Graduate Student Texas Southern University Houston, Texas, United States
Purpose: Gastrointestinal side effect is a serious concern of mycophenolic acid (MPA), an active metabolite of the prodrug mycophenolic mofetil (MMF) that is used as an immunosuppressive agent in clinical settings. Upon oral administration, MMF undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, acyl mycophenolic acid glucuronide (AcMPAG) and mycophenolic acid glucuronide (MPAG). Several studies have showed that decreasing the intestinal and colonic exposure of MPA can mitigate MMF-induced diarrhea. The purpose of this study is to evaluate the gender differences in MPA tissue drug exposure in rats using a sensitive and robust validated LC-MS/MS method to quantitate MMF and its metabolites; MPA, MPAG and AcMPAG. Methods: MMF (60 mg/kg, p.o. once daily for 5 days) was administered to both male and female F344 rats for pharmacokinetic (PK), tissue biodistribution studies and fecal condition monitoring. Plasma PK profiles and tissue drug distribution were determined after day three and six, respectively. On day six, the rats were sacrificed and tissues were collected (liver, small intestine (SI), colon and kidney) and washed in saline, then stored in -80 °C until when used. Thereafter, the collected plasma and tissue was processed and quantified using the validated LC-MS/MS method. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Results: The method has been shown to be reproducible, with intra- and inter-day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Significant differences in MMF-induced diarrhea severity were observed with the female rats as they experienced greater GI side effects than the male rats, which was measured by weight loss and diarrhea score. Following the PK profile of MPA and MPAG, the concentration of MPA and MPAG on day 6 was higher than on day 3, and MPA was shown to undergo enterohepatic recycling as seen with double peaks in the plasma concentration-time plot. Also, the tissue drug concentration for both male and female genders were quantified for MPAG, AcMPAG, MPA and MMF respectively. The concentrations shown to be in these sequence for the different compounds, MPAG; SI > colon > kidney > liver; AcMPAG; SI > colon > liver > kidney; MPA and MMF; SI > colon, respectively. In the female rat, there was a 4-fold increase of MPA drug exposure in the small intestine and colon as compared to the male rat and this could be attributed to increased enterohepatic recycling and gut efflux transporter functionality. Conclusion: This novel LC-MS/MS method comprehensively quantified the concentrations of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. These findings suggest that MPA exposure in the intestinal region is gender-dependent as it could be affected by intestinal UGT activities and blood esterase activities, correlating with the severity of MMF-induced diarrhea. Modulating intestinal MPA concentration and gut UGT expression should be the focus of future studies to alleviate MMF-induced diarrhea.
Acknowledgements: This work was supported by a grant from the Cancer Prevention Research Institute of Texas (CPRIT, RP190672) and National Institute of General Medical Sciences (1R15GM126475-01A1) for Song Gao.
MPA drug concentration in small intestine and colon of F344 rats.
MPA Plasma Concentration-time profile showing day 3 and 6
MMF, MPA, MPAG and AcMPAG Accuracy and Precision data
