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.

Copies of author's previously published articles inserted / Describes the exploratory clinical development of tucaresol, consisting of three studies performed on humans and subsequent in vitro and animal studies investigating the possible effects on the immune system. Demostrates that rational drug design may be an efficient way of selecting potential therapeutic candidates. / xxi, 265 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995