In-Vitro and In-Vivo Models of Bile Acid Metabolism and Transport

All biomedical research is conducted in animal models first. In addition, the Food and Drug Administration requires extrapolation from animal data to predict human responses. There are ongoing scientific and regulatory challenges translating interspecies comparisons and predictions. Metabolic pathways are a cornerstone to understanding drug metabolism and toxicities and the liver is a key organ in this process. Bile acids (BAs) play a central role in the hepatobiliary toxicities of chemicals, toxins, and biological reagents. BAs have many physiological functions including regulation of genes involved in cholesterol and glucose metabolism and BA homeostasis. However, BAs also have several pathological effects including carcinogenicity and liver toxicity. Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. Several metabolic pathways including sulfation by sulfotransferase (SULT), glucuronidation by UDP-glucuronosyltransferases (UGTs), and oxidation by Cytochrome-P450 (CYP450) enzymes participate in the direct detoxification, enhance the elimination of BAs, and help maintain their homeostasis. In addition, influx and efflux transporters at both the sinusoidal and basolateral membranes play an important role in determining intracellular BA concentration, and therefore their hepatotoxicity. There are known species differences in BA metabolism and transporter. There are known species differences in the composition of the BA pool, the toxicity of BAs, and drug-induced hepatotoxicity related to BAs. These species differences can prevent extrapolation of toxicity profiles of xenobiotics between species causing a serious disconnect between preclinical safety findings in rodent and canine animal models and safety finding at clinical stages. In this thesis, we compare the metabolic profile of representative BAs between several species including humans, chimpanzee, monkeys, minipigs, hamster, rabbits, dogs, rats, and mice. The metabolic profile was characterized by the identification of BA metabolites and by quantifying the kinetics of their formation in hepatocyte S9 in-vitro system. The relative contributions of individual metabolic pathways were determined. LC-MS/MS was used for the qualitative and quantitative analysis of BAs and their metabolites. A mixture of stable-isotope labeled (2H4) and unlabeled BAs were used to facilitate the identification of all minor and major metabolites. Major species differences were found in the metabolism of BAs. Amidation with taurine and glycine was the major pathway in all species. Sulfation was predominant in humans, whereas oxidation and glucuronidation were predominant in rodents and dogs, respectively. Glucuronidation and amidation of BAs are exclusive, where glucuronidation only takes place for unamidated BAs. In vitro-In vivo extrapolation (IVIVE) is performed to establish the correlation of the in vitro results and the bile acid profile in vivo. These results explain, at least in part, the dissociation between preclinical toxicity data in various in vitro and in vivo models and toxicities observed in humans. Furthermore, more relevant species are suggested based on the similarity to the human BA metabolism. In addition, we also screened different bile acids as a potential biomarker for transporter activity using cynomolgus monkey as preclinical model. This resulted in identification of key bile acid sulfates as biomarker for transporter mediated drug-drug interactions

Characterization of CDK(5) Inhibitor, 20-223 (aka CP668863) for Colorectal Cancer Therapy

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 – now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was ~3.5-fold and ~65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average \u3e2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy

Simultaneous LC–MS/MS analysis of eicosanoids and related metabolites in human serum, sputum and BALF

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra- and inter-day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC-MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials