Biotransformation and pharmacokinetic evaluation of PF-5190457, a novel drug candidate for alcoholism

Alcohol use disorder is a chronic condition characterized by an inability to control alcohol intake. Recent research has shown that appetite-regulating hormones, ghrelin, and leptin, may play a role in alcohol craving. In addition, it is known that food and alcohol craving have overlapping receptors in the brain reward system. Ghrelin, a naturally occurring hormone in the human body, is produced mainly by ghrelinergic cells of the gastric mucosa. Once the hormone crosses the blood-brain barrier, it binds to growth hormone secretagogue receptor (ghrelin receptor) in the hypothalamus and stimulates the appetite and food intake. Leptin, a hormone produced from adipose tissue has an opposite effect to that of ghrelin and reduces hunger. Leptin and ghrelin work in tandem to maintain energy homeostasis in the body. It was demonstrated that antagonizing ghrelin receptor reduces alcohol craving thus making it a potential pharmacological target to treat alcohol use disorders. PF-5190457 is a highly selective, orally bioavailable ghrelin receptor inverse agonist developed by Pfizer. In order to measure the concentration of the drug and its metabolite a liquid chromatography tandem mass spectroscopy method (LC-MS/MS) was developed and validated as described in Manuscript I. The manuscript describes a new LC-MS/MS method developed for this purpose and the assay was fully validated according to the FDA guidance document for bioanalytical methods. The analytes of interest were extracted from plasma using a simple protein precipitation technique, and tacrine was used as the internal standard. All the bioanalysis method parameters, i.e., sensitivity, specificity, linearity, accuracy, precision, matrix effect, recovery, and stability were within the recommended FDA guidelines. Incurred sample reanalysis (10% of the patient plasma samples) was performed to evaluate the repeatability of the new method and was found to be within the acceptance criteria. Manuscript II describes the biotransformation of PF-5190457 (molecular weight: 513). Before this work in our lab, no information on the metabolism of the PF-5190457 in humans was available. Extensive LC-MS/MS profiling of plasma from Phase 1b study showed many circulating metabolites. In parallel, in vitro experiments were conducted in human hepatic microsomes, cytosolic fractions and hepatocytes to characterize the drug metabolism enzymes responsible for the formation of these metabolites. Aldehyde oxidase (AOX1) is gaining increased attention for its role in the metabolism of new drug candidates. A significant variability in the levels of the AOX1 protein expression in the human liver has been reported in published studies. We assessed the AOX1 levels in a novel repository of human liver using mass spectroscopy as described in Manuscript III. The levels of AOX1 were estimated using traditional data-dependent acquisition and upcoming SWATH-MS method. A strong and significant correlation (Spearman r-0.72, P\u3c0.0001) was observed between the two methods making a case for the use of SWATH based methods in the field of drug metabolism. Expression levels of AOX1 determined in our liver bank using MaxQuant (proteomics data analysis software) were found to be well within the literature reported range. Further, we evaluated the effect of demographic factors (gender, age, ethnicity, drug usage, alcohol consumption, and smoking) and disease conditions (diabetes, non-alcoholic fatty liver disease) on the AOX1 expression. Manuscript IV outlines the pharmacokinetics data via non-compartmental analysis of PF-5190457 and its metabolite. The concentrations of analytes from phase 1b trials were evaluated in N=12 patients at placebo, 50 and 100 mg doses using validated LC-MS/MS method as described in Manuscript I. Additionally, the rate of formation of metabolite was studied in 10 human liver samples. (Abstract shortened by ProQuest.