CENTER FOR DRUG EVALUATION AND
`RESEARCH
`
`
`
`APPLICATION NUMBER:
`
`206439Orig1s000
`
`PHARMACOLOGY REVIEW(S)
`
`
`
`
`
`
`

`

`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 23, 2014
`
`From: Lois M. Freed, Ph.D.
`Supervisory Pharmacologist
`
`Subject: NDA 206-439 (Namzaric; memantine HCl ER and donepezil HCl; Forest
`Laboratories, Inc.)
`________________________________________________________________
`
`NDA 206-439 is a 505(b)(2) application, submitted on February 26, 2014, to
`support marketing approval for Namzaric for treatment of moderate to severe
`Alzheimer’s disease. Namzaric is a combination product, containing memantine
`HCl (MEM) ER and donepezil HCl (DPZ). Doses recommended by the sponsor
`are 28 mg MEM and 10 mg DPZ (14 mg MEM and 10 mg DPZ for patients with
`severe renal impairment). Clinical development of the combination was
`conducted under IND 109,763.
`
`In support of this application, the sponsor cross-referenced two previously
`approved NDAs for MEM (NDA 21-487 for Namenda; NDA 22-525 for Namenda
`XR) and stated a reliance on FDA’s previous findings of safety and effectiveness
`for Aricept (DPZ; NDA 20-690). In addition, the following nonclinical study reports
`were provided:
`
` Two pharmacology studies of the combination in rodent (MEM-PH-10;
`MEM-PH-14)
` acute dose study of the combination in female rat (MEM-TX-29)
` 28-day neurotoxicity study of the combination in rat (MEM-TX-27)
` TK/MTD study of the combination in rat (MEM-TX-30)
`
`These studies were reviewed by Dr. Hawver (cf. Pharmacology/Toxicology NDA
`Review and Evaluation, NDA 206-439, David B. Hawver, Ph.D., 10/25/2014). Dr.
`Hawver notes that the studies MEM-TX-27 and MEM-TX-29 have previously
`been submitted and reviewed; therefore, his review focused on the
`pharmacology and TK/MTD studies. Based on his review, Dr. Hawver has
`concluded that the NDA is approvable, from a pharmacology/toxicology
`standpoint.
`
`Reference ID: 3662616
`
`1
`
`

`

`I concur with Dr. Hawver’s recommendation on the approvability of the
`application and his conclusion that the pharmacology data provided do not
`support the sponsor’s claims regarding any synergistic effects of MEM and DPZ
`on brain acetylcholine levels or on cognitive function (sponsor’s labeling, Section
`12.2). Additional comments on labeling will be provided separately.
`
`Reference ID: 3662616
`
`2
`
`

`

`---------------------------------------------------------------------------------------------------------
`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
`11/23/2014
`
`Reference ID: 3662616
`
`

`

`
`
`
`
`
`
`
`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:
`Applicant’s letter date:
`CDER stamp date:
`Product:
`Indication:
`
`Applicant:
`Review Division:
`Reviewer:
`Supervisor:
`Acting Division Director:
`Project Manager:
`
`206-439
`1
`February 26, 2014
`February 26, 2014
`Memantine HCl ER and Donepezil HCl
`Moderate to severe dementia of the Alzheimer’s
`type
`Forest Laboratories, Inc.
`Neurology Products
`David B. Hawver, Ph.D.
`Lois M. Freed, Ph.D.
`Billy Dunn, M.D.
`Teresa Wheelous
`
`
`Disclaimer
`
`Except as specifically identified, all data and information discussed below and
`necessary for approval of NDA 206-439 are owned by Forest Laboratories or are data
`for which Forest Laboratories has obtained a written right of reference.
`Any information or data necessary for approval of NDA 206-439 that Forest
`Laboratories 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 206-439.
`
`Reference ID: 3648408
`
`1
`
`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`TABLE OF CONTENTS
`
` EXECUTIVE SUMMARY .................................................................................... 3 
`1.1 
`INTRODUCTION .................................................................................................... 3 
`1.2 
`BRIEF DISCUSSION OF NONCLINICAL FINDINGS ...................................................... 3 
`1.3  RECOMMENDATIONS ............................................................................................ 5 
` DRUG INFORMATION ....................................................................................... 6 
`2.1  DRUG ................................................................................................................. 6 
`2.2  RELEVANT INDS AND NDAS ................................................................................. 7 
`2.3  DRUG FORMULATION ........................................................................................... 7 
`2.4  COMMENTS ON NOVEL EXCIPIENTS ....................................................................... 7 
`2.5  COMMENTS ON IMPURITIES/DEGRADANTS OF CONCERN ......................................... 7 
`2.6 
`PROPOSED CLINICAL POPULATION AND DOSING REGIMEN ...................................... 7 
`2.7  REGULATORY BACKGROUND ................................................................................ 8 
` STUDIES SUBMITTED ..................................................................................... 10 
`3.1 
`STUDIES REVIEWED ........................................................................................... 10 
`3.2 
`STUDIES NOT REVIEWED ................................................................................... 10 
`3.3 
`PREVIOUS REVIEWS REFERENCED ...................................................................... 10 
` PHARMACOLOGY ........................................................................................... 11 
`4.1 
`PRIMARY PHARMACOLOGY ................................................................................. 11 
`5 TOXICOLOGY ...................................................................................................... 29 
`MEMANTINE/DONEPEZIL: TOXICOKINETIC/MAXIMUM TOLERATED DOSE STUDY IN RATS ..... 29 
`6 SPONSOR’S PROPOSED PACKAGE INSERT LABELING ............................... 33 
`8.1 
`PREGNANCY ..................................................................................................... 33 
`8.3  NURSING MOTHERS ........................................................................................... 34 
`8.4 
`PEDIATRIC USE ................................................................................................. 34 
`12.1  MECHANISM OF ACTION ..................................................................................... 36 
`12.2  PHARMACODYNAMICS ........................................................................................ 37 
`13.1  CARCINOGENESIS, MUTAGENESIS, IMPAIRMENT OF FERTILITY ............................... 38 
`13.2  ANIMAL TOXICOLOGY AND/OR PHARMACOLOGY ................................................... 39 
`
` 1
`
` 
`
`2 
`
`3 
`
`4 
`
`
`
`Reference ID: 3648408
`
`2
`
`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`1 Executive Summary
`1.1
`Introduction
`This submission is a 505(b)(2) NDA for a once-daily oral capsule fixed dose
`combination of two drugs already approved for marketing in the U.S. for the
`treatment of patients with Alzheimer’s disease: memantine HCl extended release
`(ER), and donepezil HCl. For nonclinical studies to support this NDA, the sponsor
`is largely relying on studies previously submitted to approved NDAs for Namenda
`(NDA 21-487) and Namenda XR (NDA 22-525), and on the Agency’s previous
`findings of safety and effectiveness for the reference listed drug (RLD), Aricept
`(NDA 20-690).
`
`
`1.2 Brief Discussion of Nonclinical Findings
`Five nonclinical studies were included in the current submission. Two of these,
`single- and repeated-dose oral neurotoxicity studies in rat, showed that the
`combination of memantine (MEM) and donepezil (DPZ) increased the incidence,
`severity, and distribution of neurodegeneration compared with memantine alone.
`These results have already been reviewed and are adequately described in the
`current labeling for memantine and donepezil products. The third combination
`toxicity study consisted of an acute oral dose-ranging study in female rats, with
`MEM at 100 and 200 mg/kg ± DPZ at 10 and 20 mg/kg, and DPZ alone at 20
`mg/kg. Combination treatment increased mortality and the incidence and severity
`of clinical signs (e.g., convulsions, acrocyanosis, tremors, prostration, ataxia,
`labored breathing, and excessive salivation) compared to MEM or DPZ alone.
`This study is reviewed in Section 5 below.
`
`The final two studies were pharmacology studies that are reviewed in detail in
`Section 4 below. The first study compared the effects of 3-week treatments with
`MEM, DPZ, and placebo on performance on the Delayed Non-Matching To
`Sample object recognition task and on hippocampal levels of acetylcholine (ACh)
`using microdialysis in rats after a partial lesion of the fimbria-fornix. Treatment of
`lesioned rats with MEM in the drinking water for 3 weeks significantly improved
`performance on the memory task compared to placebo, but levels of brain ACh
`were not significantly affected. This experiment also included an acute treatment
`with MEM or DPZ after the 3-week treatments. However, all results reported in
`this study were difficult to interpret and unreliable due to the lack of concurrent
`acute placebo controls, insufficient numbers of animals per group, and/or the
`absence of individual animal data to allow independent analyses.
`
`The second pharmacology study explored the effects of 3-month treatments with
`sucrose (control), MEM, DPZ, or MEM + DPZ on performance in the Morris
`Water Maze and brain Aβ levels in triple transgenic 3xTg-AD mice age 6 to 9
`months (young mice); and the same parameters, as well as brain levels of APP,
`C99, C83, HT7-reactive tau, PHF-1-reactive phosphor-tau, and AT8-reactive
`phospho-tau, in 3xTg-AD mice age 15 to 18 months (old mice). Treatment of
`
`Reference ID: 3648408
`
`3
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`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`young mice with the combination of MEM + DPZ improved performance on the
`MWM, but did not change brain levels of APP, soluble Aβ40 or Aβ42, or insoluble
`Aβ40 or Aβ42 compared to controls. Treatment of old mice with the combination
`MEM + DPZ also improved performance on the MWM, but increased brain levels
`of AT8-reactive phospho-tau and insoluble Aβ40 and Aβ42 compared to controls.
`These pathophysiological changes would seem to be in the opposite direction of
`those expected for an effective treatment for Alzheimer’s disease, according to
`most current theories. No explanation was provided for the apparent mismatch
`between effects on performance and effects on AD-related pathophysiology. No
`statistical comparisons were made between the groups treated with the
`combination of MEM + DPZ and the groups treated with MEM or DPZ alone.
`Finally, individual animal data were not provided to allow independent analyses,
`so all conclusions based on these data cannot be verified.
`
`
`Reference ID: 3648408
`
`4
`
`

`

`NDA 206-439
`
`1.3 Recommendations
`
`
`
`David B. Hawver, Ph.D.
`
`1.3.1 Approvability
`
`From a Pharmacology/Toxicology perspective, this application is approvable.
`
`1.3.2 Additional Non Clinical Recommendations
`
`None
`
`1.3.3 Labeling
`The following changes should be made to sections of the sponsor’s proposed
`labeling that contain nonclinical information:
`
`1. References to the maximum recommended human dose (MRHD) should be
`revised such that they refer to the MRHD of 28 mg/10 mg NAMZARIC rather
`than to the MRHD of MEM or DPZ.
`
`2. Dose comparisons of NOAEL doses of DPZ in animals to the MRHD should
`be based on 10 mg/day DPZ in the MRHD of 28 mg/10 mg, rather than on
`.
`
`3. Descriptions of the nonclinical studies in Section 8.4 Pediatric Use in the
`current label for Namenda XR should be inserted into the same section of
`the label for Namzaric.
`
`4.
`
`5. In Section 13.2 Animal Toxicology and/or Pharmacology, the heading
`
`Detailed labeling recommendations are located in Section 5 of this review.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Reference ID: 3648408
`
`5
`
`(b) (4)
`
`(b) (4)
`
`(b) (4)
`
`

`

`
`
`David B. Hawver, Ph.D.
`
`Namzaric
`
`NDA 206-439
`
`2 Drug Information
`2.1 Drug
`Brand Name:
`
`Generic Name:
`
`
`
`Code Name:
`
`
`Chemical Name:
`Memantine HCl: 1-amino-3,5-dimethyladamantane hydrochloride
`Donepezil HCl: (±)-2, 3-dihydro-5, 6-dimethoxy2-[[1-(phenylmethyl)-4
`piperidinyl]methyl]-1H-inden-1-one hydrochloride
`
`Memantine HCl Extended Release (ER) and
`Donepezil HCl Fixed Dose Combination Capsules
`
`MDX-8704
`
`
`Molecular Formula:
`Memantine HCl: C24H29NO3•HCl
`Donepezil HCl: C12H21N•HCl
`
`
`Molecular Weight:
`Memantine HCl: 215.77
`Donepezil HCl: 415.95
`
`
`Structure or Biochemical Description:
`
`Memantine HCl:
`
`
`
`
`
`
`
`Donepezil HCl:
`
`
`
`
`Pharmacologic Class:
`Memantine HCl: N-methyl-D-aspartate (NMDA) receptor antagonist
`Donepezil HCl: Acetylcholinesterase inhibitor
`
`
`
`
`
`
`Reference ID: 3648408
`
`6
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`

`

`NDA 206—439
`
`David B. Hawver, PhD.
`
`2.2
`
`Relevant INDs and NDAs
`
`IND 109763 Memantine HCI ER and Donepezil HCI Fixed Dose Combination for
`Moderate to Severe Dementia of the Alzheimer’s Type
`NDA 21—487 Memantine HCI (Namenda) for Moderate to Severe Dementia of the
`Alzheimer’s Type
`NDA 22-525 Memantine HCI ER (Namenda ER) for Moderate to Severe
`Dementia of the Alzheimer’s Type
`NDA 20-690 Donepezil HCI (Aricept) for Mild to Severe Dementia of the
`Alzheimer’s Type
`
`2.3
`
`Drug Formulation
`
`Size 1 blue opaque oral capsules containing 28 mg memantine HCI ER/10 mg
`donepezil HCI.
`
`Size 2 green opaque oral capsules containing 14 mg memantine HCI/10 mg
`donepezil HCI.
`
`Memantine HCI is formulated as
`
`, consisting of sugar sphere
`
`(b) (4)
`
`(5) (4)
`
`Donepezil HCI is formulated as
`
`“m
`
`2.4
`
`Comments on Novel Excipients
`
`No novel excipients were used in the clinical formulation.
`
`2.5
`
`Comments on Impurities/Degradants of Concern
`
`No concerns.
`
`2.6
`
`Proposed Clinical Population and Dosing Regimen
`
`Namzaric 28 mg/10 mg is indicated for the treatment of patients with moderate to
`severe dementia of the Alzheimer’s type who are currently stabilized on
`memantine HCI (10 mg twice daily or 28 mg extended release once daily) and
`donepezil (10 mg once daily).
`
`Namzaric 14 mg/10 mg is indicated for the treatment of patients with moderate to
`severe dementia of the Alzheimer’s type who have severe renal impairment and
`are currently stabilized on memantine HCI (5 mg twice daily or 14 mg extended
`release once daily) and donepezil (10 mg once daily).
`
`Reference ID: 3648408
`
`

`

`
`
`David B. Hawver, Ph.D.
`
`NDA 206-439
`
`
`2.7 Regulatory Background
`This is a 505(b)(2) NDA submission for a fixed dose combination oral capsule
`formulation for the treatment of moderate to severe dementia of the Alzheimer’s
`type. As such, it relies on the Agency’s previous findings of safety and
`effectiveness for the RLD, Aricept (NDA 20-690), and references approved NDAs
`for Namenda (NDA 21-487) and Namenda XR (NDA 22-525) previously
`submitted by the current sponsor, Forest Laboratories, Inc. IND 109763
`
`Donepezil HCl (oral tablets; 5 mg, 10 mg) was approved on November 25, 1996,
`for the treatment of mild to moderate dementia of the Alzheimer’s type
`(NDA 20-690 Aricept, Eisai America, Inc.). The indication was expanded to
`include severe Alzheimer’s disease on October 13, 2006. Donepezil HCl 23 mg
`(oral tablet) was approved on July 23, 2010, for the treatment of moderate to
`severe dementia of the Alzheimer’s type (NDA 22-568 Aricept 23 mg, Eisai, Inc.).
`
`Memantine HCl (oral tablet; 5 mg, 10 mg) was approved on October 16, 2003, for
`the treatment of moderate to severe dementia of the Alzheimer’s type
`(NDA 21-487 Namenda, Forest Laboratories, Inc.). Memantine HCl XR (oral
`capsule; 7 mg, 14 mg, 21 mg, 28 mg) was approved on June 21, 2010, for the
`treatment of moderate to severe dementia of the Alzheimer’s type (NDA 22-525
`Namenda XR, Forest Laboratories, Inc.).
`
`IND 109763 was submitted by Adamas Pharma, Inc., on September 15, 2010, to
`support the development of ADS-7803 (memantine HCl and donepezil HCl)
`capsules for the treatment of moderate to severe dementia of the Alzheimer’s
`type. The initial proposed clinical protocol was allowed to proceed.
`
`At an End of Phase 2 meeting with Adamas Pharma, Inc., on October 13, 2011,
`the only nonclinical issue discussed was the need for a single-dose oral
`neurotoxicity study in rats with memantine alone, donepezil alone, and both
`drugs in combination: “We continue to recommend that the study be conducted
`concurrent with Phase 3 clinical trials; however, if the study is not available at the
`time of NDA submission, it will be a post-marketing requirement, unless we have
`determined that you no longer need to conduct the study.” This study was
`submitted as a post-marketing requirement to NDA 22-525, and has also been
`submitted to the current NDA.
`
`At a Type C meeting with Adamas Pharmaceuticals, Inc. on June 20, 2013, no
`nonclinical issues were discussed.
`
`IND 109763 was transferred from Adamas Pharma, Inc. to Forest Research
`Institute, Inc., a subsidiary of Forest Laboratories, Inc., on July 12, 2013.
`
`At a Pre-NDA meeting with Forest Research Institute, Inc. on November 19,
`2013, the Division agreed that the completed pharmacology and toxicology
`
`Reference ID: 3648408
`
`8
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`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`program that led to FDA’s previous finding of safety and effectiveness for the
`RLD, Aricept, reference to the completed pharmacology and toxicology program
`submitted for the approval of Namenda, as well as the additional studies
`described in the Pre-NDA briefing package, supported the review and potential
`approvability of MDX-8704 (ADS-7803) for the indication of moderate to severe
`dementia of the Alzheimer’s type.
`
`NDA 206-439 Namzaric (memantine HCl ER and donepezil HCl) for the
`treatment of moderate to severe dementia of the Alzheimer’s type was submitted
`on February 26, 2014, by Forest Laboratories, Inc.
`
`
`Reference ID: 3648408
`
`9
`
`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`3 Studies Submitted
`3.1 Studies Reviewed
`Combined Effect of Donepezil and Memantine on Hippocampal Acetylcholine
`Release and Recognition Memory in Freely Moving Rats
`(Study MEM-PH-10)
`
`Therapeutic Administration of Donepezil and Memantine in the Triple Transgenic
`Mice: Evaluation for Treatment of Established Neuropathology and Cognitive
`Impairments
`(Study MEM-PH-14)
`
`Memantine/Donepezil: Toxicokinetic/Maximum Tolerated Dose Study in Rats
`(Non-GLP Study MEM-TX-30)
`(Not previously submitted or reviewed; results were used to select doses for GLP
`Study MEM-TX-29)
`
`
`
`3.2 Studies Not Reviewed
`
`Memantine/Donepezil: A 28-Day Oral Toxicity Study in Rats
`(GLP Study MEM-TX-27)
`(Previously reviewed under IND
`
`)
`
`
`
`Memantine/Donepezil: A Single Oral Dose Toxicity Study in Female Rats
`(GLP Study MEM-TX-29)
`(Previously reviewed under IND
`
`)
`
`
`3.3 Previous Reviews Referenced
`IND
` Memantine for AD Pharmacology/Toxicology Review dated October
`12, 2012, David B. Hawver, Ph.D.; Single Dose Oral Combination
`Neurotoxicity Study in Rat
`IND
` Memantine for AD Pharmacology/Toxicology Review dated June 03,
`2010, David B. Hawver, Ph.D.; 28-Day Oral Combination Neurotoxicity Study
`in Rat
`NDA 22525 Namenda ER for AD Pharmacology/Toxicology Review dated June
`15, 2010, David B. Hawver, Ph.D.
`NDA 21487 Namenda for AD Pharmacology/Toxicology Review dated October
`09, 2003, Kathy Haberny, Ph.D.
`NDA 20690 Aricept for AD Pharmacology/Toxicology Review dated July 24,
`1996, Barry N. Rosloff, Ph.D.
`
`
`
`Reference ID: 3648408
`
`10
`
`(b) (4)
`
`(b) (4)
`
`(b) (4)
`
`(b) (4)
`
`

`

`NDA 206—439
`
`David B. Hawver, Ph.D.
`
`4
`
`Pharmacology
`
`4.1
`
`Primary Pharmacology
`
`Two primary pharmacology studies were submitted in support of the sponsor’s
`proposed changes to the mechanism of action section of the labeling for Namzaric from
`the language used in the current labels for each component drug; neither study was
`needed to support approval of NBA 206-493. These studies are reviewed below.
`
`Combined effect of donepezil and memantine on hippocampal acetylcholine
`release and recognition memory in freely moving rats
`
`(Study MEM-PH-10; conducted by
`
`Methods
`
`Final report May 22, 2008)
`
`“9‘"
`
`(ll) (4)
`
`A total of 49 male Wistar rats (National laboratory animal center,
`)
`weighing 400—550 9 (age 32 weeks) at the beginning of the experiment were used for
`the study. The animals were first trained to delayed non-match to sample object
`recognition task performance (DNMS task). The training phase was continued until the
`animal reached a criterion of 80% or more correct choices on three consecutive testing
`days (pre—lesion performance). Thereafter the rats were divided into the following 5
`treatment groups:
`
`Group A (sham lesion + placebo + acute MEM 5.0 mg/kg), 13 rats
`Group B (FF-lesion + subchronic MEM 30 mg/kg/day + acute DPZ 2.5 mg/kg), 8
`rats
`
`Group C (FF-lesion lesion + subchronic DPZ 2.5 mg/kg/day + acute MEM 5.0
`mg/kg), 14 rats
`Group D (FF-lesion + placebo + acute MEM 5.0 mg/kg), 6 rats
`Group E (FF-lesion + placebo + acute DPZ), 8 rats
`
`Each animal was randomly assigned to sustain either sham lesion or a partial fimbria-
`fornix lesion. The lesion group received bilateral electrolytic lesions, produced by
`passing 500 pA anodal current for 40 sec through a tungsten electrode (0.1 mm in
`diameter, un-insulated about 0.75 mm at the tip of the electrode). The lesion
`coordinates were 1.5 mm posterior to bregma; 0.7 mm and 1.6 mm lateral to midline;
`4.4 mm and 4.5 mm below dura for fornix and fimbria, respectively. The sham-lesioned
`group was treated identically, but the electrode tip was only lowered 2.0 mm below dura
`and no current was applied. Finally, the microdialysis guide cannula (
`“m
`) was implanted just above the right dorsal
`hippocampus (mm from bregma: AP -4.4 mm; L — 2.5 mm; V — 1.6 mm).
`
`One day prior to initiation of microdialysis experiments, the microdialysis probe was
`inserted through the guide cannula and the hippocampus was continuously perfused
`with the Ringer solution (145 mmol/L NaCl, 2.7 mmol/L KCI, 1.2 mmol/L CaClz, and 1.0
`mmollL MgClz) at a rate of 0.5 uUmin for 18 h (
`"’"°
`
`Reference ID: 3648408
`
`1 1
`
`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`). The next day, the perfusion speed was increased to 2 μL/min and 750 nM
`neostigmine was added to the perfusion fluid to prevent hydrolysis of ACh during
`sample collection. The perfusion was continued for the next 2.5 h before sampling the
`dialysate. The total sampling time was 2 h 30 min and included 12 samples (each 10
`min, 20 μL).
`
`Delayed non-match to sample object recognition task
`The rectangular apparatus was made of perspex glass (41 x 27 x 35 cm; length, width
`height) and was divided into two compartments. On the opposite side of the apparatus
`(goal area) was a separate hole-board (23 x 13 x 1 cm) that had six drilled food wells
`(2.0 cm in diameter and 1.0 cm in depth) in two evenly spaced parallel rows. The
`objects were glued onto a square, thin metal plate (4 x 4 cm). The rats were able to
`easily remove the objects from the top of the food well.
`
`Pre-training: During the first days of pre-training, each animal was handled and allowed
`to explore test apparatus for 20 min. The animals learned to displace the object above
`the food consistently within 3-5 days.
`
`Training: On the second week of training, the delayed non-match to sample (DNMS)
`protocol was introduced. Each animal received 20 trials per day. The training phase
`continued until the animal reached a criterion of 80% or more correct choices on three
`consecutive sessions (pre-lesion performance). The rats reached the criterion
`approximately after 5.5 weeks of training.
`
`Test 1: Test 1 took place 17 days after fimbria-fornix lesion. The animals were pre-
`trained on the task three days before Test 1 was performed. The result of Test 1 was
`calculated as the mean score on two testing days.
`
`Test 2: Test 2 took place one day after Test 1. The animals were treated acutely either
`with memantine (5.0 mg/kg) or donepezil (2.5 mg/kg) 1 h before task performance.
`
`ACh levels in microdialysis samples were determined using LC-MS methods validated
`according to the FDA guideline on bioanalytical method validation. The limit of
`quantification was 0.15 nM (1.5 fmol injected) and linearity was maintained over the
`concentration range of 0.15 – 73 nM. Minimum sample size was 15 μL. The accurate
`placement of microdialysis cannulae, the size of the fimbria-fornix lesion, and AChE
`staining density were verified in all animals by histological analysis after histochemical
`staining of sections for AChE.
`
`Results
`Part A. Object recognition task
`Model validation
`Prior to lesioning of the fimbria-fornix, 5.5 weeks of training on the DNMTS task was
`sufficient for all groups to reach the pre-specified criterion of 80% or greater correct
`choices on three consecutive testing days.
`
`
`Reference ID: 3648408
`
`12
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`(b) (4)
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`

`
`
`David B. Hawver, Ph.D.
`
`NDA 206-439
`
`Bilateral electrolytic lesion of the fimbria-fornix resulted in impaired performance on the
`DNMTS object recognition task compared to sham-lesioned animals and compared to
`pre-lesion performance, as well as a decrease in the number of AChE-positive neurons
`in the dorsal hippocampus of more than 50% compared to sham-lesioned rats.
`
`Subchronic drug effects
`Oral administration of MEM for 3 weeks significantly improved performance of lesioned
`rats on the DNMTS task compared to treatment with placebo, whereas treatment with
`DPZ resulted in a modest improvement that was not statistically different from treatment
`with placebo.
`
`Acute drug effects
`No significant differences were observed in DNMTS performance among the following
`groups: sham lesioned + 3 wks placebo + acute MEM; lesioned + 3 wks MEM + acute
`DPZ; lesioned + 3 wks DPZ + acute MEM; lesioned + 3 wks placebo + acute MEM; and
`lesioned + 3 wks placebo + acute DPZ. No statistical comparisons were provided for
`values in Figure 6 vs those in Figure 7. In view of the lack of concurrent control groups
`receiving acute placebo treatment, no conclusions can be drawn from these data.
`
`Part B. Hippocampal acetylcholine release during object recognition task
`Model validation
`As shown in the sponsor’s Figure 8 below, hippocampal extracellular ACh levels
`measured using in vivo microdialysis increased from a baseline of ~20 nM to peak of
`~47 nM during exploration of the empty holeboard, then to successive peaks of ~65 nM
`during the two trials of the DNMTS object recognition task. Though not explicitly stated,
`it seems likely that the values in Figure 8 were obtained from a single representative
`animal, since no error bars were included and the peak values are different from the
`mean baseline, holeboard, and task values presented in Figure 9 below. The sponsor
`notes that “The task-induced fluctuation in ACh release was similar in all treatment
`groups,” which is reflected in Figure 9.
`
`
`Reference ID: 3648408
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`13
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`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`
`
`Figure 8. Hippocampal acetylcholine release during the DNMS task performance. Baseline
`(samples 1-3), empty holeboard (sample 4), task phases (samples 7 and 10). Each sample was
`collected during a 10-minute interval.
`
`
`Subchronic drug effects
`As shown in the sponsor’s Figure 9 below, no significant differences were observed
`between groups in the hippocampal extracellular ACh levels measured at baseline,
`during holeboard exploration, or during task performance. The sponsor notes that the
`lack of reduction in ACh release due to the fimbria-fornix lesion (i.e. lesion + placebo vs.
`sham + placebo), most likely reflects a compensatory increase in ACh release from the
`remaining cholinergic terminals, since the fimbria-fornix lesion was intentionally only
`partial. The sponsor cites two earlier studies showing no change in hippocampal ACh
`release after similar fimbria-fornix lesions (Erb et al., 1997, Neurosci Lett 231(1):5-8;
`and Lapchak et al., 1991, J Neurosci 11(9):2821-2828). The group treated with MEM for
`3 weeks showed an apparent modest increase in ACh levels at all three timepoints, but
`the increases were not statistically significant compared to the lesion + placebo group.
`
`
`Reference ID: 3648408
`
`14
`
`

`

`NDA 206-439
`
`
`
`
`David B. Hawver, Ph.D.
`
`
`
`
`
`Figure 9. Effects of subchronic drug administration and task phase on hippocampal acetylcholine
`release in male Wistar rats. Data are given as means ± SEM. ANOVArm: F(3,34)=0.8, P=0.49
`(between groups); F(2,31)=57.8, P<0.001 (task phase).
`sham = sham lesion + placebo
`MEM = fornix lesion + MEM (30 mg/kg/day p.o.) for 3 weeks
`DPZ = fornix lesion + DPZ (2.5 mg/kg/day p.o.) for 3 weeks
`placebo = fornix lesion + placebo
`
`
`Acute drug effects
`As shown in the sponsor’s Figure 10 below, acute administration of MEM or DZP
`resulted in some statistically significant differences in the level of extracellular ACh in
`the hippocampus among the groups tested. However, once again, the lack of
`appropriate concurrent control groups makes these differences difficult to interpret. For
`example, ACh levels were significantly increased in rats receiving MEM for 3 weeks
`followed by acute DPZ compared to those receiving DPZ for 3 weeks followed by acute
`MEM (P=0.001). It is unclear whether this difference is due to the differences in
`subchronic treatment, acute treatment, or both. The groups receiving acute MEM after
`subchronic DPZ or subchronic placebo showed higher ACh levels than those receiving
`acute DPZ after subchronic MEM or subchronic placebo (P=0.003); while this may be
`due to the acute MEM, the difference in subchronic treatments complicates the issue.
`Even a direct comparison of the two subchronic placebo groups (placebo + acute MEM
`vs. placebo + acute DPZ) would not be as informative as comparing each of those to a
`group receiving subchronic placebo + acute placebo. Finally, the difference in ACh
`levels between the DPZ + acute MEM group and the placebo + acute MEM group was
`not statistically significant (P=0.11). Whether this means that the subchronic DPZ
`treatment did not meaningfully change the hippocampal response to acute MEM, or that
`
`Reference ID: 3648408
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`15
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`
`
`David B. Hawver, Ph.D.
`
`NDA 206-439
`
`the number of animals in each group was too small (N=7-8) to confirm that such a
`change may have occurred is unclear. The sponsor also notes, correctly, that acute
`DPZ would not be expected to increase ACh levels under the conditions tested,
`because AChE is already maximally inhibited by the 750 nM neostigmine present in the
`microdialysis perfusion medium to prevent breakdown of ACh before it can be
`measured.
`
`
`
`
`Figure 10. Effect of acute drug injection and task phase on hippocampal acetylcholine release.
`Data are given as means ± SEM.
`
`Overall statistics:
`– ANOVArm: F(4,31)=5.0, P=0.003 (between groups); F(2,30)=58.9, P<0.001 (task phase).
`– One-way ANOVA: baseline P=0.021; holeboard P=0.001; OR task P=0.008 (between groups)
`
`Selected group comparisons:
`– acute MEM groups (D+M and 0+M) vs. acute DPZ groups (M+D and 0+D): ANOVArm:
`F(1,25)=10.5, P=0.003 (between groups); t-test: baseline P=0.012, holeboard P=0.001, OR
`task P=0.009.
`– comparison between acute MEM groups (D+M vs. 0+M): ANOVArm: F(1,13)=2.9, P=0.11
`(between groups).
`– order of administration D+M vs M+D: ANOVArm F(1,12)=18.09, P=0,001 (between groups).
`
`sham 0+M = sham lesion + placebo + acute MEM (5 mg/kg i.p.)
`M+D = fornix lesion + MEM (30 mg/kg/day p.o.) for 3 weeks + acute DPZ (2.5 mg/kg i.p.);
`D+M = fornix lesion + DPZ (2.5 mg/kg/day p.o.) for 3 weeks + acute MEM (5 mg/kg i.p.);
`0+M = f