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`Page 1 of 2382
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`BG00012: A 1 Year Chronic Toxicity Study of
`Dimethyl Fumarate Suspension Administered
`by Nasogastric Intubation to Cynomolgus
`Monkeys
`Testing Facility Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
`
`Final Report
`
`
`
`Charles River Laboratories
`Preclinical Services Nevada
`587 Dunn Circle
`Sparks, NV 89431
`
`Biogen Idec
`14 Cambridge Center
`Cambridge, MA 02142
`
`
`June 21, 2007
`
`
`Testing Facility:
`
`
`Study Sponsor:
`
`Study Completion
`Date:
`
`
`
`
`
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`Page 1 of 11
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`Biogen Exhibit 2276
`Coalition v. Biogen
`IPR2015-01993
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`Page 2 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`I. Abstract
`The objectives of this chronic toxicity study were to determine the potential
`chronic toxicity of Dimethyl Fumarate when administered by nasogastric
`intubation to cynomolgus monkeys for at least 52 weeks, and to evaluate
`recovery of animals from any effects of the test article over a treatment-free
`period of at least 4 weeks.
`article,
`control
`the
`and
`(DMF),
`Dimethyl
`Fumarate
`0.8% Hydroxypropylmethylcellulose (HPMC), were given orally once daily for
`52 consecutive weeks to 48 experimentally naive cynomolgus monkeys at
`0 (control), 5, 25, or 75 mg/kg (6/sex/group). One male animal in the
`5 mg/kg/day dose group was euthanized on Day 197 because of a dosing
`accident, and one female monkey in the 25 mg/kg/day dose group was
`incorrectly transferred from the recovery necropsy to the main study necropsy.
`Thirty-two animals (4/sex for Groups 1 and 4, 3 males/4 females for Group 2,
`and 4 males/5 females for Group 3) were euthanized one day after the last dose
`(Day 365). The remaining 15 animals (2/sex for Groups 1, 2 and 4,
`2 males/1 female for Group 3) were continued on study without further
`treatment, and were terminated approximately 4 weeks after the last dose
`(Day 393).
`There was no test article-related mortality or moribundity, nor were there any
`test article-associated findings in electrocardiographic evaluations, ophthalmic
`evaluations, hematological parameters, coagulation parameters, or urinalysis
`parameters.
`Test article-associated findings included increased incidence of low food
`consumption and initial reduction in mean body weight gain (Week 1 of
`treatment) in the 75 mg/kg/day treated monkeys. Test article-associated serum
`chemistry findings consisted of decreased BUN in the 25 and 75 mg/kg/day
`monkeys of both sexes and decreased creatinine values in a few female
`monkeys. Reductions in serum phosphorus levels were observed in some
`monkeys in the 75 mg/kg/day dose group. Reductions in BUN, creatinine, and
`phosphorus were generally resolved by the end of the recovery period.
`Test article-associated pathologic findings at terminal necropsy were limited to
`the kidney. The macroscopic alterations included pale discoloration, increased
`size, and watery consistency in the kidneys of some male and female animals
`in the 25 and 75 mg/kg/day dose groups. Test article-related increases in
`kidney weight, kidney to brain, and kidney to body weight ratios were noted in
`females in the 75 mg/kg/day dose group. Histologic alterations consisted of
`single cell necrosis and regeneration of the cortical tubular epithelial cells in
`monkeys in the 25 and 75 mg/kg/day dose groups, with a higher incidence and
`severity in the 75 mg/kg/day dose group. At the recovery necropsy, pale
`discoloration in a male and female animal in the 75 mg/kg/day dose group, and
`increased kidney weight, kidney to brain, and kidney to body weight ratios were
`noted
`for male animals given 75 mg DMF/kg/day.
` Microscopic
`test
`article-related changes observed at the recovery necropsy in 25 and 75 mg/kg
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`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`animals were similar to those seen at the terminal necropsy; however the
`incidence and/or severity of single cell necrosis and regeneration of the tubule
`epithelium were reduced in some dose groups, suggesting a trend towards
`recovery. Additionally, both male monkeys in the 75 mg/kg/day recovery group
`had mild to moderate interstitial fibrosis, a morphologic indication of irreversible
`loss of tissue and function, accompanied by tubular atrophy. Renal fibrosis was
`associated with increased BUN and creatinine in one of these animals at weeks
`38, 52 and 56.
`After once daily dosing of DMF in cynomolgus monkeys, Cmax values of MMF
`increased in a dose proportional manner from 5 to 75 mg/kg. AUC0-24hr values
`increased in a dose proportional manner from 5 to 25 mg/kg (Excluding Day 1
`values, mean values ranged from 1.99-2.73 µg*hr/mL in the 5 mg/kg group and
`12.21-15.58 µg*hr/mL in the 25 mg/kg group), and in a slightly greater than
`dose-proportional manner from 25 to 75 mg/kg (Excluding Day 1 values, mean
`values ranged from 43.63-58.41 µg*hr/mL in the 75 mg/kg dose group). There
`was no statistically significant accumulation of MMF at any tested dose levels.
`There was no significant gender effect on MMF exposure in any tested dose
`levels. The terminal half-life was slightly prolonged at higher doses at 25 and
`75 mg/kg (mean T1/2 values ranged from 0.46-0.73 hr in the 5 mg/kg group,
`0.42-0.89 hr in the 25 mg/kg group and 0.81-1.04 in the 75 mg/kg group,
`respectively), while the total clearance was slightly decreased.
`Levels of methanol in vehicle treated monkeys ranged from near the detection
`limit of the assay (5 µg/mL) to a value of 39 µg/mL in a male monkey at
`Week 52. With DMF treatment, there were observed increases in methanol
`concentration at early time points after dosing, that were comparable to the high
`level of 39 µg/mL observed in vehicle controls. AUC0-24 hr values calculated for
`DMF and vehicle control groups (178.52 to 233.05 µg*hr/mL in male and
`181.6 to 212.84 µg*hr/mL in female control groups) demonstrated a statistically
`significant increase in the 25 mg/kg males (261.33 to 471.18 µg*hr/mL) and
`75 mg/kg treated males (199.92 to 409.34 µg*hr/mL) and females (229.33 to
`335.35 µg*hr/mL) at the Week 26 collection interval. At the Week 52 interval,
`the mean AUC 0-24 hr ranged from 337.64 to 341.81 µg*hr/mL in vehicle control
`treated monkeys, with no increase observed after any dose level of DMF.
`Formic acid AUC0-24hr levels among different dosing groups were not statistically
`significant, except on Week 26 in the 75 mg/kg dose group (a 30% decrease).
`The formic acid AUC0-24hr in the control group in Week 26 was 57.71 to
`109.79 µg*/hr/mL in male animals, and 41.53 to 87.35 µg*/hr/mL in female
`animals. Formic acid AUC0-24 levels in the 75 mg/kg dose group at Week 26
`was 16.36 to 58.19 µg*/hr/mL in males, and 40.27 to 51.15 µg*/hr/mL in
`females. Formic acid levels, as a product of methanol metabolism, were not
`changed appreciably at any interval for any dose group during the 1 year DMF
`treatment
`In summary, test article associated clinical observations were limited to low
`food consumption in animals given 75 mg DMF/kg/day. Initial decreases in
`body weight gain were observed in both genders during the first week of dosing
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`Page 4 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`at 75 mg/kg. Clinical chemistry changes included decreased BUN in both
`genders and creatinine
`in a
`few
`females (25 and 75 mg DMF/kg/day).
`Reductions in serum phosphorus levels were observed in some monkeys in the
`75 mg/kg/day dose group. At terminal necropsy, gross and histologic findings
`related to treatment were limited to the kidney and consisted of pale
`discoloration, increased size (to include absolute and relative kidney weight
`increases in the highest dose group), and watery consistency at the 25 and
`75 mg/kg/day dose levels. Histologically, this correlated with single cell
`necrosis and regeneration of tubular epithelial cells. At necropsy following a
`four week treatment free period, similar findings were noted in the kidney with a
`trend towards recovery in some dose groups. However, both male monkeys in
`the 75 mg/kg/day recovery group had mild to moderate interstitial fibrosis
`accompanied by tubular atrophy and, in one animal, an increase in BUN
`(42-77% increase from baseline value) and creatinine (22-56% increase from
`baseline value).
`In consideration of these findings, the no-observed-effect level (NOEL) for
`dimethyl fumarate under the conditions of this study was 5 mg DMF/kg/day.
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`Page 8 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`III. List of Abbreviations
`
`American College of Veterinary Pathologists
`Analysis of variance
`Code of Federal Regulations
`Diplomate, American Board of Toxicology
`Diplomate, American College of Laboratory Animal Medicine
`Diplomate, American College of Veterinary Internal Medicine
`Diplomate, American College of Veterinary Pathologists
`Deionized
`Dimethyl Fumarate
`Electrocardiogram
`Food and Drug Administration
`Good Laboratory Practice
`Hydroxypropylmethylcellulose
`Institutional Animal Care and Use Committee
`Institute for Laboratory Animal Resources
`Ministry of Health, Labor, and Welfare
`Material Data Safety Sheet
`Organisation for Economic Cooperation and Development
`Quality Assurance Unit
`Standard operating procedures
`Toxicokinetic
`United States Department of Agriculture
`
`ACVP
`ANOVA
`CFR
`DABT
`DACLAM
`DACVIM
`DACVP
`DI
`DMF
`ECG
`FDA
`GLP
`HPMC
`IACUC
`ILAR
`MHLW
`MSDS
`OECD
`QAU
`SOP
`TK
`USDA
`
`
`
`
`
`
`
`
`
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`Page 9 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
`
`IV. Regulatory Compliance and Signatures
`Study No.:
`EBA00176
`Title:
`BG00012: A 1 Year Chronic Toxicity Study of Dimethyl
`Fumarate Suspension Administered
`by Nasogastric
`Intubation to Cynomolgus Monkeys
`
`A. Regulatory Compliance
`
`1. Testing Facility-conducted Study Elements
`All portions of this study performed by the Testing Facility adhered to the
`protocol and the Testing Facility Standard Operating Procedures (SOP) and
`were conducted in compliance with international Good Laboratory Practice
`(GLP) standards currently in effect. These included:
`• The Nonclinical Laboratory Studies Good Laboratory Practice
`Regulations issued by the U.S. Food and Drug Administration (Title 21 of
`the Code of Federal Regulations (CFR), Part 58; effective June 20,
`1979);
`• The Organisation for Economic Cooperation and Development (OECD)
`Principles on Good Laboratory Practice (C[97]186/Final; effective 1997);
`and
`• The Japanese Good Laboratory Practice Standards for Safety Studies on
`Drugs (Ordinance No. 21 of the Pharmaceutical Affairs Bureau, Ministry
`of Health, Labor and Welfare (MLHW), Japan; effective April 1, 1997).
`Various prestudy activities (i.e., cage side observations; body weight
`measurements; ophthalmic examinations; electrocardiographic evaluations;
`and blood collection for evaluation of clinical pathology indices, including
`serum chemistry, hematology, and coagulation; and acclimation to the
`nasogastric dosing procedure) were performed prior to the availability of a
`signed Final Protocol. The Study Director and Testing Facility Management
`authorized these prestudy activities prior to their initiation. The memo
`documenting this authorization is filed in the study records. Per current GLP
`regulations, study activities conducted prior to finalization of the protocol are
`not GLP compliant. In addition, Biogen Idec did not provide the expiration
`date
`for
`the
`analytical
`reference
`standard,
`BG00012,
`Lot
`Number 1102642 33004998 used for dose suspension analysis; therefore,
`this was also not considered to be GLP compliant.
`
`2. Testing Facility-enlisted Subcontractor Study Elements
`Interpretation of electrocardiogram (ECG) recordings (Section VI.D.4) was
`performed by a Testing Facility-enlisted consultant
`
` This work was conducted
`in adherence to the study protocol and the SOPs of the subcontractor and
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`Page 10 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`complied with the U.S. Food and Drug Administration (FDA), OECD, and
`MHLW GLP regulations. In addition, the ECG evaluation records were
`audited by the Testing Facility Quality Assurance Unit (QAU).
`An ECG interpretative report was prepared
` and reviewed by the
`Testing Facility QAU. The manner in which the raw ECG data and records
`were derived at the Testing Facility, the evaluation of these data
`,
`and the transfer and retention of these data were compliant with current FDA,
`OECD, and MLHW GLP regulations.
`
`performed
`
`by Biogen
`
`Idec
`
`3. Biogen Idec-conducted Study Elements
`The following portions of the study were conducted by Biogen Idec or a
`Biogen Idec-enlisted subcontractor:
`• Prestudy analyses of the test article was performed by Biogen Idec
`(Section VI.A.1)
`interpretations was
`• Toxicokinetic
`(Section VI.F.1)
`• Methanol and formic acid analysis was performed by
`
`• Histopathology peer review was performed by Biogen Idec (Section VI.G.6)
`
`
`
`Except as noted below, this project was conducted in compliance with the
`following regulations:
`• U.S. Food and Drug Administration Good Laboratory Practice Regulations;
`Final Rule. 21 CFR Part 58
`• Japanese Ministry of Health, Labor and Welfare (1997), Good Laboratory
`Practice Standard for Safety Studies on Drugs, MHLW Ordinance
`Number 21, 1997
`• Organisation for Economic Co-operation and Development (1998), The
`Revised OECD Principles of Good Laboratory Practices [C(97)186/Final]
`The toxicokinetic interpretations and histopathology peer review conducted by
`Biogen Idec and methanol and formic acid analysis
`
`were not performed in compliance with GLP guidelines;
`however, the analyses were performed according to Good Scientific Practices
`and appropriate facility SOPs. Prestudy analyses of the test article
`(Section VI.A.1) generally adhered to Good Manufacturing Practice (GMP)
`standards. The Testing Facility Study Director and QAU maintained oversight
`of other applicable study elements via review of Inspection Reports and
`Summary Statements provided by
`the appropriate QAU, and by
`communication with Biogen Idec’s Study Monitor.
`
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`Page 11 of 2382
`Study No. EBA00176
`Biogen Idee Study No. P00012-05-08
`
`4. Guidelines for Study Design
`The design of this study was based on consideration of the study objectives in
`· relation to the overall product development strategy.
`In addition, the following
`specific guidelines were considered in the design of this study:
`• OECD Guideline 409
`• Repeat Dose Oral Toxicity Study in Non-Rodents
`
`B. Study Director Signature
`No circumstance occurred during Study No. EBA00176 that affected the overall
`quality or integrity of the data. All protocol and SOP deviations were
`in
`the study records, and salient deviations are
`listed
`in
`documented
`Appendix A.
`
`.. .;
`
`~ ~
`
`~~s - ~770rz~···
`Stephen TichenoGhD
`Study Director
`Charles River Laboratories
`Preclinical Services Nevada
`
`2( Jun 2£v?
`Date
`
`C. Signatures of Other Responsible Personnel
`
`~ . GmiS:EiJi()C ~ PhD:DACVP
`
`2- ( ,yC{k 7-0Cll(cid:173)
`Date
`
`Principal Director, Clinical Pathology
`Charles River Laboratories
`Preclinical Services Nevada
`
`Report Review:
`
`?__ l J u.n .2 ()() 1-
`
`Date
`
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`Page 12 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`V. General Information
`
`A. Study Dates
`Experimental Start Date:
`First Day of Dosing:
`
`Terminal Necropsy:
`
`Recovery Necropsy:
`
`Experimental Completion Date:
`
`January 10, 2006
`January 17-20 & 27, 2006 (Sets A-D, and
`E, respectively)
`January 16-19, 2007 (Sets A-D,
`respectively)
`February 13-16 & 23, 2007 (Sets A-D, and
`E, respectively)
`June 21, 2007
`
`B. Responsible Personnel
`Testing Facility
`Stephen Tichenor, PhD
`Study Director:
`Tonya Hack, BS
`Study Supervisor:
`Brian Logan, BS
`Lead Research Technicians:
`Kyle Santti
`
`Ann-Marie Shank
`
`Barbara Klipfel, MBA, BA, BS
`Senior Manager, Formulations:
`Lane Meyer, BS
`Lead Formulations Technician:
`Michael M. Laffins, DVM
`Attending Veterinarian:
`Glenn S. Elliott, DVM, PhD, DACVP
`Clinical Pathologist:
`Elizabeth Gioja, BS
`Clinical Pathology Technician:
`David Jensen, BS, BA
`Statistician:
`Patrick M. Tam, BS
`Quality Assurance Auditor:
`Gary B. Baskin, DVM, DACVP, DACLAM
`Study Pathologist:
`Testing Facility-enlisted Subcontractor
`Principal Investigator, Cardiology: Larry P. Tilley, DVM, DACVIM
`Physical Address:
`Vetmed Consultants, Inc.
`Comparative Cardiology Division
`3600 Cerrillos Road
`Suite 201B
`Santa Fe, NM 87507
`Mailing Address:
`Vetmed Consultants/Dr. Tilley &
`Associates, Inc.
`1704-B Llano Street
`Suite 279
`Santa Fe, NM 87505
`
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`Page 14 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`D. Experimental Design Overview
`
`1. General Description
`A total of 48 cynomolgus monkeys were assigned to dose groups as shown in
`the table below.
`Number Necropsied:
`Dose Level
`Number of
`Group
`Week 53 Week 57
`(mg/kg)
`Males/Females
`No.
` 4/4
`2/2
`0 (control)
`6/6
`1
` 3/4
`2/2
`5
`6/6
`2
` 4/5
`2/1
`25
`6/6
`3
` 4/4
`2/2
`75
`6/6
`4
`All animals were dosed via nasogastric intubation once daily for at least
`52 consecutive weeks. The first day of dosing was designated Day 1. The
`animals were evaluated for clinical signs (twice daily), and changes in food
`consumption (once daily), body weight (prior to the first dose and weekly
`thereafter), electrocardiograms (prestudy and in Weeks 12, 24, 38, and 52),
`and ophthalmic examinations (prestudy and in Weeks 12, 24, 38, and 52).
`Blood samples
`for evaluation of serum chemistry, hematology, and
`coagulation parameters were collected from all animals twice prestudy and
`near the end of Weeks 2, 6, 12, 24, 38, 52, and 56. Urine samples (as
`available, up to 5 mL) were obtained by drainage from special stainless-steel
`cage pans prestudy, and in Weeks 2, 6, 12, 24, 38, 52, and 56. Blood
`samples were collected for toxicokinetic analyses on Day 1 (collected
`predose; and at 0.5, 1, 4, 8, and 24 hours post dose), and dosing Weeks 4,
`26, and 52 (collected predose; and at 15, 30, and 45 minutes; and 1, 2, 4, 8,
`and 24 hours post dose), and in Weeks 13 and 39 (collected prior to dosing
`and at 1 hour post dose). Blood samples were collected for methanol and
`formic acid analyses on Day 1 (collected prior to dosing, and at 0.5, 1, 4, 8,
`and 24 hours post dose), Weeks 4, 26, and 52 (collected predose; and at 15
`and 45 minutes; and 2, 8, and 24 hours post dose), and in Weeks 13 and 39,
`(collected prior to dosing and at 1 hour post dose).
`One animal in the 25 mg/kg/day dose group was euthanized on Day 197, and
`one female in the 25 mg/kg/day dose group was incorrectly transferred from
`the recovery necropsy to the main study necropsy. Thirty-two animals
`(4/sex/group, 3 males/4 females for Group 2, and 4 males/5 females for
`Group 3) were euthanized one day after the last dose (Day 365). The
`remaining 15 animals (2/sex/group, 2 males/1 female for Group 3) were
`continued on study without
`further
`treatment, and were
`terminated
`approximately 4 weeks after the last dose (Day 393).
`
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`Page 15 of 2382
`Study No. EBA00176
`Biogen Idec Study No. P00012-05-08
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`2. Rationale for the Study
`Studies in laboratory animals provide the best means of assessing the toxicity
`of prospective pharmaceuticals intended for human use. According to Biogen
`Idec, this study was necessitated by the absence of appropriate non-animal
`alternatives and is required by regulatory agencies. The information obtained
`from this study did not duplicate the results of previous studies and could not
`have been obtained by other means. This study is needed to support
`planned clinical trials and was requested by the FDA.
`
`VI. Materials and Methods
`
`A. Test and Control Articles
`
`1. Test Article
`Identification:
`Structural
`Category:
`Receipt:
`
`Physical Form:
`Composition/
`Purity:
`
`Storage
`Conditions:
`Stability:
`
`Dimethyl Fumarate (DMF), Lot No. 1102643 33004999
`
`Dimethyl Fumarate powder
`Supplied by Biogen Idec; received on November 22,
`2005
`for Study EBA00174, and
`transferred
`to
`Study EBA00176 on January 12, 2006, upon Biogen Idec
`authorization
`Off-white fine crystals
`
`Documented by Biogen Idec and communicated to the
`Testing Facility in the form of a Certificate of Analysis,
`which is included in the study records and Appendix D
`
`Ambient temperature
`According to Biogen Idec, the stability of the test article
`under various storage conditions, including conditions
`similar to those that were employed in this study, has
`been characterized, and information is on file with Biogen
`Idec.
`
`2. Control Article
`Identification:
`
`Receipt:
`Physical Form:
`
`(HPMC), Batch
`0.8% Hydroxypropylmethylcellulose
`Nos. TH06012N13, TH13012N11, UI25012N01 and
`RF10012N11
`Supplied by the Testing Facility
`White powder
`
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