Study On Pharmacokinetics And Chemical Stability Of Anti-HIV Nucleoside Prodrugs

Acquired immunodeficiency syndrome (AIDS), caused by the humanimmunodeficiency virus (HIV), has become a global epidemic. Although themorbidity and mortality of AIDS patients have been reduced and the survival time hasbeen prolonged, the present treatments would not eradicate HIV and cure AIDS.NRTIs/NtRTIs (nucleoside/nucleotide reverse transcriptase inhibitors) havedemonstrated wide-spread utility as antiviral and anti-cancer therapeutics. However,the clinical application of AZT, d4T is limited by their bone marrow toxicity andsuppression, low therapeutic index, low localization in brain, and a short half-life inblood. Thus, in an attempt to reduce or abolish its side-effects, to improve on thetherapeutic potential, research has focused on the discovery of pro-drugs.Administration of the nucleoside 5'-phosphates plays an important role in pro-drugapproaches. Unfortunately, the effects of pronucleotide design on the mechanism ofnucleotide release have only been systematically studied in a few cases.Therefore, in the present dissertation, the in vivo potency, plasma pharmacokineticshave been determined with an original combination of organic chemistry, analyticalchemistry and preclinical in vivo pharmacokinetics, for the candidate compounds thatdemonstrated great in vitro anti-HIV activity.In the present dissertation, research on hydrolytic stability and analysis ofhydrolysis metabolites of a highly successful candidate, d4T-D-E, were developed byvirtue of modern analytical approaches, such as HPLC, ³¹P NMR, HPLC/MS,ESI-MSⁿ. It was originally found that d4T-D-E kept the chemical structure underweak acidic condition, had a half-life time 12.5 hours in neutral pH 7.23, anddecomposed completely under alkaline or strong acidic condition. Three possiblehydrolysis pathways were as follows: D group was cleaved accompanied by phosphiteelimination giving the nucleoside d4T, or nucleoside d4T was cleaved directlyaccompanied by phosphite elimination, or d4T-D-E was oxidated, then accompaniedby phosphate elimination giving the nucleoside d4T. A rapid, sensitive HPLC/ESI-MS/MS method using multiple reaction monitoring(MRM) was originally built to determine the plasma concentration of d4T, d4T-D-E.The method was validated and the specificity, linearity, lower limit of quantification(LLOQ), precision, accuracy, recoveries and stability were determined. This validatedmethod was employed to the pharmacokinetic studies of d4T, d4T-D-E.It was shown that d4T-D-E was cleaved to d4T promptly in plasma after oraladministration to mice, which correlates to the structural friability to endogenousalkaline phosphatase. Compared to d4T, pharmacokinetics of d4T-D-E presented ashorter elimination t_1/2, a low relative bioavailability 40.31%.The candidatecompound was short of clinical potency due to poor pharmacokinetics in spite ofnotable in vitro activity.In the present dissertation, six phosphorous-containing derivatives of AZT weresynthesized with one-pot procedure.A rapid, robust, simple HPLC/ESI-MS/MS method using multiple reactionmonitoring (MRM) was originally built to determine the plasma concentration of the6 pro-drugs of AZT. The method was validated and the specificity, linearity, lowerlimit of quantification (LLOQ), precision, accuracy, recoveries and stability weredetermined. This validated method was employed to the pharmacokinetic studies ofthe 6 pro-drugs.The overall pharmacokinetics of six derivatives of AZT in rats were originallyperformed, and it was found to associate with structure of the compounds, themagnitude of which varied considerably with the nature of the Y substituent. Inparticular, strongly electron-withdrawing substituents correlate with their completeconversion to AZT in rat plasma.In the present dissertation, it was originally brought forward that the overallpharmacokinetics of 6 pro-drugs improved greatly compared to AZT. The pro-drugsof AZT proved the tendency of sustained release of AZT, longer residence time invivo and increased plasma concentration of AZT. The 6 pro-drugs exhibited successin vivo potency.