The Synthesis Of (5R)-5-hydroxytriptolide By Microbial Transformation And Its Clinical Pharmacokinetic Study

BACKGROUND (5R)-5-Hydroxytriptolide, a semisynthetic structural analog of triptolide, has potency in vitro and in vivo anti-inflammatory and immunosuppressive activities. The compound is undergoing Phase I clinical trials.(5R)-5-Hydroxytriptolide is synthesized by two-step chemical reactions of triptonide with a yield of31%.1. Biotransformation of triptolide to (5R)-5-hydroxytriptolideOBJECTIVE To synthesize (5R)-5-hydroxytriptolide by microbial transformation of triptolide.METHOD Among10strains of Cunninghamella screened, Cunninghamella elegans ATCC36112was selected as a suitable bioconverter. To calculate the transformation yield, the concentration of (5R)-5-hydroxytriptolide in the fermentation fluid was determined by HPLC. Factors influencing biotransformation system were optimized to increase the yield of (5R)-5-hydroxytriptolide.RESULTS The optimized conditions were pH4.5,400μg·mL-1of substrate concentration and10d of transformation. A yield of>70%was achieved.CONCLUSION Biotransformation of triptolide to (5R)-5-hydroxytriptolide by Cunninghamella elegans ATCC36112was proved to be a new approach for the synthesis of (5R)-5-hydroxytriptolide.2. Quantitative analysis of (5R)-5-hydroxytriptolide in human urine and the applications of LC-MS/MS in Phase I pharmacokinetic studiesOBJECTIVE To develop and validate a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of (5R)-5-hydroxytriptolide in human urine. The validated method of determination of (5R)-5-hydroxytriptolide in human plasma and urine was applied to Phase I pharmacokinetic studies.METHOD The method was based on chemical derivatization from benzylamine. For quantification in human plasma, urine and feces,(5R)-5-hydroxytriptolide and the internal standard (triptolide) were extracted with ether-dichlormethane (3:2, v/v) and then derivatized with benzylamine for1h at80℃. The analytes were separated on a Gemini C18(50mm×2.0mm,5μm) column, using a gradient elution program with a solvent consisting of acetonitrile and0.77μM ammonnium hydroxide (pH10.0). An API4000tandem mass spectrometer equipped with an electrospray ionization source and operated in positive ion mode was used as detector. The mass transition ion-pairs were followed as m/z484.5â†'192for (5R)-5-hydroxytriptolide derivative and m/z468.5â†'192for the IS derivative.RESULTS The linear calibration curves for the determination of (5R)-5-hydroxytriptolide in human urine were obtained in the concentration range of0.030-100ng-mL"1. The lower limit of quantification was0.030 ng·mL-1. The intra-day relative standard deviation over the entire concentration range was less than9.4%. The accuracy was in the range of3.5%to2.8%in terms of relative error. Each sample was chromatographed within6.5min. There was a good correlation between AUC, Cmax and dose after a single oral administration of0.25-2mg (5R)-5-hydroxytriptolide tablets. The accumulated excretion in urine was less than11%in every patient for each dose level. The food-effect study compared the effect in fed state to the effect in a fasted state on the absorption of a single dose of (5R)-5-hydroxytriptolide. The meal increased the extent of absorption relative to the fasted state in the terms of Cmax and AUC. Other parameters were no relevance to the meal and the order of drug administration. The multiple administration of (5R)-5-hydroxy-triptolide indicated similar systemic exposure to the single dose.CONCLUSION The quantitative determination by liquid chromatography coupled with tandem mass spectrometric analysis in human plasma and urine yield an accurate and sensitive method for the quantification of (5R)-5-hydroxytriptolide. The method was suitable for Phase I clinical pharmacokinetic studies.