A novel monovalent FGFR1 antagonist: Preclinical safety profiles in rodents and non-human primates.

Blocking Fibroblast Growth Factor Receptor 1 (FGFR1) is an attractive therapeutic option for treatment of cancer subtypes with amplification and over-expression of FGFR1. Selective targeting of FGFR1 can be achieved using an antibody-based approach, as small molecule inhibitors may not discriminate between FGFR1, 2, 3 and 4 due to their highly homologous kinase domain. However, development of classical bivalent FGFR1 directed antibodies has failed due to non-tolerated body weight decreases in preclinical species. M6123 is a novel IgG-like monovalent FGFR1 specific binder with enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) effector function and inhibits tumor growth significantly in FGFR1-dependent human xenograft models without reduced body weight in tumor-bearing mice. Toxicology studies reported here characterized the safety profile of M6123 in mouse, rat, and monkey. There were significant differences among animal species under similar M6123 exposure levels. Rats showed metastatic mineralization with an imbalance in serum phosphate at low doses, while mineralization was not found in mice or monkeys, even though hyperphosphatemia was detected in mice. Subtle differences in calcium/phosphate homoeostasis feedback loops may trigger the susceptibility to mineralization among animal species; nevertheless, the exact mechanism remains unknown. Monkeys showed marked, but reversible, decreases in peripheral blood NK cells and neutrophils. The latter was associated with considerably increased neutrophilic infiltrates in the liver sinusoids and red pulp of the spleen. These effects in monkeys are likely related to the enhanced ADCC activity of M6123. Overall, M6123 showed a superior safety profile in animals compared to bivalent FGFR1 antagonists or pan-FGFR inhibitors.

l-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. METHODS: In several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. METHODS: In several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

Nonclinical safety, pharmacokinetics, and pharmacodynamics of atacicept.

Atacicept, a soluble recombinant fusion protein of the human immunoglobulin (Ig) G(1) Fc and the extracellular domain of the human transmembrane activator and calcium modulator and cyclophylin ligand interactor receptor, acts as an antagonist of both B lymphocyte stimulator and a proliferating-inducing ligand. Here we determined the nonclinical safety, pharmacokinetics and pharmacodynamics of atacicept in mice and cynomolgus monkeys. Subcutaneous atacicept treatment (twice weekly in cynomolgus monkeys, three times weekly in mice) was generally safe and well tolerated safe and well tolerated with dosing up to 10 mg/kg every other day for up to 39 weeks or up to 80 mg/kg when dosed for 4 weeks. At a dose of 1 mg/kg subcutaneous (sc) bioavailability of atacicept in mice and monkeys was 76 and 92%, with a mean serum t(1/2) of 44 and 179 h, respectively. In accord with its anticipated mechanism of action, repeated administration of atacicept decreased serum IgG concentrations up to 50%, IgM concentrations >99%, and circulating mature B-cell concentrations up to 60%. These effects were dose-related but reversible, as determined in a 25-week follow-up period. Microscopically, B cells numbers were reduced in the follicular marginal zone of the spleen and the mantle surrounding germinal centers of the lymph nodes. These data confirm the preclinical safety and the pharmacological activity of atacicept and support its clinical development.