Reviewer: Anthony L Parola, PhD
`
`NDA 22,341
`
`liraglutide increased absolute and relative weight of pancreas at all doses in both sexes in 28—day and 52-
`week studies (HEM 3 1X). In 5 mg/kg high dose monkeys in the 52-week study, increased pancreas
`weight was due to an increase in the mass of pancreas exocrine cells and ducts, but liraglutide had no
`effect on beta cell mass.
`
`Postmarketing cases of acute pancreatitis have been reported in exenatide treated patients. There '
`was a numerical imbalance in the number of reported pancreatitis cases in clinical trials of liraglutide with
`a larger number of cases and a higher rate in liraglutide treated groups.
`
`Other Effects
`In the 104-week carcinogenicity study in mice, liraglutide caused tubular cystic hyperplasia in
`thymus in males at all doses (0.03, 0.2, l, 3 mg/kg/day, HEM 3 0.2X) and in females at 3 0.2 mg/kg/day
`(HEM 3 2X). In the 52-week toxicity study in monkeys, group mean relative thymus weight (normalized
`to body weight) was 21.5 — 26.8% lower than controls in males at all liraglutide doses (0.05, 0.5, 5
`mg/kg/day) (HEM 3 1X) with correlative microscopic findings of atrophy.
`In the 104 week mouse carcinogenicity study, centrilobular hypertrophy of hepatocytes and
`diffuse centrilobular hepatocyte vacuolation occurred in males at all liraglutide doses (0.03, 0.2, l, 3
`mg/kg/day, HEM 3 0.2X)). In the same study, femoro-tibial degenerative joint disease occurred in male
`and female mice at all doses (HEM 3 0.2X).
`
`Genetic Toxicity
`Liraglutide was not mutagenic or clastogenic, with or without metabolic activation, in an Ames
`bacterial mutagenicity assay or chromosomal aberrations assay in human peripheral blood lymphocytes.
`Liraglutide did not induce micronuclei in bone marrow polychromatic erythrocytes from rats treated with
`up to 30 mg/kg liraglutide for 4 days or up to 1 mg/kg (HEM 11X) for 28 days.
`
`Carcinogenicity
`In 2—year life—time exposure carcinogenicity studies in mice and rats, liraglutide was a non-
`genotoxic, multisex, multispecies rodent carcinogen causing thyroid C—cell tumors in male and female
`rats and mice and fibrosarcomas on the dorsal skin and subcutis in male mice.
`
`In the mouse carcinogenicity study, the NOAEL for neoplastic findings was 0.2 mg/kg/day
`liraglutide (safety margin 2) based on thyroid C-cell adenomas in males and females and combined C-cell
`adenomas and carcinomas in females at 3 1 mg/kg/day liraglutide (HEM 3 10X). Liraglutide caused focal
`C-cell hyperplasia, a preneoplastic lesion, at 3 0.2 mg/kg/day in males and females (HEM 3 2X). C-cells
`secrete calcitonin, and in mice, plasma calcitonin increased at 3 0.2 mg/kg/day and it was a biomarker for
`increased C-cell focal hyperplasia and tumors. In males, fibrosarcomas on the dorsal skin and subcutis
`occurred at 3 mg/kg/day liraglutide. Equivocal finding of dose-related dorsal skin and subcutis
`rhabdomyosarcoma and injection site fibrosarcomas in males, and incidences in the 3 mg/kg/day group
`were above the historical control range for both tumors, but the increased incidence for either finding
`never reached statistical significance in any liraglutide group. Dorsal skin and subcutis tumors were likely
`related to the high local concentration of liraglutide at or near injection sites, so comparison of systemic
`exposure is not relevant for risk assessment. The liraglutide concentrations in high dose drug formulation
`was 0.6 mg/mL, lO-times lower than the 6 mg/mL concentration in the clinical formulation.
`In the rat carcinogenicity study, liraglutide dose-dependently caused thyroid C—cell adenomas at 3
`0.25 mg/kg/day in males (HEM 3 2X) and at 3 0.075 mg/kg/day in females (HEM 3 0.5K), C-cell
`carcinomas at 0.75 mg/kg/day in males (HEM 8X), and combined C-cell adenomas or carcinomas at 3
`0.25 mg/kg/day in males (HEM 32X) and at 3 0.075 mg/kg in females (HEM 3 0.5X). The incidence of
`C—cell carcinomas, a rare tumor in rats, was above concurrent and historical controls at 3 0.075 mg/kg/day
`liraglutide in males (HEM 3 0.5X) and at 3 0.25 mg/kg/day in females (HEM 3 2X).
`’
`To determine human relevance of liraglutide-induced rodent thyroid C-cell tumors, Novo Nordisk
`proposed a novel mode-of-action based on drug-induced, GLP-lR-mediated calcitonin secretion and
`synthesis driving C-cell hyperplasia with progression of hyperplasia to tumors, and they performed an
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`
`NDA 22,341
`
`extensive series of mechanistic studies to evaluate it. However, mechanistic studies did not adequately
`support the proposed mode of action. CDER’s Executive Carcinogenicity Assessment Committee
`(December 2008 meeting) and a large majority of members from a April 2009 Advisory Committee
`convened to evaluate the safety of liraglutide both concluded there was insufficient evidence to determine
`that liraglutide-induced C-cell tumors in mice and rats are not relevant to humans.
`Until recently, liraglutide was the only investigational or marketed drug known to cause C-cell
`tumors in both mice and rats, but data from other marketed and investigational GLP- 1R agonists suggest
`persistent GLP-lR activation cause C—cell tumors in both species. The human relevance of liraglutide-
`induced thyroid C-cell tumors in rodents is unknown.
`
`Reproductive and Developmental Toxicity
`
`Reproductive Toxicity
`In a definitive combined fertility and embryofetal developmental toxicity study in rats, the
`NOAEL for reproductive toxicity was 1 mg/kg liraglutide in males (HEM 11X) and 0.25 mg/kg in
`females (HEM 2X). .Although liraglutide decreased the absolute weight of seminal vesicles, prostate, and
`epididymis at 3 0.25 mg/kg (estimated HEM _>_ 2X), it had no effect on reproductive performance.
`Furthermore, in a 13—week toxicity study in rats, up to 1 mg/kg/day liraglutide (HEM 14X) did not
`increase abnormalities of eosin—stained sperm from cauda epididymis. Four weeks of treatment with 1
`mg/kg/day liraglutide decreased CYP2A1 (hepatic steroid hydroxylase, testosterone 7 oc-hydroxylase
`activity) ~2-fold in liver of male rats, but a relationship between decreased liver CYP2A1 activity and
`decreased weight of male reproductive organs in rats was not established. Decreased testosterone
`hydroxylase activity should increase circulating levels of testosterone. In the definitive combined fertility
`and embryofetal development study, liraglutide increased early embryonic deaths in females at 1 mg/kg
`(HEM 11X).
`In the 104-week mouse carcinogenicity study, lymphocyte infiltration occurred in seminal
`vesicles at 2 0.03 mg/kg/day liraglutide (HEM 3 0.2X).
`
`Developmental Toxicity
`Distribution and excretion studies show rat and rabbit fetuses were exposed to liraglutide in utero
`and liraglutide was excreted intact in milk of lactating rats. Although intact liraglutide was secreted in
`milk, since it’s a lipidated peptide, oral bioavailability was likely to be very low in nursing pups. In a
`definitive combined fertility and embryofetal development toxicity study in rats, the maternal NOAEL
`was 0.25 mg/kg liraglutide (safety margin 2) due to clinical signs of toxicity (hunched posture, rolling
`gate) at 1 mg/kg (HEM 11X). The NOAEL for fetal toxicity was < 0.1 mg/kg liraglutide based on fetal
`abnormalities of displaced kidneys, displaced azygous vein, and small additional ossified area within the
`cranial structure or fontanel at 3 0.1 mg/kg. A more complete state of ossification in fetuses from
`liraglutide treated groups compared to controls was noted. Major fetal abnormalities were misshaped
`oropharynx and/or narrowed opening to the larynx at 0.1 mg/kg and umbilical hernia at 0.1 and 0.25
`mg/kg, but they occurred without relation to liraglutide dose.
`In a definitive embryofetal development toxicity study in New Zealand White rabbits, all tested
`doses yielded estimated liraglutide plasma AUC0-241, below clinical exposure. The maternal NOAEL was
`0.05 mg/kg, the highest dose tested. Reduced food consumption, body weight gain, and body weight were
`considered pharmacologic effects in pregnant females, but reduced maternal weight may have impacted
`fetal development andreducing fetal weight. The NOAEL for fetal toxicity was < 0.01 mg/kg liraglutide
`based on decreased fetal weight compared to controls, dose—related increased fetal and litter incidence in
`total fetal malformations (2.1%, 3.7%, 5.7%, and 7.6% of fetuses and 18%, 30%, 35%, and 32% of litters
`affected by major abnormalities at 0, 0.0], 0.025, and 0.05 mg/kg/day liraglutide, respectively), fetal
`malformations (microopthalmia with or without retinal fold, forelimb flexure, right kidney represented by
`small area of tissue with attached cyst, curved scapula), and minor abnormalities (bilobed or bifurcated
`gall bladder, intermediate lung lobe absent, jugals fiised to maxilla, superior angle or lamina of axis
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`
`
`NDA 22 341
`
`incompletely ossified, slight downward pelvic shift, slight asymmetric alignment of pelvic bones) at 0.01
`mg/kg or Z 0.01 mg/kg. Fetal malformations occurred at 0.025 mg/kg (2 fetuses from 2 different litters
`with hydrocephaly, hepatic duct diverticulum, displaced or herniated umbilica, branchyury, dilated
`pulmonary trunk with incomplete aortic arch and malrotated heart, displaced umbilicus with part of the
`intestines fused to the umbilical vein, and split sternum). A minor abnormality of an additional liver lobe
`within the median cleft also occurred at 0.025 mg/kg. At 0.05 mg/kg, Malformations were connected
`parietal bones in 5 fetuses from 1 litter and dilated ascending aorta with narrow pulmonary trunk. Minor
`abnormalities at 0.05 mg/kg were corneal opacity, esophageal cyst, and kinked tail.
`Fetal abnormalities occurred in rats and rabbits at low multiples of human exposure (based on
`estimated AUC in pregnant rats or rabbits). Herniated umbilica, a malformation, occurred in both rats and
`rabbits, but the incidence was not related to liraglutide dose in either species. Increased fetal bone
`ossification was likely due to liraglutide treatment.
`
`Prenatal and Postnatal Development Toxicity
`Prenatal and postnatal toxicity of liraglutide was assessed in a multigenerational study in rats. The
`F0 maternal NOAEL was < 0.1 mg/kg liraglutide (HEM < 1X) based on clinical signs of toxicity at all
`.
`doses. The NOAEL for F0 reproductive toxicity was < 0.1 mg/kg liraglutide (HEM < 1X) based on a
`dose—related increased incidence of continuing gestation to day 22 (33%, 58%, 67%, and 96% of litters
`delivered on day 22 at 0, 0.1, 0.25, and 1 mg/kg liraglutide, respectively) with increased gestation
`duration from 21.3 to 22.0 days at 1 mg/kg (HEM 11X). Liraglutide had no effect on maternal behavior or
`F1 pup survival, post-natal development prior to weaning (physical development, functional development,
`or sexual maturation), or post—weaning sexual maturation rate. The NOAEL for postnatal toxicity in the
`F1 generation was < 0.1 mg/kg based on dose-dependent decreased body weight from lactation day 7 to
`day 21. In the postweaning period, body weight of F1 rats was lower than controls at all liraglutide doses
`(3 0.1 mg/kg) in males from postpartum day 7 to week 16 and in females at 0.1 and 1 mg/kg from
`postpartum day 7 to week 10. In weaned F] rats, clinical observations of greasy coat occurred at 3 0.1
`mg/kg in males and females. Bleeding scab and agitated behavior occurred in weaned 1 mg/kg F1 males
`and an increased incidence of scabs in males and females treated with 1 mg/kg liraglutide was noted at
`necropsy. The NOAEL for fertility, mating performance, or reproductive performance of F1 rats was 1
`mg/kg liraglutide (administered to F0 rats only). The NOAEL for development toxicity of F2 rats was 1
`mg/kg (administered to F0 rats only).
`
`Qualification of Impurities and Excipients
`Liraglutide, the active pharmaceutical ingredient (API)1n Victoza, is produced by acylating
`recombinant human Arg34GLP—1(7-37), produced1n Saccharomyces cerevisiae, with hexadecanoic acid
`(palmitic acid) at lysine 26 using a glutamate linker to yield liraglutide (Naé-(N--h—exadecanoyl—L-'Y-
`g1utamyl)—3Arg4GLP-1(7-3—7)). Impurities1n liraglutide were aw “ related impurities, "~— r-elated
`impurities, (impurities
`
`impurities.
`Liraglutide—related impurities were categorized as liraglutide-related impurities A, B, or C, or
`other ;-—-————————-
`related impurities based on HPLC elution characteristics relative to
`liraglutide.'W In
`
`degradation studies, elevated temperatures and humidity1ncrease' ~v
`related impurities
`(/ fold) and “related impurities/ fold), category B impurities / —— fold), and total
`impurities (/ fold). Exposing the drug substance (packaged1n glass vials) to light fl
`
`
`molecular weight protein/ fold), other
`impurities (/. fold), other
`impurities
`/ fold), and total impurities (/ fold), but there were no unique photo-degradation products. ChangesIn
`the drug substance manufacturing process during development resulted1n some changes1n the impurity
`profile. Repeat—dose toxicity of drug substance impurities in late—stage development batch that had
`undergone forced ‘fi 5 were assessed in a 4—week study in rats.
`
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`
`NDA 22,341
`
`Process—related impurities were not detected in the drug substance. The/' step manufacturing
`process includes fermentation (steps / ), recovery (steps / ), purification of the liraglutide precursor
`(steps / J), acylation (step
`), and purification and —-- . (steps _ ,). The acylating agent is /
`
`W’
`
`‘
`_ _
`7
`pilot and production scale batches tested (see Chemistry Review 1, page 37).
`~-———_—~—-—-—_=—u-==-
`Levels of these process-related impurities are below the threshold of toxicological concern for genotoxic
`impurities (1.5 mcg/daily dose).
`The final drug product, liraglutide injection, is an aqueous formulation of 6 mg/mL liraglutide,
`1.4 mg/mL disodium phosphate dihydrate, 14 mg/mL propylene glycol, and 5.5 mg/mL phenol at pH 8.15
`
`in an ' 1 _
`_ ‘filled glass cartridge containing 3 mL liraglutide solution. The glass cartridge is inserted
`
`into a multiple dose pen injector for subcutaneous injection. The drug product is stable for
`
`. Liraglutide is light sensitive and should be stored in the
`capped pen injector protected from light. There were no unique impurities in the drug product, so
`impurities qualification are the same as for the drug substance. Excipients are qualified by existing safety
`data.
`
`_ /
`
`Toxicity of impurities in the final to—be-marketed formulation, formulation 4,containing 6.0
`mg/mL liraglutide in solution at pH 8.15 was adequately assessed in subcutaneous repeat-dose toxicity
`studies, but not in genetic toxicity studies. In chronic repeat-dose toxicity studies, liraglutide caused
`irreversible injection site reactions in monkeys using drug formulations that were at least 3-fold more
`dilute than the clinical formulation. Fibrosarcomas occurred in the dorsal skin and subcutis of male mice.
`This carcinogenic effect of liraglutide at or near the injection site in mice may not be GLP- lR—mediated
`and it occurred using a liraglutide dosing formulation that was lO-times less concentrated than the clinical
`formulation. Genetic toxicity of liraglutide impurities at levels consistent with drug substance and drug
`product acceptance criteria should be evaluated.
`
`Unresolved Toxicology Issues
`
`1. Human relevance of liraglutide-induced thyroid C-cell tumors in rats and mice is unknown.
`
`2. Local toxicity after repeat dosing with liraglutide was not adequately assessed in nonclinical
`studies because liraglutide concentrations in nonclinical formulations used in repeat—dose toxicity
`and carcinogenicity studies were substantially lower than the liraglutide concentration in the
`clinical formulation.
`
`3. Genetic toxicity of some liraglutide impurities were not adequately assessed in vitro.
`
`Recommendations: Not approvable. Please see ‘Recommendations of Approvability’ on page 1.
`
`Suggested Labeling: Please see ‘Recommendations on labeling’ starting on page 1.
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`
`NDA 22,341
`
`APPENDICIES
`
`Appendix A: Mouse Carcinogenicity Study Review
`
`
`
`NNC 90—1 170 Tumor Findings in Male Mice
`
`Historical
`Organ/Tissue
`Incidence
`Parameter
`Neoplasm
`.
`incidence (%)
`
`o-cell adenoma
`
`Dorsal skin &
`.
`subcutis
`
`fibrosarcoma
`
`Injection site
`
`Equivocal
`(+ dose response,
`.
`lacking statistically
`significant increase in Dorsal .skm &
`at least the HD group)
`SUbCUtIS
`Equivocal
`(- dose response.
`statistically significant
`increase in at least 1
`dose group)
`
`Vascular (all
`sites)
`
`fibrosarcoma
`
`mabdomyosarwma
`
`hemangioma or
`hemangiosarcoma
`
`0
`.
`.
`____¥I}.°_,’SETEE_(..{‘EZ.
`p—value
`
`incidence (%)
`
`> 0.05 0.001 > 0.05 0.036
`pvalue
`
`Underlined values considered positive based on trend analysis p—value for rare (p < 0.025) or common (p < 0.005) tumors, p-value for
`pairwise comparison to the control group for rare (p < 0.05) or common (p < 0.01) tumors, and the incidence in the historical control group.
`
`NNC 90-1170 Tumor Findings in Female Mice
`
`Result
`
`Organ/Tissue
`
`Neoplasm
`
`Historical
`.
`Incidence
`
`c—cell ad enoma
`
`Positive
`
`.
`TherId
`
`,
`c-cell carcrnoma
`
`ccell adenoma or
`carcrnoma
`
`Parameter
`
`incidence (%)
`p—value
`
`incidence (%)
`p—value
`
`incidence (%)
`
`Underlined values considered positive based on trend analysis p—value for rare (p < 0.025) or common (p < 0.005) tumors,
`p-value for pairwise comparison to the control group for rare (p < 0.05) or common (p < 0.01) tumors, and the incidence in
`the historical control group.
`
`Key study findings:
`0
`Subcutaneously injected NNC 90-1170 (dorsal surface) was a non-genotoxic carcinogen in male
`and female mice with treatment related neoplasms occurring in thyroid c-cells (males and
`females) and dorsal skin and subcutis (males).
`The NOAEL for neoplastic findings was 0.2 mg/kg/day NNC 90-1170 (uncorrected SM 1.8)
`based on increased incidence of thyroid c-cell adenomas in males and females and combined 0—
`cell adenomas / carcinomas at 3 1 mg/kg/day NNC 90-1170. Focal thyroid c-cell hyperplasia, a
`preneoplastic finding, occurred at 2 0.2 mg/kg/day.
`
`0
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`NDA 22 341
`
`o NNC 90-1170 dose-dependently increased the incidence of focal thyroid c-cell hyperplasia, a
`preneoplastic lesion, at 3 0.2 mg/kg/day in males and females, dose-dependently increased the
`incidence of thyroid c-cell adenomas at 3 1 mg/kg/day in males and females (uncorrected
`human exposure multiple (uHEM) 10), and increased the incidence of combined c-cell adenomas
`/ carcinomas at 3 1 mg/kg/day in females (uHEM 10).
`0 Between weeks 25 and 104, plasma calcitonin levels increased > 2 fold at 3 mg/kg/day in
`males and females.
`
`0
`
`0
`
`0 A positive finding of dorsal skin and subcutis fibrosarcomas at 3 mg/kg/day NNC 90-1170 in
`males (uHEM 45). There was an equivocal finding of dose-related dorsal skin and subcutis
`rhabdomyosarcoma and injection site fibrosarcomas in males, and incidences in the 3
`mg/kg/day group (uHEM 45) were above the historical control range for both tumors, but the
`increased incidence for either finding never reached statistical significance in any NNC 90—1 170
`group. The sponsor’s analysis of tumor incidence data grouping total sarcomas dorsal surface
`skin and subcutis was statistically significant for trend (p < 0.001) and pair-wise analysis
`compared to controls at 3 mg/kg/day NNC 90-1170 in males (p < 0.001, uHEM 45). In control
`group females, there was a high incidence of total sarcomas in the skin and subcutis.
`Equivocal findings in males occurred in the vasculature (hemangiomas / hemangiosarcomas at
`all sites at 0.2 mg/kg/day), but the increased incidence was not dose related.
`The NOAEL for non-neoplastic findings was < 0.03 mg/kg/day. Non—neoplastic findings
`occurred in thyroid (inflammatory cell infiltrate at 2 0.03 mg/kg/day in males and at 0.03 and 3
`mg/kg/day in females; focal c-cell hyperplasia, considered a preneoplastic lesion, at 3 0.2
`mg/kg/day in males and females), liver (pigmented Kupffer cells (attributed to hemosiderin
`accumulation) at 3 0.03 mg/kg/day in males and at 3 0.2 mg/kg/day in females, centrilobular
`hypertrophy, diffuse centrilobular hepatocyte vacuolation at 2 003 mg/kg/day in males), spleen
`(hemosiderin accumulation at 2 0.03 mg/kg/day in females), femoro-tibial joint (degenerative
`disease at 3 0.03 mg/kg/day in males and at 0.03, 1, and 3 mg/kg/day in females), seminal
`vesicles (lymphocytic infiltration at Z 0.03 mg/kg/day and inflammation at 0.03 and 3 mg/kg/day
`in males), and thymus (tubular cystic hyperplasia at 3 0.03 mg/kg/day in males and at 3 0.2
`mg/kg/day in females).
`0 Methodological / Protocol isues:
`0 Due to low survival of control group females in the main study group, termination of the
`78 week interim sacrifice group was cancelled and treatment was continued for 104
`weeks. Tumor analysis was performed after combining results from both main study and
`week 78/ 104 groups.
`0 Actual NNC 90— 1170 concentrations were up to 3 fold lower than the nominal
`concentration for the 0.03 mg/kg/day dosing solution. However, human risk assessment is
`based on comparative exposure.
`0 Mice used for assessment of anti-liraglutide antibodies in week 104 survived 104 weeks
`of treatment (week 78/104 week group with treatment of 78 week interim sacrifice group
`extended to week 104), but these mice were sacrificed 10 days after the last dose to
`washout residual liraglutide that could potentially interfere with the anti-liraglutide
`antibody assay. These mice were included in the carcinogenicity assessment.
`Ophthalmoscopic examinations were not performed.
`0 Validation of the commercial rat plasma immunoradiometric assay to measure mouse
`plasma calcitonin was not submitted in the NDA, although the report references 2 assay
`validation reports (reports 205089 and restandardization report 205189).
`0 Although transient weight loss and food consumption occurred in the first weeks of the
`study, a pharmacodynamic effect of NNC 90-1170 was not sustained over the entire
`study period.
`
`0
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`
`NDA 22,341
`
`Adequacy of the carcinogenicig study and appropriateness of the test mode]:
`Mice are pharmacologically responsive to subcutaneously administered NNC 90-1170
`(transiently decreased body weight and food consumption in CD-l mice, lowered blood glucose in
`diabetic ob/ob and diabetic db/db mice, and increased beta cell mass in db/db mice) and in the
`carcinogenicity study, mice did not mount a neutralizing antibody response. Protein binding of NNC 90-
`1170 is slightly higher in mice than in humans. There are no major metabolites of lipid—labeled 3H-[Pal]—
`liraglutide in humans, but metabolism of 3H-[Pal]-liraglutide is similar in vivo and in vitro in mice and
`humans. In vitro metabolism of peptide-labeled 3H—[tyr]-liraglutide is similar in mice and humans, but in
`vivo metabolism was not characterized in either species.
`
`Evaluation of tumor findings:
`Treatment-related neoplastic lesions occurred in thyroid (c—cell adenomas at 3 1 mg/kg/day in
`males and females, c—cell adenomas / carcinomas at 2 1 mg/kg/day in females) and dorsal skin and
`subcutis (fibrosarcomas at 3 mg/kg/day in males).
`
`CAC concurrence:
`
`- The Committee concurred that the study was acceptable based on tumor findings in males and females.
`~ The Committee concurred that thyroid C-cell adenomas, C-cell adenomas or carcinomas (combined),
`and dorsal skin and subcutis fibrosarcomas were drug related. Liraglutide significantly increased the
`incidence of thyroid c-cell adenomas at 3 1 mg/kg in males and females, C-cell adenomas and carcinomas
`(combined) at 3 1 mg/kg in females, and dorsal skin and subcutis fibrosarcomas at 3 mg/kg in males.
`
`Study no.: 204229 (sponsor), 457274 5”
`Submission, Module, and page #: N000 42.3.4.1.1, pages 1 — 3096
`Conducting laboratory and location:
`~W
`Date of study initiation: 23 November 2004
`Study ending date: 1 December 2007
`GLP compliance: Yes (OECD compliance claimed)
`QA report: yes (X) no ( )
`Drug, lot #, and % purity: NNC 90-1170 lots shown in the table below. Purity of 97.1% by RP—HPLC
`reported for lot PQ50365 only (certificate of analysis in Appendix B).
`
`Test new
`Batch No.
`lfiuiis
`Arrival Date
`Expiry Date
`
`PQSQHB
`934
`(3? October 2004
`12 August 2005
`
`m4)
`
`NNC 9ft-I 1 m
`6.25 or 6.0 mgfml limglutide
`
`I’QSOSGS
`3‘)
`l (i-June»0§
`l LMarchJI)?
`
`PQSOM?
`541$
`21 July 3005
`,
`14 March 2001‘:
`____.____.
`18 January 3006
`l 1 September 2006
`
`P050365
`
`500
`
`PQSOIGS
`
`25.0
`
`06 July 2006
`
`ll September 2007
`
`3.90
`RQfil)S 74
`
`
`()6 September 2006
`
`‘28 March 300?
`
`Typical Certificates of Analysis for a batch of test item and vehicle used are presented in
`Appendix B and Appendix (I
`
`[N000 4.2.3.411 P15]
`
`Methods
`
`Doses: 0 (vehicle), 0.03, 0.2, 1.0, 3.0 mg/kg/day NNC 90—1170
`Basis of dose selection (MTD, MFD, AUC etc. 1: AUC ratio > 25 in males and females.
`Species/strain: CD-1 mice (Crl:CD—1TM(ICR)BR)
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`
`NDA 22,341
`
`Number/sex/group (main study 1: 50 /sex/dose main study. Due to mortality in main study control
`females, treatment of week 78 interim sacrifice group was extended to 104 weeks (29/sex/ 0 or
`3.0 mg/kg/day and 17/sex/ 0.03, 0.2, or 1.0 mg/kg/day)
`
`
`Animal Numbers
`Main Study
`Week 758
`(treatment extended to
`
`104 Weeks)
`Males
`Females
`Males
`Females
`
`1
`Central
`1)
`1'50
`251-300
`501—529
`610—638.
`
`Low
`0.03
`51.1110
`301-350
`5304346
`639-655
`2
`
`
`
`
`
`
`
` 3 Intermediate 1 02 101-150 351-401) 547—563 656672
`4
`Intemwdime II
`1.0
`[SE-201}
`401-450
`564-580
`673-65?)
`
`5 6905.718 High 3.0 201-250 451400 581~6Cv9
`
`
`
`
`
`
`Extra animals were numbered sequentially From 7 i 9 onwards.
`
`Treatment
`
`(mgikgfday)
`
`Group
`
`[NOOO 4.2.3.4.1.1 P18]
`
`
`Route formulation volume: subcutaneous injection (dorsal surface, rump and between scapula),
`6.0 — 6.25 mg/mL NNC 90-117 solution diluted in vehicle (1.4 mg/mL monosodium phosphate
`dehydrate, 14 mg/mL propylene glycol, 5.5 mg/mL phenol), 5.0 mL/kg
`Freguency of dosing: once a day
`Satellite groups used for toxicokinetics or special groups:
`78 week interim sacrifice group was planned, but treatment continued for 104 weeks due to
`decreased survival in main study control females.
`
`Satellite groupfor toxicokz'netics andplasma calcitonz‘n consisting of 17/sex/dose in each group
`terminated on weeks 26, 52, and 104.
`
`Animal Numbers
`Treamaem
`
`
`Week 26
`Week 52
`Week 104
`Group
`.
`(mgikgiday)
`Males
`Fenmles
`Males
`1~ exnales
`Males
`Femates
`
`Control
`‘
`0
`1001-1017
`1086—1102
`1171.113?
`1256-1272
`13414357
`1426-1442
`.E
`2
`Low
`'
`0.03
`1018—1034
`1103-1119
`USS-£204
`1273-1289
`1358-1374
`1443-1459
`
`1460-1476
`3
`Intermediate}
`0.2
`1035:1051
`1 l20«1136
`1205—1221
`12904306
`1375-1391
`Intermediateil
`1.0
`1052-1068
`1137-1153
`1222—1238
`1307-1323
`13924408
`14774493 12.4.2
`
`14944510
`1409-1425
`13244340
`1239—1255
`1154-1}70
`1069-1035
`3.0
`High
`A further 10 males (Numbers 151 H 520) and 10 femaies (1521»1530) were used for pretrial
`antibody level assessment. Extra animals were numbered from the last number used.
`[NOOO 4.2.3.4.].1 P18]
`Age and weight: 5 weeks on arrival with males weighing 27 .8-42.9g, and females weighing
`20.7-39.6 g at the start of the study
`Animal housing: One male and 2 or 3 females per cage by dose group were housed in suspended
`polypropylene cages (48 x 15 x 13 cm) with solid bottoms and stainless steel grid tops (including
`integral food hopper), sterilized white wood shavings (analysis revealed no significant
`contaminants), and a polycarbonate water bottle with a stainless steel nozzle (page 17).
`Restriction paradigm for dietaryflstriction studies: None.
`Drug stabilifl/homogeneity: Two different SOPs were used to determine stability of NNC 90—
`1170 solution for subcutaneous injection. Drug concentrations in O, 0.2, 1.0, and 3.0 mg/kg/day
`dosing solutions were analyzed using method 434-1018 and the 0.03 mg/kg/day dosing solution
`was analyzed using method 878-LP-08005. Dose solution samples were taken in week 1, week
`39, and week 103 on days 1 and 7.
`Dual controls employed: No, but controls included in satellite TK/calcitonin and 78 week interim
`sacrifice groups
`
`301 of513
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA 22,341
`
`Interim sacrifices: Due to reduced survival in main study control group females, dosing was
`continued to week 104 for 78 week interim sacrifice group.
`Deviations from original study protocol:
`Week 78 interim sacrifice group mice were rescheduled for sacrifice in week 104 with treatment
`continued to week 104 because of reduced survival in main study control group females.
`Therefore, a week 78 interim sacrifice group report was not issued.
`
`Sixteen males in group 5 were under-dosed on 17 February 2005 and 6 females in group 4 were
`underdosed on 28 April 2005. Both dosing errors were corrected by giving an additional dose
`volume to give the correct dose.
`
`The following TK group mice were dosed twice during toxicokinetic sampling in study week 52:
`Srwpfim mm $33}
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`
`[N000 4.2.3.411 P24]
`Due to scheduling errors, no samples were taken for the following study doses/time points in
`week 26:
`
`Group 2 predose
`Group 4 2 hour
`
`Additional thyroid processing in week 104 was going to be based on results from thyroid
`histology from mice from week 78 interim sacrifice group, but .this group was sacrificed in week
`104.
`
`The following summary table lists mice replaced during the first 2 weeks of dosing.
`Due m :1 numbered" deaths and c-lmical signed [1198 l'nilowing nlzimnl: rcgflummemsa were impale during
`the first [we weeks ufihc sandy.
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`
`The following summary table lists toxicokinetic and antibody satellite group mice replaced during
`the first week of dosing.
`
`302 0f513
`
`

`

`Reviewer: Anthony L Parola, PhD
`
`NDA 22,341
`
`The following mtin‘ml replacmnmts were $90.1an during Week 1 orthe staggered satellite and
`antibody snugly.
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`mooo 4.2.3.4.1.1 P19]
`
`Observation times
`
`Mortalig: Twice a day.
`Clinical signs: Main study mice checked twice a day with detailed examination performed once a week.
`Starting in week 83, clinical signs and detailed were recorded for week 78/104 group mice using the same
`schedule as main study group mice. Palpations for masses were performed beginning in week 13 for all
`mice. Satellite TK/calcitonin group mice and antibody group mice were examined daily for welfare
`purposes.
`Body weights: Recorded once a week prior to starting treatment, then daily during treatment. Body
`weight of main study mice was reported once a week.
`Food consumption: Quantity of food consumed by main study group mice (per cage) was recorded
`weekly until the end of week 13, then monthly afterward. For week 78/104 group mice, food consumption
`over a 4 week period (per cage) was recorded starting in week 83.
`Water consumption: Water consumption was monitored by visual inspection, but it wasn’t quantified.
`Anti-NNC 90-1170 antibody: Up to 1 mL orbital sinus blood samples from isoflurane anesthetized
`antibody study group mice (lO/sex/dose prior to initiating treatment, 5/sex/dose during treatment) was
`taken to determine if anti-NNC 90-1170 antibodies occurred after treatment. Mice were bled 3 days after
`their last dose in week 26 and 6 days after their last dose in weeks 52 and 78. Samples for antibody
`analysis were taken from week 104 satellite group mice bled 10 days after their last dose in week 97.
`Samples for antibody analysis from week 78 satellite group mice reassigned for termination in week 104
`were bled 10 days after the last dose. After bleeding mice were sacrificed and necropsied. The assay is a
`radioimmunoassay precipitating immunoglobulin bound 125I —liraglutide after overnight incubation of 125]
`—liraglutide with mouse plasma.
`In the absence of NNC 90-1170, the sensitivity of the anti—NNC 90-1170 antibody
`radioimmunoassay was 25 — 50 ng/mL. The assay sensitivity decreased to > 1 mcg/mL antibody at 3 10
`nM NNC 90-1170 in plasma. To decrease interfer