Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Fatemeh Akhlaghi

Abstract

Pharmacokinetic variability is an important consideration in pharmacotherapy to ensure safety and efficacy of medications, thus the understanding of the sources of variability in drug concentrations in the body is imperative. The goals of this dissertation were: (1) To use in vitro drug metabolism tools to characterize the influence of non-alcoholic fatty liver disease (NAFLD) on Cytochrome P450 2B6 (CYP2B6)-mediated hydroxylation of bupropion in human liver microsomes; and (2) To use population pharmacokinetics to characterize the pharmacokinetics of PF-5190457, an inverse agonist of the growth hormone secretagogue receptor (hGHSR1a). This work has been organized in two parts. Part one is made up of manuscripts I-III; and addresses the first objective. Part two, on the other hand, is made up of manuscript IV, and addresses the second objective. The manuscripts are briefly described below:

Manuscript I: Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the presence of steatosis in more than 5% of hepatocytes with little or no alcohol consumption. The physiological and biochemical changes associated with NAFLD may result in altered expression and activity of drug metabolizing enzymes (DMEs) or transporters. Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 are more consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas the activity of CYP2E1 and the efflux transporter MRP3 are up-regulated. However, it is not clear how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug variability in patients and could have serious implications for the safety and efficacy of xenobiotics. In the first manuscript, we reviewed the effects of NAFLD on the regulation, expression and activity of major drug metabolizing enzymes and transporters. We also discussed the potential mechanisms underlying these alterations.

Manuscript II: Diabetes is strongly associated with NAFLD. However, tools for predicting the diabetic status of human liver tissues (HLTs) is lacking. Manuscript II was aimed to establish a model-based approach for predicting the diabetic status of donors of HLTs. The liver tissue as well as demographic and anthropometric information were supplied by Xenotech LLC. Histopathological examination was conducted to characterize NAFLD lesions. HLTs were homogenized and levels of feeding-related hepatic neuroendocrine peptides (active amylin, insulin, c-peptide, glucagon, ghrelin, active GLP-1, GIP, PP, PYY, leptin and MCP-1) determined. The association between diabetes, and these covariates was modeled using multiple logistic regression. The statistically validated model was used to predict new diabetic classes of HLTs. A multiple logistic regression model adequately described the association between diabetes, NAFLD lesions and the neuroendocrine peptides. Liver weight, cpeptide, leptin, PYY, Amylin (active) and steatosis were significant predictors of diabetes. The final model had an AROC curve of 0.89, accuracy of 80%, sensitivity of 82.4% and specificity of 77%. The new diabetic classes showed that hepatic GLP-1 (active) level was 1.4 higher in non-diabetic livers compared to diabetic ones. In addition, the logistic regression model can be used as a tool to verify the diabetic status of HLTs which are used for drug metabolism studies.

Manuscript III: Despite the initial belief that Cytochrome P450 (CYP) 2B6 is of minor significance, it is now recognized as a clinically relevant drug metabolizing enzyme. The impact of non-alcoholic fatty liver disease (NAFLD) on drug metabolism has been identified; however, it is still unclear how it influences CYP2B6. We used in vitro approaches in human liver microsomes (HLM) and HepaRG cells to investigate the effect of NAFLD on CYP2B6-mediated formation of hydroxybupropion. The presence of NAFLD increased the km significantly (p < 0.04) and reduced CYP2B6 intrinsic clearance by 2-fold. The results from the HepaRG cells qualitatively recapitulated findings in the HLMs. Fatty acid accumulation in hepatocytes seems to be involved with the alteration. This investigation contributes to our current knowledge on the influence of NAFLD on CYP2B6 in vitro kinetics and offers a basis for clinical trial in this patient population.

Manuscript IV: PF-5190457 is an inverse agonist of the growth hormone secretagogue receptor (hGHS-R1a), that is undergoing clinical trial for treatment of alcohol use disorder. The purpose of this study was to describe the population pharmacokinetics (PK) of PF-5190457 and to identify demographic and biochemical characteristics that influence its PK variability. Data on drug dosage, sampling times and plasma concentrations were collected retrospectively from two studies: Phase 1a and Phase 1b. Thirty five (35) healthy volunteers were enrolled in the Phase 1a, and 12 non-treatment seeking alcoholic subjects in the Phase 1b trial. The log-transformed concentration and time points were modeled in NONMEM. The influence of patients' demographic and biochemical characteristics were evaluated; and the accuracy and precision of the model parameters determined using bootstrapping. The predictive performance of the final model was checked using percentile visual predictive checks.

The pharmacokinetics of PF-5190457 was best described by a one-compartmental model with first order absorption after oral administration. The estimated typical pharmacokinetic parameters included the absorption rate constant (ka, 3.6 h-1), oral clearance (CL/F, 80 Lh-1) and apparent volume of distribution (V/F, 575 L). Inclusion of body weight and serum albumin as covariates on V/F reduced the interindividual variability (IIV) associated with V/F by ~28%. Increasing body weight increased V/F, whereas increasing serum albumin levels reduced it. We anticipate that this model would serve as a guide in designing dosage regimen for future clinical trials with PF-5190457.

Conclusion. This work demonstrates that in vitro drug metabolism in human liver microsome has the potential to explain the effect of NAFLD on CYP2B6-mediated hydroxybupropion formation. Similarly, population pharmacokinetic modeling in NONMEM has the capability to elucidate the influence of body weight and serum albumin on the pharmacokinetics of PF-5190457.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Monday, April 20, 2020

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