HYDROMORPHONE-3-GLUCURONIDE - BIOCHEMICAL SYNTHESIS AND PRELIMINARY PHARMACOLOGICAL EVALUATION

Hydromorphone-3-glucuronide (H3G) was synthesized biochemically using rat liver microsomes, uridine-5'-diphosphoglucuronic acid (UDPGA) and the substrate, hydromorphone. Initially, the crude putative H3G produc t was purified by ethyl acetate precipitation and washing with acetoni trile, Final purification was achieved using semi-preparative high-per formance-liquid-chromatography (HPLC) with ultraviolet (UV) detection. The purity of the final H3G product was shown by HPLC with electroche mical and ultraviolet detection to be > 99.9% and it was produced in a yield of approximate to 60% (on a molar basis). The chemical structur e of the putative H3G was confirmed by enzymatic hydrolysis of the glu curonide moiety using P-glucuronidase, producing a hydrolysis product with the same HPLC retention time as the hydromorphone reference stand ard. Using HPLC with tandem mass spectrometry (HPLC-MS-MS) in the posi tive ionization mode, the molecular mass (M+1) was found to be 462 g/m ol, in agreement with H3G's expected molecular weight of 461 g/mol. Im portantly, proton-NMR indicated that the glucuronide moiety was attach ed at the 3-phenolic position of hydromorphone. A preliminary evaluati on of H3G's intrinsic pharmacological effects revealed that following icy administration to adult male Sprague-Dawley rats in a dose of 5 mu g, H3G evoked a range of excitatory behavioural effects.including che wing, rearing, myoclonus, ataxia and tonic-clonic convulsions, in a ma nner similar to that reported previously for the glucuronide metabolit es of morphine, morphine-3-glucuronide and normorphine-3-glucuronide.