ cervix) Vagina X X X X Zymbal gland (not processed) X (w/external ear) X, histopathology performed *, organ weight obtained Other Tissues examined: 6M Rat: pineal gland 6/12 M Monkey: pineal gland Rat Carcinogenicity: femur, preputial gland, clitoral gland, ureters Mouse Carcinogenicity: femur, preputial gland, clitoral gland, ureters 187 Reference ID: 3399188 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------MELISSA K BANKS-MUCKENFUSS 10/30/2013 LOIS M FREED 10/30/2013 Reference ID: 3399188 PHARMACOLOGY/TOXICOLOGY FILING CHECKLIST FOR NDA NDA/BLA Number:205,677 Drug Name: tasimelteon Applicant: Vanda Pharmaceuticals NDA Type: Priority Stamp Date: 5/31/13 On initial overview of the NDA/BLA application for filing: 1 2 3 4 Content Parameter Is the pharmacology/toxicology section organized in accord with current regulations and guidelines for format and content in a manner to allow substantive review to begin? Is the pharmacology/toxicology section indexed and paginated in a manner allowing substantive review to begin? Is the pharmacology/toxicology section legible so that substantive review can begin? Are all required (*) and requested IND studies (in accord with 505 b1 and b2 including referenced literature) completed and submitted (carcinogenicity, mutagenicity, teratogenicity, effects on fertility, juvenile studies, acute and repeat dose adult animal studies, animal ADME studies, safety pharmacology, etc)? Yes X No Comment X X X Generally, the standard studies appear to have been conducted; however, a standard safety pharmacology battery was not conducted and a pivotal study (6month rat; see taj0007-98348) lacks a separate, signed pathology report (as requested in the pre-NDA meeting minutes, dated 3/22/13). While the major human metabolites were not measured in the nonclinical studies, the sponsor conducted PK studies including the metabolites after oral administration in rats (29 days; parent, M3, M9, M12, M13, and M14 ; note that rat does not make M11), mice (29 days; parent and M11), pregnant rabbits (19 days; parent and M11), and monkeys (single dose only). There is a problem with the measurement of plasma M3 in rat. 5 If the formulation to be marketed is different from the formulation used in the toxicology studies, have studies by the appropriate route been conducted with appropriate formulations? (For other than the oral route, some studies may be by routes different from the clinical route intentionally and by desire of the FDA). X In most studies, tasimelteon was administered to animals by oral gavage (in PEG-400). No studies used the clinical formulation. N205677_PTFilingMemo_tasimelteon Reference ID: 3341349 PHARMACOLOGY/TOXICOLOGY FILING CHECKLIST FOR NDA 6 7 8 Content Parameter Does the route of administration used in the animal studies appear to be the same as the intended human exposure route? If not, has the applicant submitted a rationale to justify the alternative route? Has the applicant submitted a statement(s) that all of the pivotal pharm/tox studies have been performed in accordance with the GLP regulations (21 CFR 58) or an explanation for any significant deviations? Has the applicant submitted all special studies/data requested by the Division during pre-submission discussions? Yes X No Comment X This statement can be found in the Nonclinical Overview (page 4, last paragraph). X The sponsor included 2 tables specifying the identifiers used for tasimelteon main metabolites (cf. 2.6.4 Pharmacokinetics Written Summary, Table 1 pg. 10 and Table 20 pg. 53). 9 Are the proposed labeling sections relative to pharmacology/toxicology appropriate (including human dose multiples expressed in either mg/m2 or comparative serum/plasma levels) and in accordance with 201.57? X The sponsor has provided labeling, the adequacy of which is a review issue. Toxicity study margins were given as "x times the human systemic exposures at the RHD" and were sometimes given only for effect levels (e.g., for the EFD studies). A no-effect level comparison was given for the fertility studies, the pre-/post-natal study and for tumors in the carcinogenicity studies. 10 Have any impurity – etc. issues been addressed? (New toxicity studies may not be needed.) X Discussion with the CMC reviewer indicated that there are no impurity issues at this time. 11 Has the applicant addressed any abuse potential issues in the submission? If this NDA/BLA is to support a Rx to OTC switch, have all relevant studies been submitted? 12 This is not Pharm/Tox purview; the CSS have been consulted. n/a IS THE PHARMACOLOGY/TOXICOLOGY SECTION OF THE APPLICATION FILEABLE? __Yes___ Please identify and list any potential review issues to be forwarded to the Applicant for the 74-day letter: 1) We request a separate, signed, and dated Pathology Report for the pivotal 6-month toxicity study in rats (Study TAJ0007-98348). N205677_PTFilingMemo_tasimelteon Reference ID: 3341349 --------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signed electronically and this page is the manifestation of the electronic signature. --------------------------------------------------------------------------------------------------------/s/ ---------------------------------------------------MELISSA K BANKS-MUCKENFUSS 07/15/2013 LOIS M FREED 07/15/2013 Reference ID: 3341349