Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutics

First Advisor

Jürgen Venitz

Abstract

Purpose. 5-HMF is a potential antisickling agent forming a Schiff-adduct with hemoglobin (Hb). In-vitro studies were designed to identify the metabolic pathways of 5-HMF in human hepatic cytosol, to assess inter-species differences in its hepatic metabolism, and to predict in-vivo PK properties. Moreover, metabolism of 5-HMF in human RBCs was investigated. Finally, in-vitro studies were done to characterize 5-HMF binding kinetics with human Hb and albumin (HSA). Methods. NAD+ reduction was monitored at 340 nm in human hepatic cytosol for 5-HMF (26 mM) and prototypical ADH and ALDH substrates in the presence or absence of their inhibitors. Furthermore, concentration-dependency studies were performed for 5-HMF (1.5-96 mM) in mouse, rat, dog, and human hepatic cytosol and fitted by Michaelis Menten (MM) model. In-vitro-in-vivo-extrapolation (IVIVE) was performed using the well-stirred model. Moreover, metabolic studies of 5-HMF (12-142 mM) in human RBCs were done under similar conditions. Time- and concentration-dependent binding studies were conducted for 5-HMF (5 µM-5 mM), in Hb (217 μM) and HSA (63 and 202 μM) solutions. Ultrafiltered 5-HMF concentrations were measured by a validated HPLC-UV assay. After correction for nonspecific binding, rate constants, binding affinity, and capacity were estimated by nonlinear regression. Results. In human hepatic cytosol, 5-HMF followed MM kinetics with Km: 218(±74) mM and was mainly inhibited by the ALDH inhibitor. In all animal species, 5-HMF exhibited millimolar Km values and is expected to have low hepatic extraction, high oral bioavailability, and first-order PK for relevant blood concentrations. The IVIVE-predicted in-vivo half-lives for 5-HMF were adequate for the mouse and dog but overestimated for humans. In RBCs, 5-HMF had Clintin-vitro of 0.34(± 0.02) ml/min/ml RBCs scaled-up to 9.9 ml/min/kg. Time-dependent binding of 5-HMF was demonstrated for both Hb and HSA. Steady-state studies revealed saturable Hb binding and non-saturable HSA binding. Conclusions. 5-HMF is an ALDH/ADH substrate in hepatic cytosol. Across animal species, 5-HMF is expected to be a low-hepatic-extraction-ratio drug with high oral bioavailability. 5-HMF is subject to RBCs metabolism in human. 5-HMF is expected to show fast association with, but slow dissociation from its drug target, Hb, which may lead to a prolonged in-vivo PD effect.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-9-2010

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