Pegbelfermin, a PEGylated FGF21 analogue, has pharmacology without bone toxicity after 1-year dosing in skeletally-mature monkeys.

Pegbelfermin (PGBF) is a PEGylated fibroblast growth factor 21 (FGF21) analogue in development for treatment of nonalcoholic steatohepatitis (NASH). Mouse models highlight potential utility of FGF21 in NASH, but also suggest negative effects on bone, though these findings are confounded by profound FGF21-related decreases in body mass/growth. This study aimed to profile PGBF-related bone effects in adult nonhuman primates after long-term, clinically-relevant exposures. Adult male cynomolgus monkeys received weekly subcutaneous PGBF (0.3, 0.75 mg/kg) or control injections for 1 year (n = 5/group). Assessments included body weight, clinical chemistry, adiponectin levels, bone turnover biomarkers, skeletal radiography, pharmacokinetics, immunogenicity, and histopathology. Bone densitometry and body composition were evaluated in vivo and/or ex vivo with dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, and biomechanical strength testing. After 1 year of PGBF administration, there was clear evidence of sustained PGBF pharmacology in monkeys (peak increase in serum adiponectin of 1.7× and 2.35× pretest at 0.3 and 0.75 mg/kg PGBF, respectively) and decreased body weight compared with control at exposures comparable to those tested in humans. At 0.75 mg/kg PGBF, pharmacologically-mediated reductions in lean mass, lean area, and fat area were observed relative to controls. There were no PGBF-related effects on bone biomarkers, radiography, densitometry, or strength. Together, these data demonstrate that PGBF did not adversely alter bone metabolism, density, or strength following 1 year of dosing at clinically relevant (0.7-2.2× human AUC[0-168 h] at 20 mg once weekly), pharmacologically-active exposures in adult monkeys, suggesting a low potential for negative effects on bone quality in adult humans.

Thymic Lymphomas in a 6-Month rasH2-Tg Mouse Carcinogenicity Study With the RORγt Inverse Agonist, BMS-986251.

BMS-986251 is a retinoid-related orphan receptor γt (RORγt) inverse agonist that was in development for the treatment of autoimmune diseases. RORγt is a nuclear hormone receptor and transcription factor that is involved in the differentiation and function of T helper 17 cells. RORγt-deficient (constitutive or conditional) mice develop thymic lymphomas with >50% mortality at 4 months, whereas heterozygous mice are normal. A 6-month study was conducted in rasH2-Tg hemizygous mice to assess the potential carcinogenicity of BMS-986251. BMS-986251 was administered once daily by oral gavage to groups of 27 mice/sex at doses of 0 (water control), 0 (vehicle control), 5, 25, or 75 mg/kg. The positive control, N-methyl-N-nitrosourea, was administered by a single intraperitoneal injection to 15 mice/sex at a dose of 75 mg/kg. There were no tumors attributed to BMS-986251 except for thymic lymphomas. Thymic lymphoma was observed in 1 male (3.7%) and 3 females (11.1%) at the mid dose, and 6 females (22.2%) at the high dose. No lymphomas were observed in the negative control groups whereas the incidence of lymphomas in the positive control group was 47-60%. The incidence of thymic lymphomas in the BMS-986251-treated groups was higher than published literature and test facility historical control data. Furthermore, increased thymic lymphoid cellularity (lymphoid hyperplasia) was observed at the mid dose in males and at all doses in females. Since lymphoid hyperplasia may represent a preneoplastic change, a no-effect dose for potential tumor induction was not identified in this study. These results led to the discontinuation of BMS-986251 and underscore the challenges in targeting RORγt for drug development.

Intermittent dosing of the transforming growth factor beta receptor 1 inhibitor, BMS-986260, mitigates class-based cardiovascular toxicity in dogs but not rats.

Small-molecule inhibitors of transforming growth factor beta receptor 1 (TGFßRI) have a history of significant class-based toxicities (eg, cardiac valvulopathy) in preclinical species that have limited their development as new medicines. Nevertheless, some TGFßRI inhibitors have entered into clinical trials using intermittent-dosing schedules and exposure limits in an attempt to avoid these toxicities. This report describes the toxicity profile of the small-molecule TGFßRI inhibitor, BMS-986260, in rats and dogs. Daily oral dosing for 10 days resulted in valvulopathy and/or aortic pathology at systemic exposures that would have been targeted clinically, preventing further development with this dosing schedule. These toxicities were not observed in either species in 1-month studies using the same doses on an intermittent-dosing schedule of 3 days on and 4 days off (QDx3 once weekly). Subsequently, 3-month studies were conducted (QDx3 once weekly), and while there were no cardiovascular findings in dogs, valvulopathy and mortality occurred early in rats. The only difference compared to the 1-month study was that the rats in the 3-month study were 2 weeks younger at the start of dosing. Therefore, a follow-up 1-month study was conducted to evaluate whether the age of rats influences sensitivity to target-mediated toxicity. Using the same dosing schedule and similar doses as in the 3-month study, there was no difference in the toxicity of BMS-986260 in young (8 weeks) or adult (8 months) rats. In summary, an intermittent-dosing schedule mitigated target-based cardiovascular toxicity in dogs but did not prevent valvulopathy in rats, and thus the development of BMS-986260 was terminated.

Adverse vacuolation in multiple tissues in cynomolgus monkeys following repeat-dose administration of a PEGylated protein.

PEGylation is considered a safe mechanism to enhance the pharmacokinetics (PK) and pharmacodynamics (PD) of biotherapeutics. Previous studies using PEGylation as a PK enhancement tool have reported benign PEG-related vacuolation in multiple tissues. This paper establishes a threshold for PEG burden beyond which there are alterations in tissue architecture that could potentially lead to dysfunction. As part of the nonclinical safety assessment of Compound A, a 12 kDa protein conjugated to a 40 kDa branched PEG molecule, monkeys were dosed subcutaneously twice weekly for 3 months at protein doses resulting in weekly PEG doses of 8, 24, 120, or 160 mg/kg. Consistent with previous reports with PEGylated biomolecules, Compound A administration resulted in intracellular vacuoles attributed to the PEG moiety in macrophages in numerous tissues and epithelial cells in the choroid plexus and kidney. Vacuolation occurred at all doses with dose-dependent severity and no evidence of recovery up to 2 months after dosing cessation. The vacuolation was considered nonadverse at PEG doses ≤120 mg/kg/week. However, at 160 mg/kg/week PEG, the vacuolation in choroid plexus, pituitary gland, kidney, and choroid of the eye was considered adverse due to significant alterations of tissue architecture that raised concern for the possibility of compromised tissue function. To our knowledge, this is the first report of potentially adverse cellular consequences of PEG accumulation in tissues other than kidney. Furthermore, the lack of reversibility of vacuolation coupled with the lack of a biomarker for intracellular PEG accumulation highlights a potential risk that should be weighed against the benefits of PK/PD enhancement for long-term administration of PEGylated compounds at high doses.

Allogeneic murine pregnancy models for assessing the developmental effects of immune-stimulating antibodies: Challenges in reproducibility.

Literature suggests that murine allogeneic pregnancy models are an alternative approach for evaluating the developmental toxicity of immune-stimulating agents. In this study, multiple syngeneic and allogeneic murine pregnancy models were used to assess the potential embryo-fetal effects of four different murine antibodies (IgG1 or IgG2 ) that activate the immune system by binding to T-cell receptors (PD-L1, LAG-3, and GITR). The pregnancy models were generated by within and between matings of five different inbred strains of mice (CBA/CaJ, DBA/2J, BALB/c, C57BL/6, and CBA/J). The antibodies were administered every 2-3 days by intraperitoneal injection (n = 12-29/group) during gestation days 6 to 14. There were no differences in embryo-fetal endpoints between the allogeneic and syngeneic pregnancies. Additionally, treatment with the antibodies had no effect on mean postimplantation loss in either the syngeneic or allogeneic pregnancies despite confirmation of pharmacologically-relevant systemic exposures. These results suggest that allogeneic murine pregnancy models need further validation and testing before they can be reliably used as an alternative approach for assessing the developmental effects of agents that stimulate the immune system.

Investigations on the Relationship Between Ovarian, Endocrine, and Renal Findings in Nonclinical Safety Studies of the γ-Secretase Inhibitor Avagacestat.

Avagacestat, a gamma (γ)-secretase inhibitor that was in development for treatment of Alzheimer's disease, produced ovarian granulosa-thecal cell tumors in rats and dogs and a glomerulopathy with profound proteinuria in female rats. This report describes the results of follow-up investigative studies, including the use of ovariectomized (OVX) rats, to further characterize these findings and determine their mechanism(s). Ovarian proliferative changes in rats likely resulted from: (1) inhibition of Notch signaling pathways regulating ovarian follicular differentiation/development, characterized microscopically as altered ovarian cyclicity and/or ovarian follicular degeneration; (2) subsequent disruption of the hypothalamic-pituitary-ovarian axis due to ovarian atrophy with decreases in serum estrogen and progesterone (as low as 0.45× and 0.21× controls, respectively); and (3) chronic gonadotropin stimulation and pituitary hypertrophy/hyperplasia in response to the absence of negative feedback. Gonadotropin stimulation in rats was confirmed by increases in serum follicle-stimulating hormone (up to 7.75× controls) and luteinizing hormone (up to 5.84×). A similar nongenotoxic mechanism was likely responsible for the ovarian findings in dogs although changes in serum hormone levels were not detected. The dose- and time-dependent glomerulopathy with progression to chronic progressive nephropathy in female rats appears to be a direct effect of avagacestat and was not ameliorated with coadministration of 17ß-estradiol or an antihypertensive (enalapril) and was not present in control OVX rats. In contrast, adrenocortical hypertrophy in female rats was considered secondary to ovarian changes based on the absence of this finding in avagacestat-treated OVX rats and no increase in adrenocorticotropic hormone staining in the pituitary.

Toxicity of Pexacerfont, a Corticotropin-Releasing Factor Type 1 Receptor Antagonist, in Rats and Dogs.

Pexacerfont is a corticotropin-releasing factor subtype 1 receptor antagonist that was developed for the treatment of anxiety- and stress-related disorders. This report describes the results of repeat-dose oral toxicity studies in rats (3 and 6 months) and dogs (3 months and 1 year). Pexacerfont was well tolerated in all of these studies at exposures equal to or greater than areas under the curve in humans (clinical dose of 100 mg). Microscopic changes in the liver (hepatocellular hypertrophy), thyroid glands (hypertrophy/hyperplasia and adenomas of follicular cells), and pituitary (hypertrophy/hyperplasia and vacuolation of thyrotrophs) were only observed in rats and were considered adaptive changes in response to hepatic enzyme induction and subsequent alterations in serum thyroid hormone levels. Evidence for hepatic enzyme induction in dogs was limited to increased liver weights and reduced thyroxine (T4) levels. Mammary gland hyperplasia and altered female estrous cycling were only observed in rats, whereas adverse testicular effects (consistent with minimal to moderate degeneration of the germinal epithelium) were only noted following chronic dosing in dogs. The testicular effects were reversible changes with exposure margins of 8× at the no observed adverse effect level. It is not clear whether the changes in mammary gland, estrous cycling, and testes represent secondary hormonal changes due to perturbation of the hypothalamic-pituitary-adrenal axis or are off-target effects. In conclusion, the results of chronic toxicity studies in rats and dogs show that pexacerfont has an acceptable safety profile to support further clinical testing.

Nonclinical Safety Assessment of the γ-Secretase Inhibitor Avagacestat.

The toxicity of avagacestat, a sulfonamide-based gamma (γ)-secretase inhibitor that was in development as a treatment for Alzheimer's disease, was evaluated in a comprehensive nonclinical toxicology program that included 6-month and 1-year repeat-dose toxicity studies in rats and dogs, respectively. There was a spectrum of mechanism-based changes attributed to inhibition of Notch signaling that regulates the differentiation and proliferation of cells throughout development and in adult tissues. In both rats and dogs, ovarian follicular degeneration and atrophy and a low incidence of granulosa cell hyperplasia and benign granulosa-thecal cell tumors were observed. Gastrointestinal (GI) findings, including goblet cell metaplasia, dilatation of intestinal crypts/glands, mucosal epithelial necrosis and regeneration, and villous atrophy, were limited to dogs that had clinical evidence of GI toxicity. Other avagacestat-related findings attributed to interference with Notch signaling included decreases in peripheral lymphocytes (T and/or B cells) and lymphoid depletion in lymph nodes and the spleen in both species, as well as epiphyseal cartilage and trabecular bone changes in rats. Pharmacologically mediated decreases in brain and cerebrospinal fluid levels of ß-amyloid (Aß) peptides Aß40 and Aß42 and decreased expression of white blood cell mRNA levels of the Notch-regulated gene hairy and enhancer of split-1 confirmed target engagement at all doses. Reductions in brain Aß peptide levels (22 to 34%) in dogs after 1 year at exposures up to the no-observed-effect level for GI toxicity of 1.1× the human plasma exposure, and reversible GI changes at a 3.2× multiple, indicated that a sustained pharmacodynamic effect was attained at exposures without dose-limiting toxicity.

In Vitro Metabolite Formation in Human Hepatocytes and Cardiomyocytes and Metabolism and Tissue Distribution in Monkeys of the 2'-C-Methylguanosine Prodrug BMS-986094 : Potential Role in Clinical Cardiovascular Toxicity.

BMS-986094, a 2'-C-methylguanosine prodrug for the treatment of chronic hepatitis C virus infection, was withdrawn from phase 2 clinical trials because of unexpected cardiac and renal toxicities. To better understand these toxicities, the in vitro metabolism of BMS-986094 in human hepatocytes (HHs) and human cardiomyocytes (HCMs) and the measurement of BMS-986094 and selected metabolites in monkey plasma and tissues were assessed. BMS-986094 was extensively metabolized by HHs and HCMs, resulting in more efficient formation and accumulation of the active triphosphorylated metabolite, INX-09114, and less efficient efflux of metabolites in HCMs. The predominant metabolism pathway (hydrolysis) in HHs and HCMs was not associated with the formation of reactive metabolites or oxidative stress. In cynomolgus monkeys dosed with BMS-986094 of 15 or 30 mg/kg/d for 3 weeks, the nucleoside metabolite M2 was the major plasma analyte (66%-68% of the combined area under the curve). INX-09114 was the highest drug-related species in the heart and kidney (2,610-4,280 ng/mL [males]; ∼2-420× the concentration of other analytes). Other analytes increased dose dependently, with BMS-986094 highest in diaphragm (≤4,400 ng/mL) followed by M2 in liver and kidney (≤1,360 ng/mL), and M7 and M8 in other tissues (≤124 ng/mL). Three weeks after the last dose, INX-09114 remained high in the heart and kidney (≤1,870 ng/mL), with low M2 (≤37 ng/mL) in plasma and tissues. Persistent high concentrations of INX-09114 in the heart and kidney appeared to correlate with toxicities in these tissues in monkeys.

Effects of BMS-986094, a Guanosine Nucleotide Analogue, on Mitochondrial DNA Synthesis and Function.

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Comparison of physiologic and pharmacologic parameters in Asian and mauritius cynomolgus macaques.

This comparative study was conducted to assess background physiologic and pharmacologic parameters of cynomolgus macaques (Macaca fascicularis) from Cambodia, from a mixed Asian source (Cambodia, Vietnam and Indonesia), and from Mauritius. This evaluation provides a comprehensive assessment of several of these parameters in a single study. Ten male and 10 female captive-bred, age-matched macaques from each source were evaluated. Criteria for evaluation included weight gain, assessment of drug metabolizing enzyme activity, metabolomic analysis, immunologic assessments (lymphocyte subsets, TDAR, and serum Ig isotyping), clinical pathology evaluations, physical (respiratory, neurologic, cardiovascular, and ophthalmologic) examinations, pathogen screening, organ weights, and gross and microscopic pathology analyses. The results of this evaluation indicate that, compared to macaques of Asian origin, macaques from Mauritius had the lowest incidence and/or severity of spontaneous pathologic findings in several organs and tissues (lymphoid organs, stomach, kidney, urothelium, heart, arteries and lung) and better testicular maturity at a given age with minimal variability in organ weights. Although slight differences were observed in other parameters, none were considered detrimental to the use of macaques of Asian or Mauritius origin in pharmaceutical candidate safety studies with the use of a consistent source, concomitant controls, and appropriate background knowledge and screening.

Carcinogenicity risk assessment supports the chronic safety of dapagliflozin, an inhibitor of sodium-glucose co-transporter 2, in the treatment of type 2 diabetes mellitus.

INTRODUCTION: Dapagliflozin is a selective inhibitor of the sodium-glucose co-transporter 2 (SGLT2) that increases urinary glucose excretion to reduce hyperglycemia in the treatment of type 2 diabetes mellitus. A robust carcinogenicity risk assessment was undertaken to assess the chronic safety of dapagliflozin and SGLT2 inhibition. METHODS: Genotoxicity potential of dapagliflozin and its metabolites was assessed in silico, in vitro, and in vivo. Dapagliflozin was administered daily by oral gavage to mice, rats, and dogs to evaluate carcinogenicity risks, including the potential for tumor promotion. SGLT2(-/-) mice were observed to evaluate the effects of chronic glucosuria. The effects of dapagliflozin and increased glucose levels on a panel of human bladder transitional cell carcinoma (TCC) cell lines were also evaluated in vitro and in an in vivo xenograft model. RESULTS: Dapagliflozin and its metabolites were not genotoxic. In CD-1 mice and Sprague-Dawley rats treated for up to 2 years at ≥100× human clinical exposures, dapagliflozin showed no differences versus controls for tumor incidence, time to onset for background tumors, or urinary bladder proliferative/preneoplastic lesions. No tumors or preneoplastic lesions were observed in dogs over 1 year at >3,000× the clinical exposure of dapagliflozin or in SGLT2(-/-) mice observed over 15 months. Transcription profiling in Zucker diabetic fatty rats showed that 5-week dapagliflozin treatment did not induce tumor promoter-associated or cell proliferation genes. Increasing concentrations of glucose, dapagliflozin, or its primary metabolite, dapagliflozin 3-O-glucuronide, did not affect in vitro TCC proliferation rates and dapagliflozin did not enhance tumor growth in nude mice heterotopically implanted with human bladder TCC cell lines. CONCLUSION: A multitude of assessments of tumorigenicity risk consistently showed no effects, suggesting that selective SGLT2 inhibition and, specifically, dapagliflozin are predicted to not be associated with increased cancer risk.

Occurrence of spontaneous pancreatic lesions in normal and diabetic rats: a potential confounding factor in the nonclinical assessment of GLP-1-based therapies.

Glucagon-like peptide 1-based therapies, collectively described as incretins, produce glycemic benefits in the treatment of type 2 diabetes. Recent publications raised concern for a potential increased risk of pancreatitis and pancreatic cancer with incretins based in part on findings from a small number of rodents. However, extensive toxicology assessments in a substantial number of animals dosed up to 2 years at high multiples of human exposure do not support these concerns. We hypothesized that the lesions being attributed to incretins are commonly observed background findings and endeavored to characterize the incidence of spontaneous pancreatic lesions in three rat strains (Sprague-Dawley [S-D] rats, Zucker diabetic fatty [ZDF] rats, and rats expressing human islet amyloid polypeptide [HIP]; n = 36/group) on a normal or high-fat diet over 4 months. Pancreatic findings in all groups included focal exocrine degeneration, atrophy, inflammation, ductular cell proliferation, and/or observations in large pancreatic ducts similar to those described in the literature, with an incidence of exocrine atrophy/inflammation seen in S-D (42-72%), HIP (39%), and ZDF (6%) rats. These data indicate that the pancreatic findings attributed to incretins are common background findings, observed without drug treatment and independent of diet or glycemic status, suggesting a need to exercise caution when interpreting the relevance of some recent reports regarding human safety.

Nonclinical toxicology assessments support the chronic safety of dapagliflozin, a first-in-class sodium-glucose cotransporter 2 inhibitor.

Dapagliflozin, a first-in-class, selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), promotes urinary glucose excretion to reduce hyperglycemia for the treatment of type 2 diabetes. A series of nonclinical studies were undertaken to evaluate dapagliflozin in species where it was shown to have pharmacologic activity comparable with that in humans at doses that resulted in supratherapeutic exposures. In vitro screening (>300 targets; 10 µmol/L) indicated no significant off-target activities for dapagliflozin or its primary human metabolite. Once daily, orally administered dapagliflozin was evaluated in Sprague-Dawley rats (≤6 months) and in beagle dogs (≤1 year) at exposures >5000-fold those observed at the maximum recommended human clinical dose (MRHD; 10 mg). Anticipated, pharmacologically mediated effects of glucosuria, osmotic diuresis, and mild electrolyte loss were observed, but there were no adverse effects at clinically relevant exposures, including in the kidneys or urogenital tract. The SGLT2-/- mice, which show chronic glucosuria, and dapagliflozin-treated, wild-type mice exhibited similar safety profiles. In rats but not dogs, dapagliflozin at >2000-fold MRHD exposures resulted in tissue mineralization and trabecular bone accretion. Investigative studies suggested that the effect was not relevant to human safety, since it was partially related to off-target inhibition of SGLT1, which was observed only at high doses of dapagliflozin and resulted in intestinal glucose malabsorption and increased intestinal calcium absorption. The rigorous assessment of supra- and off-target dapagliflozin pharmacology in nonclinical species allowed for a thorough evaluation of potential toxicity, providing us with confidence in its safety in patients with diabetes.

Cutaneous lesions in the rat following administration of an irreversible inhibitor of erbB receptors, including the epidermal growth factor receptor.

CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, including the epidermal growth factor (EGF) receptor. Various inhibitors of the EGF receptor, including CI-1033, have resulted in cutaneous toxicity in humans as a common adverse event. In a chronic toxicity study in rats, CI-1033 produced cutaneous lesions with morphologic characteristics similar to that reported in man. Here the authors describe in detail the dermal changes observed, along with other noteworthy findings of that study. Male and female Wistar rats (15/sex/group) were administered CI-1033 for 27 weeks at 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m(2), respectively) by gavage. Control animals (15/sex) received vehicle alone (aqueous 0.5% methylcellulose) in a dose volume of 5 mL/kg. Six animals/sex/dose were included for toxicokinetic evaluations. Skin lesions were the primary drug-related toxicity and occurred at > or = 2.5 mg/kg in a dose-dependent fashion. The major gross lesions were papules that evolved into crusts and scales that were first observed in weeks 1 and 3, respectively. Alopecia developed in conjunction with the papular eruptions. Skin changes were most pronounced in females, possibly due to higher drug levels. In week 13, CI-1033 plasma AUC(0-24) values were 527 to 1980 ng.h/mL in males and 844 to 2920 ng x h/mL in females at 2.5 to 10 mg/kg. Microscopic changes could be described as 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration. Areas of hyperplasia on the tail were often associated with the development of new hair follicles. Pattern 2 was characterized by a suppurative to pyogranulomatous infundibular folliculitis. Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis; this correlated with alopecia. Elevations in bone marrow myeloid counts correlated with a peripheral leukocytosis, consistent with inflammatory changes in the dermis. In addition, hepatic cholestasis and epithelial atrophy in the gastrointestinal tract and vagina occurred at > or = 2.5 mg/kg. In conclusion, CI-1033 produced cutaneous lesions involving the epidermis and hair follicle, and the morphologic characteristics were similar to that reported in clinical studies with various inhibitors of the EGF receptor. These changes are consistent with pharmacologic inhibition of the EGF receptor in these tissues and demonstrate that the rat can serve as an animal model for investigating the mechanisms for this toxicity.

Pharmacodynamic and toxicokinetic evaluation of the novel MEK inhibitor, PD0325901, in the rat following oral and intravenous administration.

The MEK-mitogen-activated protein kinase (MAPK) signal transduction pathway is involved with numerous cellular processes including cell growth and differentiation. Phosphorylation of MAPK (pMAPK) by MEK results in activation of this pathway. In various solid tumors, the MEK-MAPK pathway is constitutively active; therefore inhibition of this pathway may provide a therapeutic strategy for treating cancer. The objective of this study was to determine the extent and duration of inhibition of pMAPK in selected normal tissues in rats following single oral or intravenous (IV) doses of the novel MEK inhibitor, PD0325901. Male Sprague-Dawley rats (9/group) received either single oral (PO) or IV doses of PD0325901 at 10, 30, or 100 mg/kg (60, 180, and 600 mg/m(2), respectively). Controls received vehicle alone which was aqueous 0.5% hydroxypropylmethyl-cellulose/0.2% Tween 80 for PO dosing and 20% beta-cyclodextran sulfobutyl ether in water (w:v) for IV dosing. Animals (3/group/day) were euthanized on Days 2, 3, and 4, at approximately 24, 48, and 72 h after dosing, respectively. The effects on pMAPK in liver and lung were determined by Western blot analysis and compared with plasma PD0325901 levels. Satellite animals (6/dose/route) received single PO or IV doses and serial blood samples were collected for determination of toxicokinetic parameters of PD0325901 and its major metabolite. In general, systemic exposure to PD0325901 was comparable between routes of administration due to high PO bioavailability (56-109%). Plasma area under the concentration-time curve values of the pharmacologically inactive carboxylic acid metabolite ranged from 18 to 40% of PD0325901. Clinical signs of toxicity occurred at 100 mg/kg PO or IV, indicating the maximum-tolerated dose had been achieved. On Day 2, pMAPK was inhibited 57-95% in liver and 86-99% in lung at all doses, irrespective of route of administration. On Day 3, lung pMAPK remained inhibited 75-91% at all IV doses and by 88% after the 100-mg/kg PO dose. Liver pMAPK remained inhibited 79 and 91% on Day 3 after 100 mg/kg by IV and PO doses, respectively. On Day 4, liver pMAPK was still inhibited 66% after the 100-mg/kg PO dose. The EC(50) and EC(90) plasma drug levels for inhibition of lung pMAPK were calculated to be 20 and 99 ng/ml, respectively. Liver pMAPK levels were inhibited at least 50% at plasma PD0325901 concentrations > or =50 ng/ml. In conclusion, single PO or IV doses of PD0325901 resulted in dose-dependent inhibition of pMAPK in liver and lung. Inhibition of pMAPK in liver was comparable between routes of administration at < or =30 mg/kg, whereas inhibition of pMAPK in lung occurred for a longer duration following IV administration. Measurement of pMAPK in normal tissues served as a means for assessing the pharmacologic activity of PD0325901 and should be included in toxicity studies to evaluate toxicity-pharmacology relationships.

Cartilage dysplasia and tissue mineralization in the rat following administration of a FGF receptor tyrosine kinase inhibitor.

PD176067 is a reversible and selective inhibitor of fibroblast growth factor receptor tyrosine kinase, and was in preclinical development as an angiogenesis inhibitor for the treatment of solid tumors. A 14-day oral toxicity study of PD176067 in young female rats (7 weeks old) was conducted at doses of 2.5, 5, and 10 mg/kg/day (15, 30, and 60 mg/m(2), respectively). Skeletal changes, and vascular and soft tissue mineralization were observed as primary drug-related toxicities. To determine if these changes are specific to young, rapidly growing animals with increased vascular and osseous development, PD176067 was administered to mature (11 months old) rats. Female rats received PD176067 by gavage for 14 days at doses of 2.5, 5, and 10 mg/kg/day and necropsied on day 15. Clinical signs of toxicity were seen at > or =5 mg/kg and one death occurred at 10 mg/kg. Physeal dysplasia (distal femur, proximal tibia, sternum) occurred in all drug-treated animals and was characterized by dose-related increased thickness of the zones of chondrocyte proliferation and hypertrophy, and marked thickening of the zone of ossification. Cartilage hyperplasia was characterized by proliferation of chondrocytes along margins of the synchondrosis and subperiosteum of sternebrae. Serum phosphorus levels increased 47% and 166% at 5 and 10 mg/kg, respectively. Mineralization of cardiac myocytes, aorta, various arteries, renal tubules, and gastric mucosa and muscularis was seen at 10 mg/kg, and consistent with the presence of calcium-phosphorus deposition. Physeal changes occurred at similar plasma PD176067 exposures in young and mature rats (AUC > or = 4.83 microg.hr/mL). PD176067 produced morphologically similar lesions in young and adult rats.

Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)(*).

CI-994 (acetyldinaline) is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 &mgr;M) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 &mgr;M CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 &mgr;M CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.