Biology and postnatal development of organ systems of cynomolgus monkeys (Macaca fascicularis).

BACKGROUND: The cynomolgus macaque has become the most used non-human primate species in nonclinical safety assessment during the past decades. METHODS: This review summarizes the biological data and organ system development milestones of the cynomolgus macaque available in the literature. RESULTS: The cynomolgus macaque is born precocious relative to humans in some organ systems (e.g., nervous, skeletal, respiratory, and gastrointestinal). Organ systems develop, refine, and expand at different rates after birth. In general, the respiratory, gastrointestinal, renal, and hematopoietic systems mature at approximately 3 years of age. The female reproductive, cardiovascular and hepatobiliary systems mature at approximately 4 years of age. The central nervous, skeletal, immune, male reproductive, and endocrine systems complete their development at approximately 5 to 9 years of age. CONCLUSIONS: The cynomolgus macaque has no meaningful developmental differences in critical organ systems between 2 and 3 years of age for use in nonclinical safety assessment.

Enzalutamide and Apalutamide: In Vitro Chemical Reactivity Studies and Activity in a Mouse Drug Allergy Model.

Enzalutamide and apalutamide are two androgen receptor inhibitors approved for the treatment of castration-resistant prostate cancer (CRPC) and nonmetastatic castration-resistant prostate cancer (nmCRPC), respectively. Apalutamide is associated with an increased incidence of skin rash above the placebo groups in the SPARTAN trial in nmCRPC and in the TITAN trial in metastatic castration-sensitive prostate cancer patients. On the contrary, the rate of skin rash across all clinical trials (including PROSPER [nmCRPC]) for enzalutamide is similar to the placebo. We hypothesized that the apalutamide-associated increased skin rash in patients could be linked to a structural difference. The 2-cyanophenyl and dimethyl moieties in enzalutamide are substituted in apalutamide with 2-cyanopyridine and cyclobutyl, respectively. In our evaluations, the 2-cyanopyridine moiety of apalutamide was chemically reactive with the thiol nucleophile glutathione, resulting in rearranged thiazoline products. Radiolabeled apalutamide, but not radiolabeled enzalutamide, was shown to react with mouse and human plasma proteins. Thiol nucleophiles decreased the extent of covalent binding to the model protein bovine serum albumin, whereas amine and alcohol nucleophiles had no effect, suggesting reactivity with cysteine of proteins. Subcutaneous administration of apalutamide dose dependently increased lymphocyte cellularity in draining lymph nodes in a mouse drug allergy model (MDAM). Enzalutamide, and its known analogue RD162 in which the cyanophenyl was retained but the dimethyl was replaced by cyclobutyl, demonstrated substantially less covalent binding activity and negative results in the MDAM assay. Collectively, these data support the hypothesis that the 2-cyanopyridine moiety in apalutamide may react with cysteine in proteins forming haptens, which may trigger an immune response, as indicated by the activity of apalutamide in the MDAM assay, which in turn may be leading to increased potential for skin rash versus placebo in patients in the SPARTAN and TITAN clinical trials.

Age- and sex-related changes in body weights and clinical pathology analytes in cynomolgus monkeys (Macaca Fascicularis) of Mauritius origin.

BACKGROUND: Clinical pathology and body weight information for the cynomolgus monkey in the literature is primarily derived from a small number of animals with limited age ranges, varying geographic origins, and mixed genders. OBJECTIVES: This study aimed to summarize the age- and sex-related changes in clinical pathology analytes and body weights in cynomolgus monkeys of Mauritian origin. METHODS: Pre-study age and body weight data were reviewed in 1819 animals, and pre-study hematologic, coagulation, and serum biochemical analytes were reviewed in 1664 animals. RESULTS: Body weights were statistically higher (P < 0.01) in males than females in all age groups (2-10 years). These measurements became prominent after 4 years of age and peaked at 7 to 8 years of age in both sexes. Sex-related differences were noted in reticulocyte (RETIC) counts, creatinine, cholesterol, and triglyceride concentrations, and alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) activities. Age-related differences were noted in RETIC and lymphocyte counts, creatinine, triglyceride, phosphorus, and globulin concentrations, and ALP and GGT activities. The youngest (2 to <3 year) age group had the fewest number of clinical pathologic analyte differences including ALP and GGT activity differences which occurred in all age groups from 2 to 10 years; they also had age-related lower globulin concentrations. There were no age- or sex-related differences in coagulation measurands. CONCLUSIONS: Sexual dimorphism in body weight was apparent for all ages from 2 to 10 years of age. The only difference in clinical pathology analytes unique to the 2 to <3 years of age group were age-related lower globulin levels.

Non-clinical combination toxicology studies: strategy, examples and future perspective.

Over the last decade, combination of drugs in all stages of pharmaceutical development has accelerated availability of promising new therapies for difficult to treat diseases. Safety assessment of combined drugs to be tested in humans can occur at a critical path prior to proceeding in clinical testing. A recent survey by The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ DruSafe) summarized member companies' approaches to combination safety strategies. In addition, feedback from Health Authorities (HAs) support a case-by-case scientific approach in assessing combination products' safety in accordance with the International Council on Harmonization (ICH) guidelines. Here, we present Pfizer's drug combination safety approach for various therapeutic areas (TA) including inflammation and immunology, metabolic, and anti-cancer products. There is no one-size-fits-all approach; rather, our main considerations include: strength of the existing clinical safety data for the individual compounds, common target organs, the potential for a synergistic effect, potential drug-drug interaction, routes of administration of each product and disease indications. No formal toxicity studies are considered necessary for anti-cancer drugs, while safety endpoints may be collected in preclinical pharmacology studies especially when the combined drugs present a novel mechanism. Combination safety studies when conducted for non-cancer indications can range from 2 to 13-weeks in duration, conducted usually in rodents, with dosages of individual molecules within clinical pharmacologic ranges. A case-by-case strategy guided by scientific rationale and in close collaboration with HAs remains the best approach to decide on the design and conduct of combination safety studies.

Myocarditis in Cynomolgus Monkeys Following Treatment with Immune Checkpoint Inhibitors.

PURPOSE: Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys. EXPERIMENTAL DESIGN: Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29. RESULTS: Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. CONCLUSIONS: We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.

Evaluation of the effects of subchronic oral administration of n-butyl maleate in Sprague-Dawley rats.

n-Butyl maleate, also referred to as monobutyl maleate, is an ester of maleic acid, which is used as a counterion in the pharmaceutical industry. While substantial published data exist on short-term treatment, maleic acid-induced renal toxicity in the rat, no toxicity data are available on the monobutyl ester. This study evaluated the oral subchronic nephrotoxicity potential of n-butyl maleate administered to Sprague-Dawley rats (10/males and females/group) at doses of 0 (vehicle control), 10, 30, or 60 mg/kg/d for 2 wk. Statistically significant elevations in organ weights were noted in males at 60 mg/kg/d and included: (a) increases in absolute heart, kidney, and liver weights; (b) increased liver to body weight ratios; and (c) increased heart, kidney, liver, spleen, and epididymides to brain weight ratios. In females, statistically significant increases in organ weights were limited to increases in adrenal to brain weights at > or = 10 mg/kg/d, kidney to brain weights at > or = 30 mg/kg/d, and kidney to body weight and liver to brain weight ratios at 60 mg/kg/d. There were no macroscopic or microscopic pathology changes observed in any of the tissues examined. Importantly, light microscopic examination of the kidney was unremarkable at the end of the 2-wk dosing period with n-butyl maleate. Although lacking a histopathological correlate, resultant increases in organ weights at 60 mg/kg/d might be considered indicative of an adverse effect. However, renal perturbation induced by n-butyl maleate was mild in comparison to maleic acid-induced renal toxicity, which manifested as impaired tubular resorption and necrosis of the proximal tubules at doses > or = 60 mg/kg/d. The no-observed-adverse-effect level (NOAEL) for the study was 30 mg/kg/d.

Effects of cyclooxygenase inhibition on the gastrointestinal tract.

Cyclooxygenase (COX) is a rate-limiting enzyme that catalyzes the conversion of arachidonic acid, an essential fatty acid present in cell membrane phospholipids and liberated by phospholipase, into prostaglandins (PGs) and prostanoids. COX has two distinct membrane-anchored isoenzymes; COX-1 and COX-2. COX-1 is a constitutively expressed and found in most normal body tissues; COX-2 is expressed in normal tissues at low levels and is highly induced by pro-inflammatory mediators in the setting of inflammation, injury, and pain. Inhibitors of COX activity include: (1) conventional non-selective non-steroidal anti-inflammatory drugs (ns-NSAIDs); (2) selective COX-2 inhibitors (COXIBs); and (3) COX-1 inhibitors. Non-selective NSAIDs, at therapeutic doses, inhibit both COX-1 and COX-2. The anti-inflammatory benefits of these drugs are primarily derived from COX-2 inhibition, while inhibition of COX-1 often elicits gastrointestinal (GI) toxicity. Therefore, COXIBs were developed to provide a selective COX-2 agent, i.e., one, that at fully therapeutic doses demonstrated comparable therapeutic benefit to non-selective NSAIDs, without the attendant COX-1-mediated GI toxicities. In this review, we evaluate available literature describing the pathophysiologic role of cyclooxygenases and the effects of their inhibition in GI system in experimental and domestic animal species.

Mechanistic Investigation of Bone Marrow Suppression Associated with Palbociclib and its Differentiation from Cytotoxic Chemotherapies.

PURPOSE: Palbociclib (PD-0332991) is the first selective cyclin-dependent kinase (CDK) 4/6 inhibitor approved for metastatic breast cancer. Hematologic effects, especially neutropenia, are dose-limiting adverse events for palbociclib in humans. EXPERIMENTAL DESIGN: Reversible hematologic effects and bone marrow hypocellularity have been identified in toxicology studies in rats and dogs after palbociclib treatment. To understand the mechanism by which the hematologic toxicity occurs, and to further differentiate it from the myelotoxicity caused by cytotoxic chemotherapeutic agents, anin vitroassay using human bone marrow mononuclear cells (hBMNC) was utilized. RESULTS: This work demonstrated that palbociclib-induced bone marrow suppression occurred through cell-cycle arrest, with no apoptosis at clinically relevant concentrations, was not lineage-specific, and was reversible upon palbociclib withdrawal. In contrast, treatment with chemotherapeutic agents (paclitaxel and doxorubicin) resulted in DNA damage and apoptotic cell death in hBMNCs. In the presence or absence of the antiestrogen, palbociclib-treated hBMNCs did not become senescent and resumed proliferation following palbociclib withdrawal, consistent with pharmacologic quiescence. The breast cancer cells, MCF-7, conversely, became senescent following palbociclib or antiestrogen treatment with additive effects in combination and remained arrested in the presence of antiestrogen. CONCLUSIONS: Palbociclib causes reversible bone marrow suppression, clearly differentiating it from apoptotic cell death caused by cytotoxic chemotherapeutic agents. This study also distinguished the cell-cycle arresting action of palbociclib on normal bone marrow cells from the senescent effects observed in breast cancer cells. These results shed light on the mechanism and support risk management of palbociclib-induced bone marrow toxicity in the clinic.

Cyclooxygenase-2 in human pathological disease.

To understand the potential role of cyclooxygenase (COX) in normal and inflammatory human diseases, we characterized the expression of COX-1 and COX-2 in biopsies of osteoarthritis, atherosclerosis, and cancer. Tissues were prepared for immunohistochemistry by standard methods, and representative cases assayed via Western blot and quantitative RT-PCR. COX-2 was not detected in normal human tissues with few exceptions. Moderate to marked COX-2 was observed in the macula densa (MD) and thick ascending limb (TAL) in human fetal kidneys, but was not detected in neonatal and adult MD and TALs. Low level, constitutive COX-2 was detected in colonic epithelium, peribronchial glands, and pancreatic ductal epithelium. Low to moderate COX-2 was detected basally in the cortex, hippocampus, hypothalamus, and spinal cord, and in reproductive tissues during ovulation, implantation and labor. No COX-2 was detected in the existing vasculature in normal tissues, and was also not expressed throughout the ductus arteriosus. COX-2 was markedly induced in human tissues of osteoarthritis, atherosclerosis and cancer. COX-2 was prominently expressed in the synovium, fibrocartilage of osteophytes, and in the blood vessels in the osteoarthritic (OA) knee joint. COX-2 was also prominently detected in the macrophages/foam cells in atherosclerotic plaques, and in the endothelium overlying and immediately adjacent to the fibrofatty lesion. Moderate- to intense COX-2 expression was consistently observed in the inflammatory cells, neoplastic lesions, and blood vessels in all epithelial-derived human cancers studied. In contrast, COX-1 was relatively ubiquitously observed in both normal and pathophysiological conditions. These data collectively imply COX-2 plays an important role in mediating a variety of inflammatory diseases, and imply COX-2 inhibitors may be effective in the prevention and/or treatment of OA, heart disease, and epithelial cancers.

Comparative juvenile safety testing of new therapeutic candidates: relevance of laboratory animal data to children.

Differences in drug response in patients of various ages including children and the elderly are common, often leading to challenges in optimizing dosages and duration of use. For example, developmental changes in renal function can dramatically alter the plasma clearance of compounds with extensive renal elimination and thus can enhance renal and systemic toxicity of these drugs. Preclinical and clinical research of new therapeutics is initially focused on adults, and provides little relevant information for children especially those who are still going through skeletal and organ development. The organ systems in the pediatric population that can be most susceptible are lungs, brain, kidneys, immune, skeletal, and reproductive systems. Considering that significant differences can exist between adult and juvenile populations that may affect drug safety, major regulatory agencies around the world are encouraging and sometimes requiring companies to generate preclinical juvenile animal data to predict for potential drug toxicity in children. However, data generated from such studies are useful only if obtained using the most appropriate species at the most relevant age considering comparability of specific organ system development in question. Other factors in the design of juvenile safety studies should include the indication, existing toxicological data and likely route of human exposure. This report will discuss these factors with a focus on reviewing species-specific developmental schedules for specific target organs and relevance of preclinical data in the design and conduct of clinical pediatric studies. Specific examples will be used to discuss the relationship of preclinical juvenile toxicity observations to risk assessment in humans.

Comparative expression and distribution of c-fos, estrogen receptoralpha (eralpha), and p38alpha in the uterus of rats, monkeys, and humans.

The uterine cellular expression and distribution of c-fos, ERalpha and p38alpha was compared in humans, nonhuman primates, and rats using immunohistochemistry. ERalpha and c-fos were present in the glandular (GE) and luminal epithelial cells (LE) of humans and nonhuman primates, with differing expression patterns evident between proliferative and secretory cycle phases. In rats, the highest and lowest expression of c-fos was present during proestrus and estrus, respectively, in the LE and GE. The most intense ERalpha staining in rats was observed during proestrus in the GE, while the least intense staining was seen in the LE during proestrus. Strong LE and GE expression of p38alpha was present in rats in all stages of the estrous cycle and during the proliferative phase in both humans and nonhuman primates. No p38alpha expression was observed during the secretory phase in either humans or nonhuman primates. Our work suggests that c-fos, ERalpha and p38alpha (a) are primarily expressed during the proliferative phase, but not the secretory phase and exhibit interspecies expression variability, and (b) rats exhibit cyclic changes in the expression of c-fos and ERalpha.