Richard A. Byrd

Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits

Pregnant Fischer 344 rats were given fluoxetine orally at dose levels of 0, 2, 5, or 12.5 mg/kg on Gestation Days (GD) 6-15; pregnant Dutch Belted rabbits were given 0, 2.5, 7.5, or 15 mg/kg orally on GD 6-18. Cesarean sections were performed on rats and rabbits on GD 20 and 28, respectively. In rats, maternal toxicity was indicated at 12.5 mg/kg by depression of weight gain and food consumption. Fetal viability, weight, and morphology were not affected at any dose level. Maternal and developmental No Observed Adverse Effect Levels (NOAELs) in the rat were 5 and 12.5 mg/kg, respectively. In rabbits, weight loss occurred at 2.5, 7.5, and 15 mg/kg. Food consumption was also depressed at 7.5 and 15 mg/kg; abortions and maternal mortality occurred secondarily to anorexia and cachexia at 15 mg/kg. Fetal viability, weight, and morphology were not affected at any dose level. A NOAEL for maternal effects was not established in the rabbit; the NOAEL for developmental effects in the rabbit was 15 mg/kg. Based on these data, fluoxetine did not exhibit any toxicity toward the developing rat or rabbit conceptus at doses that were maternally toxic.

The selective estrogen receptor modulator, raloxifene: segment II studies in rats and rabbits.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company primarily as a therapeutic agent for postmenopausal osteoporosis. Two Segment II studies were conducted that examined maternal reproductive parameters and fetal outcome following gestational exposure to raloxifene. Pregnant CD rats (25/group) and New Zealand white rabbits (20/group) were dosed once daily by oral gavage with 0, 0.1, 1, or 10 mg/kg on Gestation Days (GD) 6 through 17 and 7 through 19, respectively. Maternal body weight and food consumption were monitored throughout pregnancy. Caesarean sections were performed on GD 20 and GD 28 for rats and rabbits, respectively, to evaluate fetal viability, weight, and morphology. In rats, maternal body weight, body weight gain, and food consumption were reduced in all raloxifene treatment groups. Fetal viability was depressed in the 10-mg/kg group and was often associated with signs of hemorrhaging from the vagina. Fetal growth retardation was indicated in the 1- and/or 10-mg/kg groups by increased incidences of fetal runts and the developmental deviations, wavy ribs and kidney cavitation. There was no evidence of treatment-related malformations in rat fetuses. In rabbits, depressions in body weight gain and food consumption occurred in the 10-mg/kg group, and a single abortion occurred in the 1-mg/kg group. Fetal viability and weights were not affected in any of the raloxifene treatment groups. The overall proportions of fetuses with malformations, deviations, or variations were not affected by treatment with raloxifene; however, one fetus each from the 0.1-, 1-, and 10-mg/kg groups had incomplete closure of the interventricular septum. Therefore, maternal and fetal no-effect levels were not obtained in this study of raloxifene.

The Selective Estrogen Receptor Modulator, Raloxifene: Reproductive Assessments in Adult Male Rats

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed for postmenopausal osteoporosis. Reproductive toxicity of raloxifene was examined in adult male CD rats after the oral administration of doses of 0, 10, 30, or 100 mg/kg/d. In the first study, males (12/group) were treated for 2 weeks followed by 2 weeks without treatment. After dose administration on Day 13, 6 males/group were cohabited with untreated females (1:2) for up to 7 d. Males were killed on Day 14 or 28 (6/group each day). Sperm were collected from the right cauda epididymis and evaluated for relative concentration, motion characteristics, and breakage. The kinetics of spermatogenesis were examined by DNA flow cytometry. The left testis and epididymis were preserved for histopathologic evaluation. Females were examined for reproductive status on Gestation Day 13. In a second study, males (20/group) were treated for 7 weeks (4 weeks prior to cohabitation during a 2-week cohabitation period, and for 1 additional week). Treated males were cohabited with untreated females (1:1). On Gestation Day 20, untreated females were examined for reproductive status and fetuses were examined for viability, weight, gender, and morphology. At necropsy, male reproductive tissues were collected, weighed, and preserved for histopathologic evaluation. In both studies, male body weight gain and food consumption were depressed at all dose levels. There was no indication in either study that raloxifene caused important changes in sperm production, sperm quality, or male reproductive performance at doses as high as 100 mg/kg/d.

Chronic Toxicity and Carcinogenicity Studies of the Long-Acting GLP-1 Receptor Agonist Dulaglutide in Rodents

The tumorigenic potential of dulaglutide was evaluated in rats and transgenic mice. Rats were injected sc twice weekly for 93 weeks with dulaglutide 0, 0.05, 0.5, 1.5, or 5 mg/kg corresponding to 0, 0.5, 7, 20, and 58 times, respectively, the maximum recommended human dose based on plasma area under the curve. Transgenic mice were dosed sc twice weekly with dulaglutide 0, 0.3, 1, or 3 mg/kg for 26 weeks. Dulaglutide effects were limited to the thyroid C-cells. In rats, diffuse C-cell hyperplasia and adenomas were statistically increased at 0.5 mg/kg or greater (P ≤ .01 at 5 mg/kg), and C-cell carcinomas were numerically increased at 5 mg/kg. Focal C-cell hyperplasia was higher compared with controls in females given 0.5, 1.5, and 5 mg/kg. In transgenic mice, no dulaglutide-related C-cell hyperplasia or neoplasia was observed at any dose; however, minimal cytoplasmic hypertrophy of C cells was observed in all dulaglutide groups. Systemic exposures decreased over time in mice, possibly due to an antidrug antibody response. In a 52-week study designed to quantitate C-cell mass and plasma calcitonin responses, rats received twice-weekly sc injections of dulaglutide 0 or 5 mg/kg. Dulaglutide increased focal C-cell hyperplasia; however, quantitative increases in C-cell mass did not occur. Consistent with the lack of morphometric changes in C-cell mass, dulaglutide did not affect the incidence of diffuse C-cell hyperplasia or basal or calcium-stimulated plasma calcitonin, suggesting that diffuse increases in C-cell mass did not occur during the initial 52 weeks of the rat carcinogenicity study.

Effects of Dulaglutide on Thyroid C Cells and Serum Calcitonin in Male Monkeys

Glucagon-like peptide-1 (GLP-1) receptor agonists, used for the treatment of type 2 diabetes, have caused hyperplasia/neoplasia of thyroid C cells in rodent carcinogenicity studies. Studies in monkeys have not identified an effect of GLP-1 receptor agonists on thyroid C cells; however, group sizes were small. Dulaglutide is a once-weekly, long-acting human GLP-1 receptor agonist recently approved in the United States and the European Union. The objective of this study was to determine whether dulaglutide altered C-cell mass in monkeys. Male cynomolgus monkeys (20 per group) were sc injected with dulaglutide 8.15 mg/kg (∼500-fold maximum human plasma exposure) or a vehicle control twice weekly for 52 weeks. Basal and calcium gluconate-stimulated serum calcitonin concentrations were obtained at 3, 6, 9, and 12 months. Thyroid glands were weighed, fixed, and sectioned at 500-μm intervals. C-cell volumes were measured using an automated image analysis. C-cell proliferation was estimated using Ki67/calcitonin colabeling and cell counting. Administration of dulaglutide 8.15 mg/kg twice weekly for 52 weeks did not increase serum calcitonin in monkeys or affect thyroid weight, histology, C-cell proliferation, or absolute/relative C-cell volume. This study represents a comprehensive evaluation of the monkey thyroid C cells after dosing with a GLP-1 receptor agonist, with a large group size, and measurement of multiple relevant parameters. The lack of effect of dulaglutide on C cells is consistent with other studies in monkeys using GLP-1 receptor agonists and suggests that nonhuman primates are less sensitive than rodents to the induction of proliferative changes in thyroid C cells by GLP-1 receptor agonists.

Developmental Toxicology Studies of Vancomycin Hydrochloride Administered Intravenously to Rats and Rabbits

Pregnant CD rats were given vancomycin intravenously in doses of 0, 40, 120, or 200 mg/kg on Gestation Days (GD) 6-15; pregnant New Zealand white rabbits were given 0, 40, 80, or 120 mg/kg intravenously on GD 6-18. Cesarean sections were performed on rats and rabbits on GD 20 and 28, respectively. In rats, maternal toxicity was indicated in the 120- and 200-mg/kg treatment groups by cortical tubular nephrosis. Maternal body weight gain and food consumption and fetal viability, weight, and morphology were not adversely affected by vancomycin. Maternal and developmental no observed adverse effect levels (NOAELs) in the rat were 40 and 200 mg/kg, respectively. In rabbits, maternal toxicity was indicated by cortical tubular nephrosis in the 80- and 120-mg/kg treatment groups; a single death and depression of body weight gain and food consumption occurred in the 120-mg/kg treatment group. Developmental toxicity was indicated by depression of fetal weight in the 120-mg/kg treatment group; fetal viability and morphology were not adversely affected by vancomycin. Maternal and developmental NOAELs in the rabbit were 40 and 80 mg/kg, respectively. Based on these data, vancomycin did not exhibit selective toxicity toward the developing rat or rabbit conceptus.

An Investigative Study of Pancreatic Exocrine Biomarkers, Histology, and Histomorphometry in Male Zucker Diabetic Fatty (ZDF) Rats Given Dulaglutide by Subcutaneous Injection Twice Weekly for 13 Weeks

Glucagon-like peptide-1 (GLP-1) receptor agonist therapy has been implicated as a possible risk factor for acute pancreatitis in patients with type 2 diabetes. Dulaglutide is a long-acting GLP-1 receptor agonist in development for treatment of type 2 diabetes. The effects of dulaglutide were evaluated in male Zucker diabetic fatty (ZDF) rats to examine whether dulaglutide may induce or modulate pancreatitis. Rats were randomized to dose groups receiving twice-weekly subcutaneously administered dulaglutide 0.5, 1.5, and 5.0 mg/kg/dose (corresponding human plasma exposures following twice-weekly dosing are 3-, 8-, and 30-fold, respectively) for 13 weeks or to vehicle control. Following termination, serially trimmed sections of pancreases were stained with hematoxylin and eosin or co-stained with an epithelial marker and a marker of either proliferation or apoptosis. Efficacious reductions in glucose and hemoglobin A1c occurred at all dulaglutide doses. Lipase activity was unaffected, and there were modest increases in total and pancreatic amylase activities at all doses without individual microscopic inflammatory correlates. Microscopic dulaglutide-related pancreatic changes included increased interlobular ductal epithelium without ductal cell proliferation (≥0.5 mg/kg), increased acinar atrophy with/without inflammation (≥1.5 mg/kg), and increased incidence/severity of neutrophilic acinar pancreatic inflammation (5.0 mg/kg). In summary, dulaglutide treatment was associated with mild alterations in ductal epithelium and modest exacerbation of spontaneous lesions of the exocrine pancreas typically found in the ZDF rat model.

Chronic Toxicology Studies of Basal Insulin Peglispro in Rats and Dogs: A Novel, PEGylated Insulin Lispro Analog with a Prolonged Duration of Action:

Basal insulin peglispro (BIL) consists of insulin lispro with a 20-kDa polyethylene glycol (PEG) moiety covalently attached to lysine B28. Because chronic parenteral administration of PEGylated proteins to animals has sometimes resulted in PEG vacuolation of tissue macrophages, renal tubular cells, and choroid plexus ependymal cells, we investigated whether chronic subcutaneous (sc) injection of BIL in rats (52 weeks) and dogs (39 weeks) was associated with systemic toxicities or other changes, including vacuolation of tissue macrophages, renal tubular cells, and ependymal cells. Rats and dogs received daily sc injections of BIL (rats: 0.17, 0.45, or 1.15 mg/kg/d and dogs: 0.025, 0.10, or 0.20 mg/kg/d) and the reference compound, HUMULIN N® (neutral protamine Hagedorn [NPH] human insulin; rats: 0.15 mg/kg/d and dogs: 0.02–0.03 mg/kg/d). Animals were evaluated for standard end points including mortality, clinical signs, body weights, toxicokinetics, glucodynamics, clinical pathology, and morphological pathology. Nonadverse injection site lipohypertrophy occurred for all BIL and NPH doses but more frequently with BIL. No BIL-related hyperplasia or neoplasia was observed. There was no vacuolation of tissue macrophages, renal tubular cells, or ependymal cells attributable to PEG. These studies demonstrate BIL is not associated with tissue vacuolation attributable to PEG at 4- to 6-fold multiple of the median clinical exposure in patients with diabetes.

Nonclinical pharmacology and toxicology of the first biosimilar insulin glargine drug product (BASAGLAR®/ABASAGLAR®) approved in the European Union

ABSTRACT Basaglar®/Abasaglar® (Lilly insulin glargine [LY IGlar]) is a long‐acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus® (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union–sourced SA IGlar (EU‐SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin‐like growth factor‐1 (IGF‐1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU‐SA IGlar was similar when tested in both insulin‐ and IGF‐1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU‐SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU‐SA IGlar in satellite animals showed comparable dose‐related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU‐SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU‐SA IGlar. HIGHLIGHTSBasaglar®/Abasaglar® (LY IGlar) is the first biosimilar insulin glargine drug product approved in the European Union.We compared nonclinical profiles of LY IGlar and European Union–sourced Lantus (EU‐SA IGlar).We found no biologically relevant differences between LY IGlar and EU‐SA IGlar.

Effects of the GLP-1 Receptor Agonist Dulaglutide on the Structure of the Exocrine Pancreas of Cynomolgus Monkeys.

Clinical and nonclinical studies have implicated glucagon-like peptide-1 (GLP-1) receptor agonist therapy as a risk factor for acute pancreatitis in patients with type 2 diabetes. Therefore, it is critical to understand the effect that dulaglutide, an approved GLP-1 receptor agonist, has on the exocrine pancreas. Dulaglutide 8.15 mg/kg (approximately 500 times the maximum recommended human dose based on plasma exposure) was administered twice weekly for 12 months to cynomolgus monkeys. Serum amylase and lipase activities were measured and 6 sections of each pancreas were examined microscopically. Ductal epithelial cell proliferation was estimated using Ki67 labeling. Dulaglutide administration did not alter serum amylase or lipase activities measured at the end of treatment compared to control values. An extensive histologic evaluation of the pancreas revealed no changes in the acinar or endocrine portions and no evidence of pancreatitis, necrosis, or pancreatic intraepithelial neoplasia. An increase in goblet cells noted in 4 of the 19 treated monkeys was considered an effect of dulaglutide but was not associated with dilation, blockage, or accumulation of mucin in the pancreatic duct. There was no difference in cell proliferation in ductal epithelium between control and dulaglutide-treated monkeys. These data reveal that chronic dosing of nondiabetic primates with dulaglutide does not induce inflammatory or preneoplastic changes in exocrine pancreas.