Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Fatemeh Akhlaghi

Abstract

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. PF-5190457 was made available for research by the National Center for Advancing Translational Sciences (NCATS) under the NIH New Therapeutic Uses Pilot Program. Initial studies by Pfizer had shown that the compound is safe and well tolerated in healthy volunteers after oral administration. To date, the compound has completed Phase 1b clinical trials in nontreatment-seeking patients with alcohol use disorder. The plasma samples obtained from the study was evaluated for the pharmacokinetics of PF-5190457 and its major hydroxy metabolite. 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 (to be submitted to Journal of Chromatography B: Biomedical Sciences and Applications). 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 with methanol 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 (to be submitted to Drug Metabolism and Disposition) 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. Subsequent analysis revealed the presence of a significant hydroxy metabolite (m/z 529) along with minor glucuronide and hydroxy glucuronide metabolites. Further exploration of the fragmentation pattern of the major hydroxy metabolite (m/z 351 and m/z 225) suggested possible hydroxylation between the two nitrogen atoms of the drug. In parallel, in vitro experiments were conducted in human hepatic microsomes and cytosolic fractions to characterize the drug metabolism enzymes responsible for the formation of metabolites. Preliminary data revealed the formation of hydroxy metabolite in the cytosol but not in the microsomes. The reaction was found to be independent of the presence of nicotinamide adenine dinucleotide phosphate (NADPH) in the incubation media. Addition of a non-selective CYP450 inhibitor (1-amino benzotriazole) inhibited the formation of all the minor metabolites but not the hydroxy metabolite. A comparative mass spectra analysis showed the presence of hydroxy metabolite in the human liver cytosol incubations which was previously found in human plasma. PF-5190457 was also incubated in hepatocytes leading to the formation of major and minor metabolites. Further addition of an aldehyde oxidase inhibitor (hydralazine) in hepatocytes completely inhibited the formation of the hydroxy metabolite indicating the involvement of aldehyde oxidase in the metabolism of the PF-5190457. The hydroxy metabolite was also found in incubations conducted with PF-5190457 and cytosol from different animal species. The aldehyde oxidase metabolite of PF-5190457 was biosynthesized, from female mouse liver cytosol and the structure of the metabolite was confirmed using NMR analysis indicating the hydroxylation position to be on the pyrimidine ring between the two nitrogen atoms. Literature shows both aldehyde and xanthine oxidase share substrate specificity. Specific inhibitors of both the enzymes were used to identify their involvement in the metabolism of the drug. Raloxifene and febuxostat were used as selective aldehyde oxidase and xanthine oxidase inhibitor, in human liver cytosol incubations with PF-5190457 respectively. The data from inhibition study suggested a role for both the enzymes in the biotransformation of the drug with aldehyde oxidase enzyme being dominant over xanthine oxidase. The kinetic parameters Km and Vmax for hydroxy metabolite formation in human liver cytosol were 42 ± 4 μM and 0.12 ± 0.003 nmol/min/mg protein, respectively.

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 (n=104) using mass spectroscopy as described in Manuscript III (to be submitted to Drug Metabolism and Disposition). 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<0.0001) 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 (2 - 49 pmol/mg total cytosolic protein). 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. We found no significant association between demographic factors with the expression of the AOX1 protein. Similarly, disease conditions were not found to influence the expression of AOX1 indicating other unidentified factors influencing the enzyme expression.

Manuscript IV (to be submitted to British Journal of Clinical Pharmacology) outlines the pharmacokinetics data via non-compartmental analysis of PF-5190457 and its hydroxy 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. The half-life of the metabolite (~13 h) was found to be longer than the parent drug (~6 h). Additionally, the rate of formation of hydroxy metabolite was studied in cytosol prepared from 10 human liver samples. The rate of formation of hydroxy metabolite was found to be highly variable with a 20.5-fold (0.02 – 0.41 nmol/min/mg protein) variation among the ten samples. Interestingly, the AOX1 protein expression for same samples showed only 2.5-fold variability (16.8 - 44.1 pmol/mg protein).

In summary, an understanding of the enzymes responsible for the metabolism of a drug is essential to avoid drug-drug interactions or the presence of polymorphic enzymes interfering with the therapeutic outcome of the drug. Collectively, we for the first time determined the clinical pharmacokinetics of PF-5190457 and its major metabolite (hydroxy metabolite) in alcoholic patients. Further studies suggested the involvement of aldehyde oxidase (major role) and xanthine oxidase (minor role) in the metabolism of the PF-5190457. Moreover, to examine the variability in the protein expression of the enzymes, we quantified AOX1 in a large liver databank. This work also makes a case for considering non-cytochrome-P450 enzymes in the early screening during drug discovery. Considering the longer half-life of metabolite than the parent, it will be warranted to investigate the pharmacological properties of hydroxy metabolite in future studies.

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