Toxicologic Pathology, XX: 1-13, 2015 Copyright # 2015 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623315581020

Utilization of the Zucker Diabetic Fatty (ZDF) Rat Model for Investigating Hypoglycemia-related Toxicities MARK TIRMENSTEIN1, JOSEPH HORVATH1, MICHAEL GRAZIANO2, RAJA MANGIPUDY1, THOMAS DORR1, KARYN COLMAN1,3, BRADLEY ZINKER4, MARK KIRBY4,5, PETER T. W. CHENG6, LAURA PATRONE1, JOHN KOZLOSKY1, TIMOTHY P. REILLY7, VICTOR WANG1, AND EVAN JANOVITZ8 1

Bristol-Myers Squibb, Drug Safety Evaluation, New Brunswick, New Jersey, USA 2 Bristol-Myers Squibb, Drug Safety Evaluation, Princeton, New Jersey, USA 3 Present affiliation: Novartis Institutes for BioMedical Research, East Hanover, New Jersey, USA 4 Bristol-Myers Squibb, Discovery Biology, Pennington, New Jersey, USA 5 Present affiliation: Lilly China Research and Development Center, Shanghai, China 6 Bristol-Myers Squibb, Discovery Chemistry, Pennington, New Jersey, USA 7 Bristol-Myers Squibb, Exploratory Clinical and Translational Research, Princeton, New Jersey, USA 8 Bristol-Myers Squibb, Discovery Toxicology, Pennington, New Jersey, USA ABSTRACT Glucokinase (GK) catalyzes the initial step in glycolysis and is a key regulator of glucose homeostasis. Therefore, glucokinase activators (GKa) have potential benefit in treating type 2 diabetes. Administration of a Bristol-Myers Squibb GKa (BMS-820132) to healthy euglycemic SpragueDawley (SD) rats and beagle dogs in 1 mo toxicology studies resulted in marked and extended hypoglycemia with associated clinical signs of toxicity and degenerative histopathological changes in the stomach, sciatic nerve, myocardium, and skeletal muscles at exposures comparable to those expected at therapeutic clinical exposures. To investigate whether these adverse effects were secondary to exaggerated pharmacology (prolonged hypoglycemia), BMS-820132 was administered daily to male Zucker diabetic fatty (ZDF) rats for 1 mo. ZDF rats are markedly hyperglycemic and insulin resistant. BMS-820132 did not induce hypoglycemia, clinical signs of hypoglycemia, or any of the histopathologic adverse effects observed in the 1 mo toxicology studies at exposures that exceeded those observed in SD rats and dogs. This indicates that the toxicity observed in euglycemic animals was secondary to the exaggerated pharmacology of potent GK activation. This study indicates that ZDF rats, with conventional toxicity studies, are a useful disease model for testing antidiabetic agents and determining toxicities that are independent of prolonged hypoglycemia. Keywords:

glucokinase activator; hypoglycemia; Zucker diabetic rats; ZDF; diabetes; type 2 diabetes.

pancreatic islets and catalyzes the conversion of glucose to glucose-6-phosphate (Matschinsky et al. 2011). In the liver, GK regulates hepatic glucose utilization and output by determining the rate of glucose uptake and catalyzing the initial step in glycolysis and glycogen synthesis. In the pancreas, GK establishes the threshold for b-cell glucose-stimulated insulin secretion and its activation leads to insulin release uncoupled from blood glucose levels (Cardenas 1995). Through these mechanisms, GK is a major regulatory enzyme in maintaining glucose homeostasis. Patients with T2DM have a decreased capacity for hepatic glucose uptake and glycogen synthesis, which may be due in part to decreases in hepatic GK activity (Pfefferkorn 2013). Mutations in GK that decrease the activity of this enzyme have been characterized in humans and are associated with diabetes, while mutations that result in pseudo-activation of hepatic GK have been associated with reduced blood glucose levels (Pfefferkorn 2013). Based on the key role of GK in glucose homeostasis, GK activators (GKa) have been targeted by the pharmaceutical industry as a potentially attractive therapeutic option for the treatment of T2DM (Matschinsky et al. 2011). Bristol-Myers Squibb GKa (BMS-820132), a partial activator of GK, was developed based on its potential to improve glucose homeostasis through a balance in the

INTRODUCTION Type 2 diabetes mellitus (T2DM) is a worldwide epidemic affecting 347 million people globally (World Health Organization 2015). Despite the existing therapeutic options, many T2DM patients do not achieve adequate glycemic control, leading to complications. Therefore, there is a need for novel therapies with improved efficacy and enhanced safety to help patients achieve their goal of adequate glycemic control (Coghlan and Leighton 2008). The enzyme glucokinase (ATP:D-glucose 6-phosphotransferase; EC 2.7.1.2; GK) is expressed primarily in the liver and The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Mark Tirmenstein, Bristol-Myers Squibb, Drug Safety Evaluation, PO Box 191, One Squibb Drive, New Brunswick, NJ 08903, USA; e-mail: [email protected]. Abbreviations: ALP, alkaline phosphatase activity; AUC, area under the curve; BMS-820132, Bristol-Myers Squibb glucokinase activator; Cmax, maximum concentration; DIO, diet-induced obese; NOAEL, no observed adverse effect level; GK, glucokinase; Gka, glucokinase activator; SD, Sprague-Dawley; T2DM, type 2 diabetes mellitus; ZDF, Zucker diabetic fatty. 1

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reduction of excess hepatic glucose production and stimulation of pancreatic insulin release via allosteric modulation of hepatic and pancreatic b-cell GK, respectively. As part of the development program for BMS-820132, industry-standard 1-mo toxicity studies were conducted in normal healthy (euglycemic) rats and dogs. The results of these studies showed that BMS-820132 was associated with prolonged and significant hypoglycemia and degenerative histopathologic lesions at BMS-820132 exposures (10 [dog] and 50 [rat] mgh/mL) that were similar to those expected to occur at clinically relevant doses (projected exposures [area under the curve (AUC)] in humans at the starting [0.3 mg] and stopping [300 mg] single doses in the phase 1 clinical trial were 0.021 mgh/mL and 21.04 mgh/mL, respectively). Therefore, it was necessary to determine if the observed histopathologic lesions were a direct result of BMS-820132 or were secondary to prolonged hypoglycemia. Adverse clinical outcomes due to hypoglycemia (pharmacologic effect of BMS-820132) could be managed much more easily in diabetics than those due to off-target effects of BMS-830132. Diabetics already have higher levels of blood glucose than euglycemic individuals and would therefore be expected to be less susceptible to hypoglycemia. In addition, levels of blood glucose could be easily measured in clinical trials with BMS-820132. The data contained in this report describe the use of Zucker diabetic fatty (ZDF) rats, which are hyperglycemic and resistant to insulin, to investigate the toxicities of BMS-820132 in normal euglycemic animals that were hypothesized to be secondary to prolonged hypoglycemia. ZDF rats were considered to be a better representation of hyperglycemic diabetic patients than normal euglycemic rats and dogs and, therefore, a useful disease model to assess the potential risks of the administration of GKa in type 2 diabetic patients. In a broader sense, this work also serves as a case study for how a disease model can be used for the risk assessment of novel therapeutic agents in human disease populations. MATERIALS

AND

METHODS

Test Substance BMS-820132 (99.8% purity) was supplied as the free base by Bristol-Myers Squibb. All analytical testing on BMS820132 was conducted by the Department of Analytical Research and Development in New Brunswick, New Jersey. The test article carrier and vehicle used in the Sprague-Dawley (SD) rat toxicity study was 50% propylene glycol (v/v), 30% Capmul PG-8 (v/v), 15% polyethylene glycol 400 (v/v), and 5% ethanol (v/v). The test article carrier and vehicle used in the ZDF rat toxicity study was 50% propylene glycol (v/v), 30% Capmul PG-8 (v/v), and 20% polyethylene glycol 400 (v/v; the ethanol component was eliminated from the formulation to reduce potential confounding gastric effects). The test article carrier and vehicle used in the toxicity study in dogs was 50% propylene glycol, 25% polyethylene glycol 400, 20% da-tocopheryl polyethylene glycol 1000, and 5% ethanol (v/v).

TOXICOLOGIC PATHOLOGY

The vehicle and test article carrier were administered at a dose of 8 mL/kg/day to SD and ZDF rats and 5 mL/kg/day to dogs. BMS-820132 dosing formulations were prepared as a solution every 5 days (rats) or 1 to 4 days (dogs) and stored in an incubator at approximately 40 to 45 C. The stability of all dosing formulations was verified under these conditions. Animals and Husbandry SD Crl:CD rats were obtained from Charles River Laboratories, Inc (Raleigh, NC). Rats were chosen on the basis of body weight and were assigned to groups using a computergenerated stratified randomization procedure. Rats in poor health or with significant abnormalities were not assigned to the study. The SD rats were approximately 9 weeks old at study start and had body weight ranges of 280 to 316 g (males) and 194 to 234 g (females). Male ZDF (ZDF/CRL-LEPR FA) rats were obtained from Charles River Laboratories, Inc. (Kingston, NY). These rats were assigned to the study using a computer-generated stratified randomization procedure based on serum glucose levels, such that all groups had approximately equivalent serum glucose levels at study start. Rats in poor health, without elevated serum glucose levels (>150 mg/dL) and/or with significant abnormalities were not assigned to the study. At the beginning of the study, the male rats were approximately 9 weeks old and weighed between 279 and 337 g. Purebred beagle dogs were obtained from Marshall Bioresources (North Rose, NY) and were chosen for the study on the basis of clinical condition and body weight. They were assigned to groups using a computer-generated stratified randomization procedure. Dogs in poor health, with body weights outside the acceptable range, or with significant abnormalities were not assigned to the study. At the start of the study, the body weight ranges were 8.1 to 10.1 kg for males and 6.1 to 7.4 kg for females, and the dogs were approximately 8 to 9 mo old. SD and ZDF rats were individually housed in stainlesssteel wire-bottom cages. The rats were offered purified and chlorinated tap water via an automated watering system ad libitum and were fed HSD/Teklad Certified Global 18% Protein Rodent Diet #2018C (Madison, WI; SD) or Purina Rodent Diet #5008 (St. Louis, MO; ZDF). The rats were housed in rooms that were monitored and maintained at a target temperature range of 64 F to 79 F, a target mean humidity range of 30 to 70% and illuminated for 12 hr daily. Dogs were housed individually in runs and were offered purified and chlorinated tap water via an automated watering system ad libitum and were fed Harlan Diet #2025C Certified Global 25% Protein Dog Diet. Study protocols and all procedures involving the use of animals were reviewed and approved by the Bristol-Myers Squibb Institutional Animal Care and Use Committee. Animal care and experimental procedures were conducted in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

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SD Rat Study BMS-820132 was administered to SD rats (15/sex/group) once daily for 1 mo by oral gavage at doses of 20, 80, or 300 mg/kg/day. A control group (15 rats/sex) received the vehicle alone. Criteria for evaluation included survival, clinical signs of toxicity, body weight, food consumption, physical, neurologic, and ophthalmic examinations, clinical pathology (including timed serum glucose analyses in satellite rats), and gross and histopathologic assessments. Necropsies were conducted at the end of the 1-mo dosing period and the 2-week post-dose recovery period. In addition, plasma exposures to BMS-820132 and serum glucose profiling were determined in satellite groups (9 rats/sex/group) following doses on days 1 and 28 (0.5, 1, 2, 4, 8, and 24 hr post-dose). ZDF Rat Study BMS-820132 was administered to male ZDF rats (15 rats/ group) once daily for 1 mo by oral gavage at doses of 10, 50, or 200 mg/kg/day. (Doses were selected to achieve similar systemic exposures to those in SD rats.) A control group (15 males) received the vehicle alone. Criteria for evaluation included survival, clinical pathology (including timed serum glucose and insulin analyses), and gross and histopathologic assessments. Necropsies were conducted at the end of the dosing period. Plasma exposures to BMS-820132 were determined following doses on days 1 and 26 (1, 2, 4, 8, and 24 hr postdose). Beagle Dog Study BMS-820132 was administered to dogs (5/sex/group) once daily for 1 mo by oral gavage at doses of 10, 60, or 120 mg/ kg/day. A control group (5 dogs/sex) received the vehicle alone. Criteria for evaluation included survival, clinical signs of toxicity, body weight, food consumption, physical, neurologic, cardiovascular, and ophthalmic examinations, clinical pathology (including timed serum glucose analyses), and gross and histopathologic assessments. Necropsies were conducted at the end of the 1-mo dosing period and the 2-week post-dose recovery period. (Based on worsening clinical condition in all high-dose [120 mg/kg/day] dogs and the death of 1 male in this group following 19 daily doses, dosing of the remaining highdose dogs was terminated following 22 days of dosing for males and 20 days of dosing for females. The 2-week postdose evaluation of these high-dose dogs [2 dogs/sex] began immediately following the interim evaluation.) In addition, plasma exposures to BMS-820132 were determined following doses on days 1, 14, and 20/22 (120 mg/kg/day dose group) or 28 (10 and 60 mg/kg/day dose groups); blood was collected at 0.5, 1, 2, 4, 8, and 24 hr post-dose. Toxicokinetics Blood samples were collected from the tail (rats) or jugular (dogs) vein in tubes containing K2EDTA and concentrations of BMS-820132 were determined using a liquid–liquid extraction

3

with subsequent analysis by liquid chromatography–tandem mass spectrometry (Shimadzu HPLC; LEAP HTC PAL autosampler; Sciex API 4000 mass spectrometer). Toxicokinetic parameter values (i.e., AUC and maximum concentration [Cmax]) were calculated using noncompartmental methods by eToolbox/Kinetica (version 2.6.1/4.4.1; Thermo Electron Corp., Philadelphia, PA). All toxicokinetic parameters were calculated from a composite mean plasma concentration versus time curve for each group (rats) or from each individual time point value (dogs). The mean AUC [0 to 24 hr] was calculated as a measure of systemic exposure for each group using the linear trapezoidal rule. Clinical Pathology Assessments Comprehensive assessments of hematology (ADVIA 2120i, Siemens Healthcare Diagnostics, Inc.; Hema-Tek Series Stainer, Miles Inc.), coagulation (STA Compact, Diagnostica Stago, Inc), and standard serum chemistry (including timed glucose measurements; ADVIA 1800, Siemens Healthcare Diagnostics, Inc.) were performed in each study. Serum insulin levels (Rat Insulin Assay Kit, Multi-Array Assay System, Sector Imager 2400, Meso Scale Diagnostics, LLC, Gaithersburg, MD) were assessed in the ZDF rats. Blood was obtained from the tail (rats) or jugular (dogs) vein. In the SD rat study, as standard practice, blood for hematology (days 29 and 43 [recovery]) and serum chemistry (days 15, 29, and 43 [recovery]) was obtained from main-study animals fasted overnight. Timed serum glucose measurements were performed on days 1, 14, and 28 (0, 0.5, 1, 2, 4, 8, and 24 hr after dosing) on non-fasted satellite rats. In the ZDF rat study, serum glucose levels were assessed 3 or 4 days prior to dosing (to establish groups with equivalent serum glucose profiles). In addition, blood for serum glucose and insulin analysis was collected from all animals prior to and 2 hr following the daily dose on days 1, 14, and 26. (This more limited serum glucose profiling [vs. SD rats] was based on the limitations on total blood volume withdrawal for toxicokinetic plus serum glucose analysis during a 24-hr period. The selection of the 2-hr post-dose glucose evaluation time was based on anticipated maximal plasma concentration of BMS820132). Rats had food removed for 2 hr prior to pre-dose blood collection and returned after the 2-hr blood collection (to minimize potential effects of food consumption on blood glucose). Blood for day 26 serum chemistry analysis (including insulin) was collected from non-fasted rats overnight. In the dog study, blood samples for scheduled clinical pathology tests were obtained from fasted animals twice prior to the first dose, once prior to a daily dose during week 3, once at the end of dosing, and once during the second week of the post-dose period. An interim evaluation of all remaining high-dose dogs was added to the study and included evaluation of hematology, serum chemistry, and coagulation on day 22/ 20. In addition, blood samples for evaluation of serum glucose were obtained from all surviving high-dose dogs at approximately 0.5, 1, 2, 4, 8, and 24 hr after dosing on day 22/20.

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On days of toxicokinetic evaluations (day 1 and during weeks 2 and 4), blood samples for evaluation of serum glucose were obtained at approximately 0.5, 1, 2, 4, 8, and 24 hr after dosing. A urinalysis was performed on urine collected over an approximate 24-hr period, prior to the first dose, during week 5, and during the second week of the post-dose period. Dogs were not fasted during the 24-hr urine collection. Anatomic Pathology Assessments Animals were euthanized by isoflurane (rats) or propofol (dogs) anesthesia followed by exsanguination. Necropsies included gross examination and weights of selected organs and tissues (adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary gland, prostate gland, spleen, testes, thyroid gland, uterus, and thymus). Representative samples of each organ and tissue and all gross lesions were collected and fixed in 10% neutral-buffered formalin (except testes and eyes which were fixed in modified Davidson fixative). Tissues were processed, sectioned at approximately 6 m, stained with hematoxylin and eosin, and examined by light microscopy for any BMS820132-related or spontaneous lesions. Statistical Analysis Mean values from body weight and food consumption (rats only) determinations, clinical laboratory tests, and absolute and relative (to body weight) organ weights of treated groups were compared with those of the control group using the two-sided Dunnett’s test in the context of a one-way analysis of variance (Dunnett 1955; Dunnett 1964, conducted on Common Technical Document Table Generator application, Nagarro Inc., San Jose, California, USA, for generation of report summary tables and individual data appendices). Statistical tests were not applied to group comparisons for clinical observations and the incidence of gross findings and microscopic lesions.

RESULTS Toxicokinetics The systemic exposures of BMS-820132 in SD and ZDF rats and dogs are shown in Table 1. In each species, systemic exposures were generally dose related and similar in males and females with no evidence of BMS-820132 accumulation following repeated daily dosing (SD rats [no NOAEL established; 50 mgh/mL or approximately 2 the anticipated exposure at the 300 mg stopping dose in the phase 1 clinical trial] and dogs [no NOAEL established; 10 mgh/mL or approximately 0.5 the anticipated exposure at the 300 mg stopping dose in the phase 1 clinical trial]). Cmax occurred between 1 and 4 hr (SD rats), at 4 hr (ZDF rats), and between 0.5 and 4 hr (dogs) after dosing. Systemic exposure levels in male ZDF rats were up to 2.2 and 7.2 the exposures in SD rats and dogs, respectively. The NOAEL exposure in the ZDF rats was 281 mgh/mL or approximately 13 the anticipated exposure at the 300 mg stopping dose in the phase 1 clinical trial.

TOXICOLOGIC PATHOLOGY

Clinical Signs In the SD rat study, there were no BMS-820132-related clinical signs at 20 mg/kg/day. However, clinical signs of toxicity were observed at the higher doses and included perigenital soiling (4/15 females at 80 mg/kg/day noted only in week 3 and 11/ 30 rats at 300 mg/kg/day starting on day 3) and mostly 1 or 2 instances of black feces (sometimes associated with occult blood) in 1 male at 80 mg/kg/day on day 30 and in 10/30 rats at 300 mg/kg/day primarily during week 3. Additional BMS820132-related clinical signs at 300 mg/kg/day, noted in most rats on 1 to 5 days during the last week of the study, included transient episodes of decreased activity and ataxia within 8 hr following dosing. One female in this group was sacrificed early on day 26 (scheduled necropsies occurred on day 29) following the loss of righting reflex (see Table 2 for a complete listing of BMS-820132-related clinical signs). BMS-820132-related clinical signs were not observed during the 2-week post-dose period, with the exception of a single rat in the high-dose group that displayed tremors during the recovery period. In dogs, BMS-820132-related clinical signs were observed at all doses including sporadic salivation in 2/10, 7/10, and 8/10 dogs at 10, 60, and 120 mg/kg/day, respectively, and black/discolored feces (sometimes associated with occult blood at 60 and 120 mg/kg/day) in all dogs starting in week 2 at 10 and 60 mg/kg/day and week 1 at 120 mg/kg/day. Additional BMS-820132-related clinical signs at the mid and high doses included decreased activity (in 7/10 dogs [starting in week 2] and 10/10 dogs [starting in week 1] at 60 and 120 mg/kg/day, respectively) within 8 hr following dosing and ataxia which was evident in only 3 dogs at 60 mg/kg/day (1 or 2 episodes mostly in week 3 or 4) and in 9/10 dogs at 120 mg/kg/day (generally 1 or 2 episodes mostly in week 3). Body weight gain and food consumption in BMS-820132-treated dogs were similar to control dogs during the dosing period of this study. One male at 120 mg/kg/day died on day 19 following overt clinical signs including vomiting within 2 hr following dosing and signs consistent with severe hypoglycemia including convulsions, decreased activity, and recumbency. Due to the compendium of clinical signs in the majority of dogs at 120 mg/kg/day, 2 males and 3 females were euthanized early on days 23 (males) and 21 (females; see Table 2 for a complete listing of BMS820132-related clinical signs). In contrast, there were no BMS-820132 associated clinical signs or deaths in ZDF rats. However, balanoposthitis (swollen glans penis and preputial tissue) was observed in both control and BMS-820132-treated ZDF rats beginning on day 19. (Incidences of chronic-active inflammation of the penis/prepuce at necropsy were 12, 8, 6, and 11 out of 15, 8, 7, and 15 animals evaluated, respectively, in the control, and BMS-820132 low-, mid-, and high-dose groups.) Body Weights and Food Consumption In the SD rat study, males administered BMS-820132 generally had dose-related reductions in body weight gains starting on day 7 (body weights were up to 11% lower than controls by

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TABLE 1.—Toxicokinetics summary for SD rats, ZDF rats, and dogs. SD rats BMS-820132 dose (mg/kg/day) 20 BMS-820132 parameters Cmax (mg/mL)

a

AUC, 0–24 hr (mgh/mL)b

80

300

Study day

Male

Female

Male

Female

Male

Female

1 28 1 28

3.06 3.54 37.1 49.7

6.52 7.44 56.8 51.4

41.1 19.5 318 162

32.4 43.4 320 273

55.1 52.0 521 535

58.4 76.1 640 424

Male ZDF rats BMS-820132 dose (mg/kg/day) BMS-820132 parameters Cmax (mg/mL)a AUC, 0–24 hr (mgh/mL)b

Study day

10

50

1 26 1 26

6.13 5.86 55.5 60.0

34.7 26.1 380 281

200 85.6 103 1,210 1,160

Dogs BMS-820132 dose (mg/kg/day) 10 BMS-820132 parameters Cmax (mg/mL)

c

AUC, 0–24 hr (mgh/mL)e

60

120

Study day

Male

Female

Male

Female

Male

Female

1 28d 1 28d

3.60 4.08 12.2 13.9

2.10 3.81 5.22 9.76

20.1 29.3 90.8 131

22.0 30.9 70.3 92.2

36.1 34.1 166 162

53.5 36.4 262 156

Note: AUC ¼ area under the curve; BMS-820132 ¼ Bristol-Myers Squibb glucokinase activator; Cmax ¼ maximum concentration; SD ¼ Sprague-Dawley; ZDF ¼ Zucker Diabetic Fatty. a Day 1 and day 26/28 Cmax values represent the mean of n ¼3 rats. b AUCs calculated from values obtained from blood collected at 0.5, 1, 2, 4, 8, and 24 hr (SD rats) or 1, 2, 4, 8, and 24 hr (male ZDF rats) post-dose time points (n ¼ 3 rats per time point). c Day 1 and day 28 Cmax values represent the mean of n  4 dogs. d Day 20 (females) and day 22 (males) at 120 mg/kg. e AUCs calculated from values obtained from blood collected at 0.5, 1, 2, 4, 8, and 24 hr post-dose time points (n  4 dogs per time point).

the end of dosing), whereas females had dose-related increases in body weight gains from the study start (up to 12% heavier than controls by the end of the dosing phase of the study). In contrast, no significant changes were noted in body weights in dogs. In ZDF rats, no significant differences in body weights were noted between BMS-820132-treated and control rats. However, body weights in all groups (including controls) peaked after 2 or 3 weeks of dosing and then declined thereafter (Figure 1). Increased food consumption was noted in SD females (10– 22%) at the mid dose of 80 mg/kg/day, and in both sexes at the high dose of 300 mg/kg/day (9–13% in males and 22–36% in females). The high-dose SD males had decreased food consumption (12–18%) throughout the recovery period. In dogs, there were no meaningful differences in food consumption in any of the BMS-820132-treated groups compared to controls. ZDF rats consistently consumed 60 to 80% more food throughout the study compared to their male SD counterparts.

Clinical Pathology Serum Glucose/Insulin BMS-820132 induced dose- and time-dependent decreases in serum glucose concentrations in both SD rats and dogs (see Figure 2 for representative glucose concentration  time curves). These decreases were associated with BMS-820132 Cmax exposures and reached their nadir on day 14. On days 14 and 28, mean serum glucose levels were frequently 50 mg/dL in each species during the 0.5- to 8-hr post-dose period following administration of the mid and high doses of BMS820132. Mean serum glucose levels in controls ranged from 130 to 170 mg/dL (SD rats) and from 64 to 127 mg/dL (dogs) throughout the study. In the ZDF rat study, non-fasted mean serum insulin levels declined from the start (ZDF rats were 9 weeks of age) to the end of the study (13 weeks of age) in controls (Figure 3). Serum insulin was also evaluated on days 1, 14, and 26 in

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TOXICOLOGIC PATHOLOGY

TABLE 2.—Clinical signs summary for SD rats and dogs. SD rats BMS-820132 dose (mg/kg/day) Number of M/F in 1-mo dosing period Sex Finding During 1-mo dosing period Early death Ataxia Decreased activity Fecal blood Feces black Feces scant Loss of righting reflex Low body posture Perigenital soiling Rough haircoat Salivation Tremor Vocalization Finding during 2-week recovery period Number of M/F on recovery Tremor

Dogs

0 15/15 M/F

20 15/15 M/F

80 15/15 M/F

300 15/15 M/F

0 5/5 M/F

10 5/5 M/F

60 5/5 M/F

120 5/5 M/F

— — — — — — — — — — — — —

— — — — — — — — — — — — —

— — — 1/0 1/0 — — — 0/4 — — — —

0/1a 8/12 11/9 3/6 3/7 0/1 0/1 1/1 6/5 3/5 — 4/0 1/1

— — — — — — — — — — — — —

— — — — 5/5 — — — — — 1/1 — —

— 2/1 4/3 4/4 5/5 — — — —

1/0 4/5 5/5 5/5 5/5 — — — —

3/4 — —

4/4 1/0 —

5/5 —

5/5 —

5/5 —

5/5 1/0

2/2 —

2/2 —

2/2 —

2/2 —

Note: — ¼ absent. BMS-820132 ¼ Bristol-Myers Squibb glucokinase activator; M/F ¼ male/female; SD ¼ Sprague-Dawley. a Incidence per number of males/number of females during the period.

BMS-820132-treated ZDF rats. There was considerable individual variation in these data with no clear effect of BMS820132 administration (data not shown). Mean serum glucose levels in non-fasted ZDF rats were well above those observed in SD rats and ranged from 424 to 524 mg/dL in controls throughout the study (Table 3). No clear evidence of BMS820132-induced glucose lowering was observed at 2 hr postdose on days 14 and 26, however, all non-fasted serum glucose values remained above 100 mg/dL throughout the study, with the exception of a single high-dose rat (71 mg/dL at 2 hr postdose on day 1). Other serum chemistry Other BMS-820132 induced changes in serum chemistries in the SD study, observed mainly at 80 and 300 mg/kg/day, included increases in triglycerides (1.99–3.49 control) and minimal elevations in serum ALT (1.48–1.96) and AST (1.31–1.87) activities. In dogs, serum ALP was increased in females given 60 or 120 mg/kg/day (2.19–2.70 control) and in individual males on day 16 (1.44–2.78 pretest) and day 22 (1.37–1.84 pretest) given 120 mg/kg/day. Serum triglycerides were increased (1.91 controls) on day 16 in males given 120 mg/kg/day. In contrast, no similar BMS-820132-related effects on serum chemistry parameters were noted in the ZDF rats. Anatomic Pathology In the SD rats, lesions were observed microscopically in the stomach, sciatic nerve, skeletal muscle, and heart (Figure 4 and Table 4). At the lowest dose, only the stomach and sciatic nerve were affected by treatment. In dogs, lesions were limited to the

stomach at all doses and in the sciatic nerve at all doses except the lowest dose (Figure 5 and Table 4). In contrast, in ZDF rats with similar or greater exposures to BMS-820132, no lesions were observed in the sciatic nerve, skeletal muscle, or heart and only a single incidence of a stomach lesion was observed, and only at the high dose. In the stomach of SD rats treated with BMS-820132, portions of the glandular mucosa were necrotic with superficial areas of hemorrhage (Figure 4A). In the adjacent glandular mucosa, the epithelium was atrophic and/or showed a regenerative hyperplasia. In the stomach of dogs treated with BMS820132, mucus depletion/foveolar atrophy and edema of the superficial glandular mucosa (all doses) and minimal hemorrhage and/or erosion of the superficial glandular mucosa (all doses except the lowest dose) were observed (Figure 5A). In the affected ZDF rat, the stomach change comprised minimal multifocal foveolar and glandular cell necrosis/degeneration with foveolar hyperplasia/regeneration. In SD rats, nerve fiber atrophy and degeneration in the sciatic nerve, characterized by swollen sheaths containing axonal remnants, were observed (Figure 4B). Schwann cells were vacuolated and hypertrophic. In skeletal muscle, as represented by the quadriceps femoris, several myofibers were degenerate or necrotic and were associated with macrophage infiltration. Regenerating myofibers, with prominent proliferation of sarcolemmal nuclei, were also observed. In the heart, foci of myofiber degeneration and loss were particularly evident in the papillary muscles (Figure 4C). These foci of degeneration/loss were associated with reactive inflammation (mainly mononuclear) and mild fibrosis. After 2 weeks of recovery, skeletal muscle changes remained at the mid and high doses but with a more regenerative response and a single high-dose male had minimal

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and spores and colonies of coccoid bacteria. (Incidences of chronic-active inflammation of the penis/prepuce at necropsy were 12, 8, 6, and 11 out of 15, 8, 7, and 15 animals evaluated, respectively, in the control, and BMS-820132 low-, mid-, and high-dose groups.) There were no anatomic pathology changes related to the administration of BMS-820132 observed at the low and mid doses (10 and 50 mg/kg/day) in ZDF rats, despite equivalent or greater systemic exposure than that in SD rats and dogs. DISCUSSION

FIGURE 1.—Effects of Bristol-Myers Squibb glucokinase activator (BMS-820132) on Sprague-Dawley (SD) and Zucker diabetic fatty (ZDF) rat mean body weight. Each point represents the mean of each treatment group (n  13 for all main study time points [study days 0–28]); (n  5 for all post-dose time points [study days 35 and 42]). Significantly different from time matched controls, *p  0.05.

myocardial fibrosis considered to be a sequel to the degeneration and loss observed at the end of treatment. In dogs, axonal degeneration similar to that described above in the SD rats was observed in the sciatic nerve at all doses except the lowest dose (Figure 5B), and although there was complete resolution of the stomach changes, the sciatic nerve changes did not recover by the end of the 2-week post-dose recovery period. In the ZDF rat study, inflamed prepuces were noted in both control and BMS-820132-treated ZDF rats; this lesion was characterized by exudate overlying hyperplastic and eroded squamous epithelium and containing fungal pseudo-hyphae

The nonclinical safety testing of BMS-820132 included 1-mo toxicity studies in healthy euglycemic rats and dogs. The pharmacologic action of BMS-820132 resulted in clinical signs of hypoglycemia and associated low blood glucose levels. Microscopically, degenerative changes in sciatic nerve, gastric mucosa, myocardium, and skeletal muscle were observed. Since most of these adverse effects occurred at systemic exposures similar to or below those planned for phase 1 clinical trials, it was necessary to determine whether they were sequelae to persistent hypoglycemia and therefore related to exaggerated pharmacology or were non–targetbased toxicities. Peripheral neuropathy characterized by axonal degeneration has been previously described in laboratory animals and humans secondary to repeated or protracted episodes of severe hypoglycemia (Mohseni 2001; Sugimoto et al. 2003; Tabata 2000; Sidenius and Jakobsen 1983). The stomach lesions seen in the BMS-820132-treated rats were considered likely to reflect an increased susceptibility of the gastric mucosa to local vehicle (ethanol component in the rat BMS-820132 dosing formulation) and/or BMS-820132 irritation under conditions of chronic hypoglycemia. Low plasma/blood glucose levels have been previously reported to alter and/or decrease glycoprotein production/synthesis within the mucus cells of the superficial gastric mucosa, thereby increasing susceptibility to gastric damage by drugs or chemical agents (Macdonald et al. 1977; Dekanski et al. 1975; Kinoshita et al. 2000). Similarly in dogs, the stomach lesions (mucus depletion/foveolar atrophy, edema/ hemorrhage/erosion of the superficial glandular mucosa) were also likely associated with the pharmacologically mediated hypoglycemia and an increased susceptibility of the gastric mucosa to local vehicle (ethanol component in the dog BMS820132 dosing formulation). The histopathologic changes in the heart (myocardial cell degeneration, fibrosis, fibroplasia, and inflammation) seen in the BMS-820132-treated rats were considered likely secondary to a protracted hypoglycemiainduced counterregulatory catecholamine response (Zhang et al. 2008; Liang et al. 1982; Khullar et al. 1989). Pettersen et al. (2014) observed similar pathology in the heart (inflammation, necrosis, arteriopathy, and subendocardial hemorrhage) following GKa administration to preclinical species. The reason for the lack of heart lesions in dogs following BMS-820132 administration is not clear, but might be related to an interspecies difference in cardiac myocyte response to

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FIGURE 2.—Effects of Bristol-Myers Squibb glucokinase activator (BMS-820132) on mean serum glucose levels in male Sprague-Dawley (SD) rats and dogs on days 1, 14, and 22/28. Serum glucose levels for male dogs receiving BMS-820132 at 120 mg/kg/day were measured on day 22. Serum glucose levels for all other male dogs were determined on day 28. Each point represents the mean of each treatment group for rats (n  2) and dogs (n  4). Significantly different from time-matched controls, *p  0.05.

hypoglycemia-induced catecholamine release. The lack of skeletal muscle lesions in dogs in the present study may be due to their unique type 1, 2A, and 2X skeletal muscle fibers. Dogs lack classical-type 2B (purely glycolytic) fibers (Snow et al. 1982) and thereby rely more on oxidative (fatty acid-based) rather than glycolytic (glucose-based) metabolism for energy production in skeletal muscle. Dog fast-twitch muscle fibers

(types 2A and 2X) exhibit an unusually high mitochondrial content and oxidative capacity (Acevedo and Rivero 2006). This unique physiology may therefore account for the lack of myotoxicity in dogs that were in a prolonged state of hypoglycemia in the 1-mo toxicity study. In dogs, the increased serum ALP in females given 60 or 120 mg/kg/day and in individual males on day 16 and day 22 given 120 mg/kg/day may

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FIGURE 3.—Serum insulin levels in control male ZDF rats. Each point represents the mean + SD (n ¼ 13). TABLE 3.—Mean serum glucose summary for male ZDF rats. BMS-820132 dose (mg/kg/day) 0

10

50

Day Time after dose (hr) Serum glucose (mg/dL)a 1 0 424 + 63 396 + 102 431 + 71 2 436 + 57 396 + 126 375 + 99 14 0 507 + 43 555 + 47* 553 + 60* 2 487 + 40 552 + 48** 509 + 42 26 0 524 + 54 546 + 48 560 + 45 2 449 + 41 514 + 51 542 + 36*

200

432 + 377 + 496 + 489 + 518 + 496 +

71 107 32 45 44 31

Note: Significantly different from time matched controls. BMS-820132 ¼ BristolMyers Squibb glucokinase activator. a Values represent the mean + SD of each treatment group (n  14). *p  0.05. **p < 0.01.

have resulted from the corticosteroid-induced ALP isoenzyme secondary to stress (Hall 2001). The increased serum triglycerides on day 16 in males given 120 mg/kg/day were possibly part of the acute phase response due to the condition of the dogs (Higgins et al. 2003; Burstein 1992). Based on this literature, we hypothesized that the degenerative lesions observed in these normal euglycemic rats and dogs were not a direct toxic effect of BMS-820132 but were rather a secondary effect caused by extended periods of hypoglycemia. To test this hypothesis, BMS-820132 was administered to hyperglycemic ZDF rats once daily for 1 mo at doses (10, 50, and 200 mg/kg/day) that produced exposures equivalent to and greater than those achieved in the pivotal SD rat and dog toxicology studies (Table 1). Despite having higher exposures

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(up to about 2- and 7-fold) than in healthy male SD rats and beagle dogs, respectively, there were no nerve, skeletal muscle, or cardiac lesions observed in any of the ZDF rats following administration of BMS-820132 for 1 mo. Other pathological changes recorded in the ZDF rat study and consistent with those seen in the 1-mo study in healthy euglycemic SD rats were only seen at the 200 mg/kg/day dose. These changes included increased adrenal gland weights at necropsy with cortical hypertrophy and/or increased cytoplasmic acidophilia (consistent with stress [Greaves 2007]), and a single rat with a minimal multifocal superficial glandular necrosis/degeneration with foveolar epithelial hyperplasia/regeneration in the stomach. This lesion, in a single rat, was minimal in severity and had a much reduced incidence and severity than the same lesion seen at all doses in SD rats in the pivotal toxicology study. Based on the stomach lesion in 1 of 15 rats and increased adrenal gland weight with cortical hypertrophy and/or increased cytoplasmic acidophilia in the high-dose rats (data not shown), the no-observed-adverse-effect-level (NOAEL) in the ZDF rat study was determined to be 50 mg/kg/day (exposure multiples approximately 13 the anticipated exposure at the 300 mg stopping dose in the phase 1 clinical trial). Balanoposthitis (swollen glans penis and preputial tissue) was observed in both control and BMS-820132-treated rats beginning on day 19. However, since there was no dose response noted in the number of observations or in the number of rats affected, balanoposthitis was not considered BMS-820132 related. ZDF rats are known to be susceptible to preputial and penile infections, presumably due the higher levels of urinary glucose found in this strain of rat (Charles River Laboratories Inc. personal communication). The decline in body weights in all ZDF groups (including controls) starting after 2 or 3 weeks of dosing was likely associated with the diabetic phenotype of this strain. An alternative explanation for our data is that male ZDF rats are inherently resistant to the pathologic effects of BMS820132 independent of their glycemic state. This alternative hypothesis is unlikely because the spectrum of lesions we observed in both SD rats and beagle dogs correlated well to BMS-821032-induced insulin secretion and subsequent hypoglycemia (observed in single-dose rat and dog BMS-820132 toxicology studies [see supplemental data]), and has been reported in a variety of species with induced hypoglycemia including mice and monkeys in addition to rats and dogs (Pettersen et al. 2014). Furthermore, the alternate hypothesis is practically untestable because ZDF rats are resistant to insulin-induced hypoglycemia (Metais et al. 2008) and because, based on a toxicokinetic study, we achieved maximal feasible exposure of BMS-820132 in male ZDF rats at the high dose of 200 mg/kg/day without inducing hypoglycemia. Male ZDF rats are a widely used genetic model of obesityassociated type 2 diabetes. Published literature indicates that young ZDF rats exhibit normal serum glucose levels but become progressively more hyperglycemic with age (Torres et al. 2009). Serum glucose levels in ZDF rats are known to increase rapidly between weeks 6 and 12 of age, with serum

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FIGURE 4.—Bristol-Myers Squibb glucokinase activator (BMS-820132) causes lesions in the stomach, sciatic nerve, and heart in Sprague-Dawley (SD) rats (A–C) but not in ZDF rats (D–F). (A) Stomach, glandular mucosa, SD rat treated with BMS-820132 at 300 mg/kg/day for 1 mo. A discrete portion of the glandular mucosa is necrotic and superficially contains an area of hemorrhage (arrows). The adjacent mucosa lacks a mucus layer and is partially atrophic. Contrast to the normal stomach of a ZDF rat treated with BMS-820132 at 200 mg/kg/day for 1 mo (D). Bar * 150 m. (B) Sciatic nerve, SD rat treated with BMS-820132 at 300 mg/kg/day for 1 mo. Nerve fiber atrophy and degeneration is characterized by swollen sheaths containing axonal remnants (arrow). Interspersed are vacuolated and hypertrophied Schwann cells. Contrast to the normal sciatic nerve of a ZDF rat treated with BMS-820132 at 200 mg/kg/day for 1 mo (E). Bar * 50 m. (C) Heart, left ventricular papillary muscle, SD rat treated with BMS-820132 at 80 mg/kg/day for 1 mo. Focal loss of cardiac myofibers is accompanied by reactive mononuclear cell inflammation and mild fibrosis. Contrast to the normal left ventricular papillary muscle of a ZDF rat treated with BMS-820132 at 200 mg/kg/day for 1 month (F). Bar * 200 m.

insulin levels peaking at 8 weeks of age and then rapidly declining thereafter (Mulder 2010). In the present study, male ZDF rats had high non-fasted serum glucose levels on day 1 (mean control glucose ¼ 424 mg/dL) that increased as the

study progressed (mean control glucose ¼ 524 mg/dL on day 26). There was no evidence of hypoglycemia in any of the control or BMS-820132-treated ZDF rats based on both clinical pathology data and clinical observations, with non-fasted

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TABLE 4.—Histopathologic summary for SD rats, ZDF rats, and dogs. SD rat BMS-820132 dose (mg/kg/day) Number of M/F at end of dose necropsy Sex Finding at the end of 1-mo dosing period Stomach (glandular) Degeneration/necrosis: superficial mucosaa Edema, lamina propria, superficial Hemorrhage: superficial glandular mucosa (rat); lamina propria, superficial (dog) Hyperplasia/regeneration: foveolar epitheliuma Nerve (sciatic) Axonal degeneration Skeletal muscle (quadriceps) Degeneration/necrosis: skeletal muscle cell Regeneration: skeletal muscle cell Heart Myocardial cell degeneration Myocardial fibrosis/fibroplasia Number of M/F at end of dose necropsy: Finding at the end of 2-week recovery period Stomach (Glandular) Degeneration/necrosis: superficial mucosaa Edema, lamina propria, superficial Hemorrhage: superficial glandular mucosa (rat); lamina propria, superficial (dog) Hyperplasia/regeneration: foveolar epitheliuma Nerve (Sciatic) Axonal degeneration Regeneration: Schwann cell Skeletal muscle (quadriceps) Degeneration/necrosis: skeletal muscle cell Regeneration: skeletal muscle cell Heart Myocardial cell degeneration Myocardial fibrosis/fibroplasia

Dog

0 10/10 M/F

20 10/10 M/F

80 10/10 M/F

300 10/10 M/F

0 5/5 M/F

10 5/5 M/F

60 5/5 M/F

120 5/5 M/F

— — 3/4 10/9

4/0b — 8/4 10/7

2/2 — 8/5 10/10

8/7 — 7/10 10/9

— — — —

— 1/0 0/1 —

— 2/0 1/1 —

— 2/3 2/3 —

—

2/2

9/10

10/9

—

—

1/0

1/2

1/0 —

1/0 —

2/0 1/0

6/8 0/1

— —

— —

— —

— —

— — 5/5

— — 5/5

2/0 — 5/5

0/2 2/1 5/5

— — 2/2

— — 2/2

— — 2/2

— — 2/2

— — 3/2 3/3

— — 2/0 4/2

— — 1/2 4/4

— — 2/2 2/2

— — — —

— — 0/1 —

— — — —

— — — —

0/1

0/1

3/5 1/0

5/5 3/2

—

—

1/0

0/1

— —

1/1 —

0/2 0/2

2/5 1/5

—

—

—

—

— —

— —

— —

— 1/0

— —

— —

— —

— —

Note: — ¼ absent; BMS-820132 ¼ Bristol-Myers Squibb glucokinase activator; M/F ¼ male/female; SD ¼ Sprague-Dawley; ZDF ¼ Zucker diabetic fatty. a One of 15 ZDF hyperglycemic rats at 200 mg/kg/day had this change. b Incidence per number of males/number of females during the period.

values remaining above 100 mg/dL at all time points evaluated at the low and mid doses of 10 and 50 mg/kg/day and above 71 mg/dL at the high dose of 200 mg/kg/day. Absence of relevant pathological changes in BMS-820132treated ZDF rats supports the hypothesis that the heart, skeletal muscle, and peripheral nerve lesions seen in the initial 1-mo studies in SD rats, and by inference in dogs, are secondary to hypoglycemia and that stomach lesions are exacerbated by hypoglycemia. Based on the results from these nonclinical toxicity studies, the most likely clinical safety concern associated with BMS-820132 is hypoglycemia. ZDF rats, with increasingly high serum glucose levels starting at 8 weeks of age, offer a useful model to assess the potential risks of the administration of GKa in diabetic patients. Based on the ZDF rat model and conventional toxicology safety assessment testing in normal euglycemic animals at lower doses, administering BMS-820132 to hyperglycemic human patients in clinical trials is very likely to be safe, particularly since blood glucose levels are easily monitored and hypoglycemia is treatable.

This study is also an example of how an animal disease model can be effectively utilized to aid in the risk assessment of novel therapeutics in human disease populations. Our results provide an example of decreased toxicity in a disease model compared to healthy animals, but examples of increased susceptibility to the toxicity of therapeutics in disease models compared to healthy animals also exist. Drugs developed for the treatment of lysosomal storage disease, for example, have been demonstrated to be more toxic in rodents with defects in lysosomal function compared to normal healthy animals (Murray et al. 2015; Morgan et al. 2013). This difference is thought to relate to the rapid breakdown of large amounts of substrate that are released following restoration of lysosomal function. While disease models are not routinely used in the safety assessment of drugs in development, this approach can provide additional information that can augment the results from toxicity studies with healthy animals. Morgan et al. (2013) and Boelsterli (2003) provide additional discussion concerning the value of disease models in toxicity testing.

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FIGURE 5.—Bristol-Myers Squibb glucokinase activator (BMS-820132) causes lesions in the stomach and sciatic nerve of dogs (A and B) as compared to vehicle control dogs (C and D). (A) Stomach, fundic mucosa, dog treated with BMS-820132 at 120 mg/kg/day for 3 weeks. Mucus depletion and foveolar atrophy are accompanied by edema of the lamina propria. Contrast to the normal stomach of a control dog (C). Bar * 200 m. (B) Sciatic nerve, dog treated with BMS-820132 at 120 mg/kg/day for 3 weeks. Axonal degeneration is characterized by multifocal dilated axon sheaths containing swollen axons or macrophages (arrow). Contrast to the normal sciatic nerve of a control dog (D). Bar * 50 m.

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