Development of a novel UPLC-MS/MS method for the simultaneous quantification of mycophenolic mofetil, mycophenolic acid, and its major metabolites: Application to pharmacokinetic and tissue distribution study in rats.

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) used to prevent rejection in organ transplant patients. The purpose of this study is to develop a sensitive LC-MS/MS method to simultaneously quantify MMF, MPA, and two major metabolites, mycophenolic acid-glucuronide (MPAG) and Acyl-mycophenolic acid-glucuronide (AcMPAG) and applied this method in a pharmacokinetic (PK) and tissue distribution study. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Protein precipitation with a mixture of methanol: acetonitrile (2:1, v:v) was used to process the plasma samples and tissue samples. Separation was achieved using an Ultra Biphenyl 5 µm column (100 × 2.1 mm) with 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases. Quantification analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. The method was linear in the range of 1.22 - 1250.00 nM for all four analytes with correlation coefficient values > 0.99. The method was reproducible, with intra- and inter-day accuracy ranging from 85.0 ± 11.2-108.3 ± 6.50 for all analytes in both plasma, liver and intestine homogenates. The extraction recovery and matrix effect of plasma sample using a mixture methanol/acetonitrile (2:1, V:V) can achieve an acceptable range (<20%), but extraction recovery and matrix effect of AcMPAG decreased to 64.10 ± 15.42 in the liver and intestine homogenates. The analytes in plasma were found to be stable under bench-top, freeze-thaw, and storage conditions. The validated method was successfully applied to quantify MMF, MPA, MPAG, and AcMPAG in a rat PK study. The PK results showed MPA was the major form exposed in the plasma in rats after oral administration of MMF, but the major metabolites in the rat's tissue disposition were MPAG.

Development of a novel UPLC-MS/MS method for the simultaneously quantification of polydatin and resveratrol in plasma: Application to a pharmacokinetic study in rats.

The purpose of this study is to develop a sensitive LC-MS-MS method to simultaneously quantify polydatin and its metabolite, resveratrol, for its application in a pharmacokinetic (PK) study and to determine polydatin hydrolysis by microflora. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Acquity BEH C18 column (2.1 × 50 mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under negative ionization mode using the multiple reaction monitoring (MRM) approach. The method was linear in the range of 9.77-1250 nM for both resveratrol and polydatin with correlation coefficient values >0.99. Themethodhas been shown to be reproducible, with intra- and inter-day accuracy and precision ±10.4% of nominal values, for both analytes. The average extraction recovery rates were 81.78-98.3% for polydatin and 86.4-103.2% for resveratrol, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench-top, freeze-thaw, and storage (-4 °C) conditions. The metabolic studies showed that polydatin can be rapidly hydrolyzed by rat fecal S9 fractions and PK studies showed that both polydatin and resveratrol were exposed in the plasma and variable tissues. This novel UPLC-MS-MS method can quantify the levels of both polydatin and its major metabolite resveratrol in biological samples.

Glucuronides Hydrolysis by Intestinal Microbial ß-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species.

Glucuronides hydrolysis by intestinal microbial ß-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl ß-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69-1.32-fold, 2.9-12.9-fold, and 0.28-1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 µmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 µM for mouse, rat, and human, respectively. The inter-individual difference was significant (4-6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant.

Developing nutritional component chrysin as a therapeutic agent: Bioavailability and pharmacokinetics consideration, and ADME mechanisms.

Chrysin is a promising naturally occurring flavonoid mainly found in honey and propolis. Although chrysin's biological activities have been demonstrated and the mechanism of actions has been determined using in vitro and in vivo models, results from the current clinical studies were largely negative. A potential reason for chrysin's low efficacy in humans is poor oral bioavailability. In this paper, we reviewed the preclinical and clinical pharmacokinetics studies of chrysin and analyzed the mechanism of poor in vivo efficacy with emphasis on its bioavailability and ADME mechanism. Low aqueous solubility, rapid metabolism mediated by UGTs and SULT, efficient excretion through efflux transporters including BCRP and MRP2 are the major reasons causing poor systemic bioavailability for chrysin. However, because of efficient enterohepatic recycling facilitated by phase II metabolism and efflux, chrysin's bioavailability in the low GI tract is high. Thus, chrysin can be ideal for treating diseases in the terminal ileum and colon (e.g., carcinoma, local infection) since it is localized in the lower GI tract with limited delivery to other organs.

Age-and Region-Dependent Disposition of Raloxifene in Rats.

PURPOSE: Raloxifene undergoes extensive glucuronidation in the gastrointestinal (GI) tract and the liver. However, the impact of age on raloxifene disposition has never been studied. The purpose of this paper is to determine glucuronidation and Pharmacokinetics (PK) profiles of raloxifene in rats at different ages. METHODS: Raloxifene glucuronidation was characterized using S9 fractions prepared from different intestinal segments and the liver of F344 rats at 4-, 11-, and 28-week. PK studies were conducted to determine raloxifene oral bioavailability at different ages. Raloxifene and its glucuronides were quantified using LC-MS/MS. RESULTS: Raloxifene-6-glucuronide and raloxifene-4'-glucuronide were detected as the major metabolites and the ratio of these two glucuronides were different ranging from 2.1 to 4.9 folds in the ileum, jejunum, liver, and duodenum, and from 14.5 to 50 folds in the colon. The clearances in the duodenum at 4-week for both two glucuronides were significantly lower than those at the other two ages. PK studies showed that the oral bioavailability of raloxifene is age dependent. The absolute oral bioavailability of raloxifene was 3.5-folds higher at 4-week compared to that at 11-weeks. When raloxifene was administered through IV bolus, its half-life was 5.9 ± 1.16 h and 3.7 ± 0.68 h at 11-and 4-week, respectively. CONCLUSION: These findings suggested that raloxifene metabolism in the duodenum was significantly slower at young age in rats, which increased the oral bioavailability of raloxifene. At 11-week, enterohepatic recycling efficiency was higher than that of 4-week. Raloxifene's dose at different ages should be carefully considered.