title: Stereospecific chemical and enzymatic stability of phosphoramidate triester prodrugs of d4T in vitro.
abstract: The phosphoramidate triester prodrug approach is widely used to deliver nucleotide forms of nucleoside analogues into target cells. We investigated the stereoselective stability of a series of prodrugs of the anti-HIV agent 2',3'-didehydro-2',3'-dideoxythymidine (d4T). Chemical stability was evaluated in phosphate buffer at pH values of biological relevance (i.e. pH 2.0, 4.6, 7.4). Enzymatic stability was tested in human plasma, in Caco-2 cell homogenates and monolayers and in rat liver. The compounds were relatively stable to chemical hydrolysis. Between 50 and 70% of unchanged prodrug was recovered after 16h incubation in human plasma, with no stereoselective preference for phosphate diastereoisomers. The p-OMe phenyl derivative, however, was an exception and only 5% of one diastereoisomer was recovered. In Caco-2 cells the stability and stereoselectivity largely depended on the experimental conditions: high enzymatic activity and stereoselectivity was observed in cell homogenates, but not in monolayers. In rat liver S9 fractions the stability profile was similar to that in Caco-2 cells and carboxyl ester cleavage appeared to be the sole mechanism of degradation in both media. The large and unpredictable differences in stereoselective metabolic rate of the pronucleotide series here presented suggest that in vivo circulating levels of intact prodrug could exert profoundly different activity or toxicity due to preferential body distribution of one diastereoisomeric form.
authors.affiliation: Pharmaceutical Cell Biology Group, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK.
authors.forename: Dario
authors.initials: D
authors.lastname: Siccardi
authors.forename: Mark
authors.initials: M
authors.lastname: Gumbleton
authors.forename: Yadollah
authors.initials: Y
authors.lastname: Omidi
authors.forename: Chistopher
authors.initials: C
authors.lastname: McGuigan
mesh_terms: D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D019380:Anti-HIV Agents / Q000097:blood / Q000737:chemistry*; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D010756:Phosphoric Acids / Q000737:chemistry* / Q000378:metabolism; D011355:Prodrugs / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018119:Stavudine / Q000097:blood* / Q000737:chemistry*; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D000577:Amides; D019380:Anti-HIV Agents; D004952:Esters; D010756:Phosphoric Acids; D011355:Prodrugs; C011067:phosphoramidic acid; D018119:Stavudine