Quality Control And Pharmacokinetic Study Of Stavudine Preparations

Stavudine, a nucleoside analogue of thymidine, is phosphorylated by cellular kinases to the active metabolite, stavudine triphosphate, which has shown powerful activity against human immunodeficiency virus（HIV）. The quality of stavudine powder and stavudine sustained-release tablet has been comprehensively investigated. Firstly, an HPLC method was developed for the determination of stavudine content and related substance, and a UV spectrophotometry method for the determination of sustained-release tablet’s drug release. Secondly, a stability test was carried out for both formulations using the developed methods. Thirdly, a reliable reversed-phase isocratic HPLC method with UV detection for the determination of stavudine in dog plasma was developed and applied to the pharmacokinetic study of stavudine in dog after administration on stavudine powder.1. The content and related substance determination of stavudine preparationsAn HPLC method was developed for the content and related substance determination of stavudine powder and stavudine sustained-release tablet. The separation was performed on a Diamonsil C₁₈ column. The mobile phase was composed of 25mmol·L^-1 ammonium acetate solution-methanol（80:20, v/v） with flow rate of 1.0mL·min^-1. The detection was set at 266nm and column temperature at ambient temperature. The method was accurate and the average recoveries of stavudine powder and stavudine sustained-release tablet were 99.7% and 99.6% with RSDs of 0.53% and 0.47%（n=9）. The precisions of content determination of stavudine perparations were 99.9% and 100.1% with RSDs of 1.03% and 0.88%（n=6）. The linear range for stavudine was 5.0～50.0μg·mL^-1（Y=0. 999 9）. The robustness of the method was demonstrated by little variations in the mobile phase composition, the columns and the flow rate. The specificity test demonstrated that the related substances and degradation products of stavudine and excipients were completely separated from stavudine and had no influence on the determination. The lower limit of detection of stavudine was 0.02μg·mL^-1. 2. The determination of drug release of stavudine sustained-release tabletA UV spectrophotometry was used for determining the drug release of stavudine sustained-release tablet. At the maximum absorption wave length（266nm） of stavudine, the excipients had no influence on the determination. The sustained-release properties of stavudine sustained-release tablet were investigated in water, 0.1mol·L^-1 hydrochloride solution and phosphate buffer solution（pH6.8） with the methodⅠof drug release ditermination. In the three dissolution mediums, the average recoveries were 99.4%（n=12）, 99.7%（n=6） and 99.9%（n=6） with RSDs of 0.32%, 0.45% and 0.47%, respectively. The linear range for stavudine was 1.0～12.0μg·mL^-1（γ=0. 999 9）. The release curves demonstrated that stavudine sustained-release tablet had good sustained-release properties and its uniformity confirmed the requirement in the Guideline for new drug development.3. The stability study on stavudine preparationsThe stress test, accelerating test and long-term stability test were carried out by using the developed methods according to Guideline of study on stability of new drug. The results of stress test demonstrated that humidity and temperature had effects on the stability of stavudine preparations. While light had no effect on the stability of stavudine preparations. Both accelerated test and long-term test demonstrated that stavudine powders and stavudine sustained-release tablets were stable under the packaging and storage conditions.4. Determination of stavudine in dog plasmaAn HPLC method with UV detection（266nm） was developed and validated for the determination of stavudine in dog plasma. Stavudine and the internal standard（dehydrate-thymidine） were extracted into ethyl acetate and separated using an isocratic mobile phase of methanol-water（20:80, v/v） on a Diamonsil C₁₈ column. The retention times for stavudine and internal standard were 9.1 and 12.3 min, respectively. No endogenous interferences were observed. This HPLC method was fully validated. The lower limit of quantification was 0.02μg·mL^-1. A linear range of 0.02～5.0μg·mL^-1（γ=0.998 9） was established. The interday and intraday RSDs were within 12.5～3.7% and 7.6～3.4%, respectively. This method developed was easily applied to the pharmacokinetic study of stavudine in dog plasma after i.g. administration stavudine powder. The pharmacodinetic parameters: C_max of 2.65±0.31μg·mL^-1, T_max of 0.58±0.13h, T_1/2 of 1.55±0.16h and AUC_0-t of 6.19±0.35μg·h·mL^-1 were observed.