`
`
`
`A 6-MONTH ORAL (GAVAGE) TOXICITY
`STUDY OF DIMETHYL FUMARATE
`IN CD®IGS RATS
`
`FINAL REPORT
`
`Volume 1 of 3
`
`Guideline
`
`OECD (408)
`
`Study Director
`
`Mark A. Morse, Ph.D., DABT
`
`Study Completed on
`
`May 12, 2006
`
`Performing Laboratory
`
`Charles River Laboratories
`Preclinical Services
`640 North Elizabeth Street
`Spencerville, OH 45887
`
`Study No.
`
`EBA00016
`
`Sponsor Study No.
`
`P00012-04-06
`
`Submitted to:
`
`Biogen Idec Inc.
`14 Cambridge Center
`Cambridge, MA 02142
`
`
`
`Page 1 of 1258
`
`Page 1 of 25
`
`Biogen Exhibit 2273
`Coalition v. Biogen
`IPR2015-01993
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(2)
`
`1. COMPLIANCE STATEMENT
`
`This study was conducted in compliance with the Food and Drug Administration
`(FDA) Good Laboratory Practice regulations (GLP) as set forth in Title 21 of the U.S.
`Code of Federal Regulations, Part 58; the Organisation for Economic Cooperation
`and Development (OECD) Principles of Good Laboratory Practice, C(97)186; and
`the Japanese Ministry of Health, Labor and Welfare (MHLW) Ordinance No. 21 ; with
`the following exceptions:
`
`Toxicokinetic analysis, performed by the Sponsor, was not performed in a GLP
`environment, but was conducted using appropriate analytical procedures and was
`reviewed for completeness and accuracy. The characterization of the test article
`was conducted by the Sponsor in a laboratory that employs current Good
`Manufacturing Practice procedures.
`
`Mark A Morse, Ph.D. , DABT
`Study Director
`Charles River Laboratories
`Preclinical Services, Ohio
`
`Date ) / /~ /o 6
`
`Page 2 of25
`
`

`

`(3)
`
`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`
`
`2. QUALITY ASSURANCE STATEMENT
`
`This study has been inspected by the Quality Assurance Unit to assure conformance
`with the Good Laboratory Practice (GLP) regulations promulgated by FDA, 21 CFR
`Part 58; OECD Principles of Good Laboratory Practice C(97)186; and Japanese
`MHLW Ordinance No. 21. Reports were submitted in accordance with Standard
`Operating Procedures as follows:
`
`
`QA INSPECTION DATES
`
`Dates Findings Submitted to:
`
`Study
`Director
`Study
`Management
`Director
`07/16/04
`07/16/04
`07/27/04
`07/27/04
`08/09/04
`08/09/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`09/02/04
`09/02/04
`09/28/04
`09/28/04
`11/02/04
`11/02/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/22/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/30/04
`11/30/04
`12/14/04
`12/14/04
`12/14/04
`12/14/04
`12/15/04
`12/15/04
`12/15/04
`12/15/04
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`
`
`
`Phase(s) Inspected
`Test Article Receipt
`Animal Receipt
`Protocol Review
`Body Weights
`Randomization
`Animal Identification
`Clinical Observations
`Dose Preparation
`Analytical Sampling
`Toxicokinetic Blood Collection
`Plasma Processing
`Dosing
`Food Consumption
`Data Audit
`Data Audit
`Retention Sample
`Protocol Amendment Review
`Clinical Pathology Blood Collection
`Biochemistry
`Hematology
`Urine Collection
`Urinalysis
`Clinical Observations
`Body Weights
`Food Consumption
`Dosing
`Dose Preparation
`Toxicokinetic Blood Collection
`Plasma Processing
`Toxicokinetic Blood Collection
`Plasma Processing
`Urine Collection
`Clinical Pathology Blood Collection
`Necropsy
`
`
`
`
`
`
`Dates of
`Inspection
`07/16/04
`07/27/04
`08/04/04
`08/16/04
`08/16/04
`08/16/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/17/04
`08/31/04
`09/28/04
`11/02/04
`11/11/04
`11/16/04
`11/16/04
`11/16/04
`11/16/04
`11/16/04
`11/23/04
`11/23/04
`11/23/04
`11/23/04
`11/30/04
`12/14/04
`12/14/04
`12/15/04
`12/15/04
`02/16/05
`02/16/05
`02/16/05
`
`Page 3 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(4)
`
`Dates of
`Inspection
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`03/23/05, 04/ 13/05,
`04/14/05, 05/03/05,
`05/05/05, 05/06/05
`08/12/05
`08/12/05, 08/ 13/05
`01 /12/06
`05/12/06
`
`Phase(s) Inspected
`Organ Weights
`Hematology
`Biochemistry
`Urinalysis
`Coagulation Analysis
`Data Audit
`
`Protocol Amendment Review
`Draft Report Review
`Revised Draft Report Review
`Final Report Review
`
`Dates Findings Submitted to:
`Study
`Director
`Management
`02/16/05
`02/16/05
`02/16/05
`02/ 16/05
`02/ 16/05
`05/27/05
`
`Study
`Director
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`02/16/05
`05/27/05
`
`08/16/05
`08/16/05
`01/12/06
`05/12/06
`
`08/16/05
`08/16/05
`01 /12/06
`05/12/06
`
`The QA Statements for the following test sites have been reviewed:
`
`Test Sites
`Charles River Laboratories, Preclinical Services,
`Massachusetts
`Charles River Laboratories, Pathology
`Associates. Ohio
`Charles River Laboratories, Preclinical Services,
`Massachusetts
`Biogen Idee Inc., Cambridge, Massachusetts
`
`QA Statement Location
`Appendix C
`
`Appendix P
`
`Appendix R
`
`Appendix S
`
`The final report has been reviewed to assure that it accurately describes the
`materials and methods, and the reported results accurately reflect the raw data.
`
`. ~) 1(~)
`!:
`
`Kelly Landin, B.S.
`Associate Quality Assurance Auditor
`
`Date _..::::6_,_/...L.;..:::...;L...L./.>...:o~k~-
`
`Page 4 of25
`
`

`

`(11)
`
`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`
`
`6. ABSTRACT
`
`The objective of this study was to evaluate the potential toxicity of Dimethyl fumarate
`(DMF) when administered daily to rats by oral (gavage) administration for six months
`with a one-month recovery period. Doses of 0 (0.8% HPMC), 25, 100, and
`200 mg/kg/day DMF were administered by daily oral gavage to male and female
`rats. There were 15/sex/group for main study animals and 5/sex/group for recovery.
`Concurrent toxicokinetics were conducted in a separate set of animals (8/sex/group)
`to evaluate systemic exposure to DMF at each dose level.
`
`There were no deaths attributable to test article administration. Test article-related
`clinical signs during the dosing period were salivation observed in the mid-and
`high-dose groups prior to dosing and salivation observed post-dose. Males treated
`with 200 mg/kg/day DMF had mean body weights lower than controls from day 7 on,
`with statistically significant differences ranging from 6.8% to 9.6% between day 112
`and day 181. There were no toxicologically meaningful treatment-related differences
`in food consumption, ocular evaluations, hematological, urinalysis or coagulation
`parameters in males or females.
`
`Gross necropsy observations, organ weight data, and histopathological findings
`indicated test article-related effects on the kidney, stomach and liver. Enlarged
`kidneys and dose-related increases in absolute and relative to brain weight kidney
`weights were observed at the end of the dosing period (day 182/183) in both males
`and females of the 100 and 200 mg/kg/day groups and in males of the 100 and
`200 mg/kg/day groups at recovery. Test-article related findings in male rats included
`an increased incidence and severity of nephropathy (100 and 200 mg/kg dose
`groups only). In addition, other renal findings not classically associated with
`nephropathy, including cortical tubular changes (diffuse dilation, hyaline droplet
`accumulation, nuclear/cellular hypertrophy of epithelial cells, segmental epithelial
`regeneration) and hypertrophy of the parietal epithelium of Bowman’s capsule were
`observed in males in all treatment groups. Test-article related findings in the
`kidneys of female rats on day 182/183 included hyaline droplet accumulation and
`hypertrophy of cortical tubular epithelial cells at the 100 and 200 mg/kg/day dose
`levels. At recovery, the character and severity of test article-related findings in the
`kidney were generally similar to those at the main study time point; however, the
`incidence was decreased in some dose groups.
`
`At the end of the dosing period, gross necropsy findings of prominent epithelials of
`the stomach were observed primarily in animals of the 100 and 200 mg/kg/day
`groups. This correlated with dose-related increases in absolute and relative
`stomach weights in both males and females of the 100 and 200 mg/kg/day groups.
`In the nonglandular stomach, test article-related findings on day 182/183 were
`
`Page 5 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(12)
`
`
`
`squamous epithelial hyperplasia and hyperkeratosis (25, 100 and 200 mg/kg/day
`groups), subacute inflammation (100 and 200 mg/kg/day groups), squamous cell
`carcinoma (one 200 mg/kg/day male), and squamous papilloma (one 100 mg/kg/day
`male). In the glandular stomach on day 182/183, subacute inflammation was
`considered test article-related at the 100 and 200 mg/kg/day dose levels. At
`recovery, test article-related findings were limited to the nonglandular stomach and
`occurred at a much lower incidence and severity than at the main study time point.
`Stomach changes at recovery consisted of minimal hyperkeratosis (100 and
`200 mg/kg/day groups), minimal squamous epithelial hyperplasia (all treated dose
`levels), and keratinized cysts (200 mg/kg/day).
`
`On day 182/183, lymphoid hyperplasia of pancreatic or abdominal lymph nodes was
`seen in a few animals receiving 200 mg/kg/day DMF, possibly secondary to stomach
`inflammation. Dose-related increases in absolute and relative liver weights were
`observed on day 182/183 in both males and females of the 100 and 200 mg/kg/day
`groups. Histopathological findings in the liver of test article-treated main study rats
`included minimal multifocal hepatic necrosis and minimal bile duct hyperplasia
`(predominantly females of the 100 and 200 mg/kg/day groups). At the end of the
`recovery period, minimal hepatic necrosis was present but at a lesser incidence in
`female recovery animals. No evidence of bile duct hyperplasia was observed in
`recovery animals, indicating complete recovery of this change.
`
`Toxicokinetics confirmed exposure in all animals tested. DMF showed rapid and
`dose-independent absorption. There was no apparent accumulation of MMF across
`dosing days. AUC increased with dose in an approximate-dose-proportional
`manner; however, Cmax did not show a consistent trend of proportionality to dose.
`There was consistent gender difference in exposure indicated by AUC values on
`day 181, which were 20-99% higher in females than in males. Elimination half-life of
`MMF was estimated to be in the range of 20 minutes to one hour.
`
`The target organs were identified as the kidney, stomach, and liver in both males
`and females. Neither a no-observed-effect level (NOEL) nor a no-observed-
`adverse-effect level (NOAEL) could be identified for male or female rats due to
`changes in the forestomach and kidney.
`
`
`Page 6 of 25
`
`

`

`(13)
`
`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`
`
`7. SUMMARY
`
`The purpose of this study was to evaluate the potential toxicity of the test article
`when administered daily to rats by oral (gavage) administration for at least six
`consecutive months with a one-month recovery period. For this purpose, rats were
`randomly assigned to four study groups as outlined in the table below:
`
`
`Concentration
`of Dosing
`Solution
`(mg/mL)
`0
`2.5
`10
`20
`
`Dosing
`Volume
`(mL/kg)
`10
`10
`10
`10
`
`No. of Animals
`(Recovery)
`[Satellite TK]
`Dose Level
`(mg/kg/day)
`Test Material
`Group
`Male
`Female
`0
`Vehicle
`15 (5) [8]
`15 (5) [8]
`1
`25
`BG00012
`15 (5) [8]
`15 (5) [8]
`2
`100
`BG00012
`15 (5) [8]
`15 (5) [8]
`3
`200
`BG00012
`15 (5) [8]
`15 (5) [8]
`4
`Note: The vehicle utilized was 0.8% HPMC in RODI water.
`
`
`The test article or vehicle control material was administered once daily, by oral
`gavage, for 26 consecutive weeks. The main study animals (Groups 1-4) were
`observed daily for overt signs of toxicity. Individual body weights and food
`consumption were recorded weekly. Selected clinical pathology parameters were
`evaluated on days 91/92, 182/183, and 210 for the main study and recovery
`animals. Ophthalmological examinations were performed for the main study animals
`once prior to in-life initiation (day -18), just prior to the end of the main study phase
`(during the last week of dosing), and just prior to the end of the recovery phase. All
`rats were subjected to a complete gross necropsy examination at the time of death
`or scheduled euthanasia (following the treatment period on day 182/183 or following
`the recovery phase on day 210). Fresh organ weights were obtained at scheduled
`euthanasia and selected tissues were preserved from all rats (except for one male
`and one female found dead). All tissues collected at necropsy were processed and
`examined microscopically.
`
` A
`
` concurrent toxicokinetic phase was conducted in a separate set of animals to
`evaluate systemic exposure to the test article and to determine if systemic
`accumulation of the test article occurred following repeated dosing.
`
`Results and Conclusion:
`
`One Group 3 male was found dead on day 91 and one Group 4 recovery female was
`found dead on day 197; neither death was attributable to test article. All other
`animals survived to the day of scheduled euthanasia.
`
`
`
`Page 7 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(14)
`
`
`
`The most clear test article-related clinical signs observed during the dosing period
`(days 0 to 182) were salivation observed prior to dosing and salivation observed
`post-dose. During the recovery period, no signs that could clearly be attributed to
`prior test article administration were observed.
`
`Males in Group 2 and Group 3 had body weights comparable to controls, whereas
`Group 4 males had body weights that were generally lower than controls throughout
`the study. Test article-treated females had mean body weights that were within 5%
`of those of controls; apparent decreases in body weights observed in females of
`Groups 2 and 4 during the recovery period appeared to be artifactual.
`
`food consumption,
`in
`toxicologically meaningful differences
`There were no
`hematological or coagulation parameters, or in urinalysis parameters among groups
`of males or females.
`
` A
`
` number of statistically significant differences in clinical chemistry parameters were
`observed throughout the course of the study. Notable changes included decreases
`in sodium and chloride in males and females of Group 3 and/or Group 4 on
`days 91/92 and 182/183, and elevations in potassium levels in Groups 4 males and
`females on day 182/183.
`
`No test article-induced ocular effects were observed in males or females.
`
`The principal test article-related findings at gross necropsy at the end of the dosing
`period were enlarged kidney and prominent epithelials of the stomach, observed
`primarily in animals of Groups 3 and 4. These findings largely resolved during the
`recovery period but were evident in a few animals in Groups 3 and/or 4 at the end of
`the recovery period.
`
`Toxicologically relevant, dose-related increases in absolute and relative to brain
`weight kidney, liver, and stomach weights were observed on day 182/183 in both
`males and females of Groups 3 and 4. At recovery, increased absolute and relative
`kidney weights were observed only in males of Groups 3 and 4.
`
`At the main study time point, test article-related findings in male rats in all treatment
`groups included an increased incidence and severity of nephropathy (100 and
`200 mg/kg dose groups only), as well as other renal findings not classically
`associated with nephropathy, including cortical tubular changes (diffuse dilation,
`hyaline droplet accumulation, nuclear/cellular hypertrophy of epithelial cells,
`segmental epithelial regeneration) and hypertrophy of the parietal epithelium of
`Bowman’s capsule. Test article-related findings in the kidneys of female rats at the
`main study time point included hyaline droplet accumulation and hypertrophy of
`
`Page 8 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(15)
`
`
`
`cortical tubular epithelial cells at the 100 and 200 mg/kg/day dose levels. At
`recovery, the character and severity of test article-related findings in the kidney were
`generally similar to those at the main study time point; however, the incidence was
`decreased in some dose groups.
`
`In the nonglandular stomach, test article-related findings in main study rats were
`squamous epithelial hyperplasia and hyperkeratosis (25, 100 and 200 mg/kg/day
`groups), subacute inflammation (100 and 200 mg/kg/day groups), squamous cell
`carcinoma (one 200 mg/kg/day male), and squamous papilloma (one 100 mg/kg/day
`group male). In the glandular stomach at the main study time point, subacute
`inflammation was considered test article-related at the 100 and 200 mg/kg/day dose
`levels. At recovery, test article-related findings were limited to the nonglandular
`stomach and occurred at a much lower incidence and severity than at the main
`study time point. Stomach changes at recovery consisted of minimal hyperkeratosis
`(100 and 200 mg/kg/day groups), minimal squamous epithelial hyperplasia (all
`treated dose levels), and keratinized cysts (200 mg/kg/day).
`
`At the main study time point, lymphoid hyperplasia of pancreatic or abdominal lymph
`nodes was seen in a few animals receiving 200 mg/kg/day. This change may have
`been secondary to stomach inflammation.
`
`In the previous 13-week study conducted with BG00012 in mice [1], the stomach
`was the primary target organ in males and females administered BG00012 at doses
`of 200 or 400 mg/kg/day. Findings included gross necropsy observations of
`prominent epithelium, foci, and thickened stomach, increased stomach weights, and
`histopathological changes in the nonglandular stomach of squamous epithelial
`hyperplasia, hyperkeratosis, keratinized cyst(s), microabscess(es), inflammation,
`focal foreign material in submucosa/serosa, ulceration, and serosal adhesion(s).
`
`At the main study time point, histopathological findings in the liver of test
`article-treated main study rats included minimal multifocal hepatic necrosis and
`minimal bile duct hyperplasia (predominantly females of the 100 and 200 mg/kg/day
`groups). Test article-related findings were not noted in the liver following recovery.
`
`The principal target organs were identified as the kidney, stomach and liver in both
`males and females. Neither a no-observed-effect level (NOEL) nor a no-observed-
`adverse-effect level (NOAEL) could be identified for male and female rats. The
`lowest observed adverse effect level in males and females was 25 mg/kg/day.
`
`
`Page 9 of 25
`
`

`

`(16)
`
`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`
`
`8. INTRODUCTION
`
`This report details the experimental procedures and results of a six-month oral
`(gavage) toxicity study of dimethyl fumarate (BG00012) in CD®IGS rats. The study
`was authorized by Biogen Idec Inc., Cambridge, Massachusetts, and was conducted
`at Charles River Laboratories, Preclinical Services, 640 North Elizabeth Street,
`Spencerville, Ohio. The laboratory rat was selected as the animal model for this
`study since it is one of the species recommended by the regulatory agencies for oral
`toxicity testing. Oral administration of the test article was selected since it is the
`proposed route of administration for clinical use. The protocol was signed by the
`Study Director on August 16, 2004 (GLP initiation date). The experimental start date
`for the study was July 27, 2004, and the experimental completion date for the study
`was May 1, 2006. The in-life phase of the main study was initiated with test article
`administration on August 17, 2004 (day 0), and concluded with final euthanasia on
`March 15, 2005 (day 210).
`
` concurrent toxicokinetic (TK) phase was conducted in a separate set of animals to
`evaluate systemic exposure to the test article and to determine if systemic
`accumulation of
`the
`test article occurred
`following repeated dosing.
` The
`experimental methods and results of the TK phase are included in separate
`appendices in this report.
`
` A
`
`9. OBJECTIVE
`
`The purpose of this study was to evaluate the potential toxicity of BG00012 when
`administered daily to rats by oral (gavage) administration for at least six consecutive
`months. Data from this study may be used in the assessment of potential human
`risk.
`
`10. MATERIALS AND METHODS
`
`10.1. Experimental Protocol
`The study protocol, protocol amendments and protocol deviations are presented in
`Appendix A.
`
`10.2. Test Article and Vehicle Control Material
`
`10.2.1. Test Article Receipt, Identification and Storage
`The test article was received from the Sponsor and identified as follows:
`
`
`
`Page 10 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(17)
`
`Identification
`
`Assigned
`Testing
`Facility ID
`
`Physical
`Description
`
`Receipt
`Date
`
`Expiration
`Date
`
`Dimethyl Fumarate (BG00012) S04.002.EBA
`Lot No. F1177170
`
`Dimethyl Fumarate (BG00012) S04.003.EBA
`Lot No. 1102642 33004998
`
`White
`crystalline
`owder
`
`White
`crystalline
`owder
`
`7/15/04
`
`08/31/07
`
`10/14/04
`
`None
`provided
`
`A one-gram sample of each lot of the test article was taken as a retention sample
`and stored at the Testing Facility. The
`from li ht.
`at room
`
`1 0.2.2. Vehicle Control Material Receipt, Identification and Storage
`The vehicle control material used in preparation of the dosing mixtures and for
`administration to control animals was 0.8% w/v hydroxypropylmethylcellulose
`(HPMC) in reverse osmosis deionized (RODI) water. The RODI water was supplied
`by the tap in the Testing Facility's pharmacy. The bulk HPMC was received at the
`Testing Facility and identified as follows:
`
`Supplier
`
`Lot
`Number
`
`Assigned
`Testing
`Facility ID
`
`Physical
`Description
`
`Receipt
`Dates
`
`Testing Facility
`Assigned
`Expiration
`Dates
`
`Sigma-Aldrich
`
`063K3683
`
`V04 .037 White powder
`
`04/26/04
`
`12/31/14
`
`Sigma-Aldrich
`
`122K0149
`
`V04 .088 White powder
`
`08/16/04
`
`12/31/14
`
`Sigma-Aldrich
`
`013K0621
`
`V04 .117 White powder
`
`11/18/04
`
`12/31/14
`
`Sigma-Aldrich
`
`013K0621
`
`V04 .127 White powder
`
`12/13/04
`
`12/31/14
`
`Sigma-Aldrich
`
`122K0149
`
`V04 .134 White powder
`
`12/27/04
`
`12/31/14
`
`Sigma-Aldrich
`
`122K0149
`
`V05 .010 White powder
`
`02/23/05
`
`12/31/15
`
`A one-gram sample of the vehicle (V04 .037, V04.088, V04.1 27, V04.134 and
`V05.010) was taken as a retention sample and stored at the Testing Facility. The
`retention samples and bulk vehicle were stored at room temperature.
`
`Page 11 of 25
`
`

`

`Study No. EBA00016
`Sponsor Study No. P00012-04-06
`
`(18)
`
`
`
`The vehicle control material was prepared by weighing a specified amount of the
`HPMC into a calibrated container, adding an appropriate amount of heated RODI
`water and stirring the mixture by hand. Room temperature RODI water was then
`added to produce a 0.8% w/v solution and the mixture was stirred overnight. The
`vehicle control material was prepared weekly and stored refrigerated.
`10.2.3. Dose Preparation
`For each test article group, an appropriate amount of BG00012 was weighed into a
`plastic weigh boat and placed in a mortar. The weigh boat was rinsed with vehicle
`(0.8% w/v HPMC in RODI water). A sufficient volume of the vehicle was added to
`the mortar, the mixture was transferred into an Erlenmeyer flask and the mortar was
`rinsed with vehicle. An appropriate quantity of the vehicle was then added to
`achieve the desired concentration and the flask was inverted several times. The
`flask was wrapped in aluminum foil and the dosing mixture was stirred for a
`minimum of 30 minutes using a magnetic stir bar. Each mixture was then stirred
`continuously and dispensed into individual amber glass containers for daily dosing.
`The dosing mixtures for Groups 1, 3 and 4 were prepared weekly and the dosing
`mixture for Group 2 was prepared daily until large batch homogeneity and 10-day
`stability were performed (a four-day preparation was made on day 17 and weekly
`preparations began on day 21). A sufficient quantity of the vehicle was similarly
`dispensed for administration to control animals. The dosing mixtures were stored
`refrigerated and allowed to warm to room temperature prior to administration. The
`mixtures were stirred continuously prior to and during dosing. The physical
`description of each dosing mixture was recorded following preparation. The vehicle
`was a clear colorless solution and each test article preparation was a white
`suspension.
`10.2.4. Analysis of Dose Preparations
`Homogeneity and concentration of the prepared formulations were verified on the
`first day of preparation. Quadruplicate samples (1 mL each) were taken from the
`top, middle and bottom of the lowest and highest concentrations. Quadruplicate
`samples were taken from the middle of the preparations for all other concentrations.
`Two samples of each quadruplicate set were shipped to Charles River Laboratories,
`Preclinical Services, Massachusetts, for analysis and the remaining samples were
`retained as backup samples at Charles River Laboratories, Preclinical Services,
`Ohio. The mean of the concentration values obtained for all locations served as the
`concentration value for that preparation.
`
`Concentration of the prepared formulations was verified weekly during the second,
`third and fourth weeks and monthly for the remainder of the study. Quadruplicate
`samples were taken from the middle of the preparations for all concentrations. Two
`samples of each quadruplicate set were shipped to Charles River Laboratories,
`Preclinical Services, Massachusetts, to be analyzed and the remaining samples
`
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`were retained as backup samples at Charles River Laboratories, Preclinical
`Services, Ohio.
`
`Stability of BG00012 in 0.8% HPMC has been established from 5 to 50 mg/mL when
`stored at room temperature for 24 hours and 2 to 8°C for 11 days under Charles
`River Laboratories, Preclinical Services, Massachusetts Study No. EBAA-0154.
`Stability was conducted with a concentration or homogeneity analysis to determine
`initial concentration. For each set of stability conditions and time points listed, a
`quadruplicate set of samples (1 mL each) was taken, shipped to Charles River
`Laboratories, Preclinical Services, Massachusetts, and stored appropriately. At the
`scheduled time points after the initial concentration analysis, duplicate samples were
`analyzed. The second set of stability samples served as backup samples, and were
`disposed of following notification of acceptable results. Stability was confirmed for
`the storage conditions and time points listed as follows:
`
`
`Storage Conditions
`Ambient
`2 to 8°C
`
`Time Points
`24 hours
`10 days
`
`
`The analytical samples were shipped overnight on ice packs to Charles River
`Laboratories, Preclinical Services, Massachusetts. The results of these analyses
`are included in Appendix C.
`
`10.3. Animals and Animal Husbandry
`Animal housing and care were based on the standards established by the
`for Assessment and Accreditation of Laboratory Animal Care,
`Association
`International (AAALAC) and the guidelines set forth in the Guide for the Care and
`Use of Laboratory Animals, NIH Publication No. 96-03, 1996.
`10.3.1. Animal Receipt, Identification and Housing
`A total of 125 male and 125 female Sprague Dawley Crl:CD®(SD)IGSBR rats were
`received in two shipments from Charles River Laboratories, Inc., Portage, Michigan.
`One hundred nine males and one hundred nine females were received on July 27,
`2004, and sixteen males and sixteen females were received on August 10, 2004. At
`the time of receipt, each animal was identified with a temporary identification number
`recorded on the cage card and in the data records. During acclimation and while on
`study, the animals were housed individually in suspended stainless steel cages. A
`metal ear tag displaying a unique identification number was used to permanently
`identify each animal assigned to the study. A cage card displaying the study, animal
`and group numbers, and sex was affixed to each cage. The cage cards were
`color-coded according to group number following randomization.
`
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`10.3.2. Acclimation
`The animals were examined upon receipt and daily thereafter for overt physical and
`behavioral abnormalities. General health/mortality and moribundity checks were
`performed twice daily, in the morning and afternoon. Individual body weights were
`recorded on the day following receipt and prior to randomization on day -1. The
`animals were acclimated to the laboratory conditions for a minimum of seven days
`prior to dosing initiation.
`10.3.3. Diet and Drinking Water
`PMI Nutrition International Certified Rodent Chow® #5002 and municipal tap water
`were provided to the animals ad libitum throughout the study, except when feed was
`withheld overnight prior to clinical pathology determinations. The feed was analyzed
`by the supplier for nutritional components and environmental contaminants. The lot
`number and expiration date of each batch of feed used during the study were
`recorded. The tap water was purified by reverse osmosis and supplied to the
`animals by an automatic watering system (or water bottles, when clinical signs
`warranted or during urine collection). Water supplying the facility is analyzed for
`contaminants according to the Testing Facility’s Standard Operating Procedures.
`The results of the feed and water analyses are maintained at the Testing Facility.
`Within generally accepted limits, there were no contaminants in the diet or drinking
`water which would interfere with the conduct of the study.
`10.3.4. Environmental Conditions
`The environmental controls in the animal room were set to maintain room
`temperature and relative humidity ranges of 65 to 79°F (18 to 26°C) and 30 to 70%,
`respectively. Environmental control equipment was monitored and adjusted as
`necessary to minimize fluctuations in the animal room environment. Light timers
`were set to maintain a 12-hour light/12-hour dark cycle and the room ventilation was
`set to produce 10 to 15 air changes per hour. The room temperature and relative
`humidity were recorded daily and ranged from 62 to 75°F (17 to 24°C) and 36 to
`62%, respectively.
`
`10.4. Experimental Procedures
`
`10.4.1. Study Design
`The study group design and dosage levels tested were as follows:
`
`
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`No. of Animals
`(Recovery)
`[Satellite TK]
`Dose Level
`Test Material
`Group
`(mg/kg/day)
`Male
`Female
`0
`Vehicle
`15 (5) [8]
`15 (5) [8]
`1
`25
`BG00012
`15 (5) [8]
`15 (5) [8]
`2
`100
`BG00012
`15 (5) [8]
`15 (5) [8]
`3
`200
`BG00012
`15 (5) [8]
`15 (5) [8]
`4
`Note: The vehicle utilized was 0.8% HPMC in RODI water.
`
`Concentration
`of Dosing
`Solution
`(mg/mL)
`0
`2.5
`10
`20
`
`Dosing
`Volume
`(mL/kg)
`10
`10
`10
`10
`
`
`10.4.2. Justification of Dose Level Selection
`The dose levels were chosen based on the results of a 90-day repeat dose
`toxicology study in CD®IGS rats dosed daily by oral gavage. The high dose
`(200 mg/kg/day) was expected to cause at least a 10% decrease in body weight
`gain compared to control animals. The mid dose (100 mg/kg/day) was used in the
`90-day study and caused moderate decreases in body weight gain as well as
`moderate toxicity of the forestomach (hyperplasia and hyperkeratosis with increased
`stomach weight). The low dose (25 mg/kg/day) was expected to cause little to no
`toxicity based on a 50 mg/kg/day dose used in the 90-day study and the mild
`hyperplasia observed in the stomach.
`10.4.3. Randomization and Group Assignment
`On day -1, the animals were weighed and examined for signs of physical disorder
`(detailed clinical observations). Animals determined to be suitable test subjects
`were arbitrarily assigned to groups using a computer randomization program. The
`program ranked the animals according to day -1 body weights and randomly
`assigned the rats to study groups. Disposition of animals not assigned to study was
`documented in the study records. At randomization, the main study animals were
`approximately eight weeks of age with body weights ranging from 266 to 344 grams
`for the males and 157 to 235 grams for the females.
`
`Group 3 female #3589 was replaced with female #5692 on day 0 since female
`#3589 died shortly after blood collection. Gross necropsy