OBJECTIVE: The aims of this investigation were to study the glucuronidation of mycophenolic acid (MPA) in human liver and kidney and to search for a compound that inhibits MPA glucuronidation among the non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: A sensitive and reproducible radiometric assay was developed to measure the rate of MPA glucuronidation in human liver and kidney microsomes. The assay employed uridine 5'-diphosphate-[U-14C]-glucuronic acid (UDPGA) and MPA-glucuronide was isolated by TLC. The final concentrations of UDPGA and MPA necessary were 1 mM (liver), and MPA concentration was 0.5 mM (kidney). The inhibition of MPA glucuronidation was studied with 18 NSAIDs and tacrolimus. RESULTS: Glucuronosyl transferase activity followed Michaelis-Menten kinetics and the Km (mean +/- SD; mM) was 0.31+/-0.06 (liver; n = 5) and 0.28+/-0.07 (kidney; n = 5; P = 0.555); the Vmax (mean SD; nmol/mg per minute) was 5.2+/-1.4 (liver; n = 5) and 10.5+/-1.2 (kidney; n = 5; P = 0.0005). The MPA glucuronidation rates (mean +/- SD; nmol/min/mg) were 3.3+/-0.9 (liver; n = 10) and 7.8+/-1.5 (kidney; n = 10; P = 0.0002). The rate of MPA glucuronidation ranged between 2.0 and 5.1 nmol/ mg per minute with a 2.5-fold variation (liver) and between 5.7 and 9.8 nmol/mg per minute with a 1.7-fold variation (kidney). The inhibition study was performed in liver and revealed that the percentage of control ranged from 8%+/-3% (niflumic acid) to 119%+/-16% (Ketoralac). The inhibition curves for MPA glucuronidation rate were determined with the four most effective inhibitors: niflumic acid, flufenamic acid, mefenamic acid and diflunisal. Their IC50 estimates (microM) were 8+/-1, 19+/-9, 63+/-8 and 109+/-15, respectively (liver), and 8+/-2, 13+/-2, 49+/-4 and 122+/-18, respectively (kidney). The IC50 estimate for niflumic acid was eightfold lower than the peak plasma levels after a single oral dose of 250 mg of this drug. CONCLUSION: The human liver and kidney are important sites of MPA glucuronidation. MPA glucuronidation was inhibited to various extents by different NSAIDs and the four most effective inhibitors were niflumic acid, flufenamic acid, mefenamic acid and diflunisal. These drugs have similar molecular structures consisting of two aromatic rings bearing a carboxylic group.
OBJECTIVE: The aims of this investigation were to study the glucuronidation of mycophenolic acid (MPA) in human liver and kidney and to search for a compound that inhibits MPA glucuronidation among the non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: A sensitive and reproducible radiometric assay was developed to measure the rate of MPA glucuronidation in human liver and kidney microsomes. The assay employed uridine 5'-diphosphate-[U-14C]-glucuronic acid (UDPGA) and MPA-glucuronide was isolated by TLC. The final concentrations of UDPGA and MPA necessary were 1 mM (liver), and MPA concentration was 0.5 mM (kidney). The inhibition of MPA glucuronidation was studied with 18 NSAIDs and tacrolimus. RESULTS: Glucuronosyl transferase activity followed Michaelis-Menten kinetics and the Km (mean +/- SD; mM) was 0.31+/-0.06 (liver; n = 5) and 0.28+/-0.07 (kidney; n = 5; P = 0.555); the Vmax (mean SD; nmol/mg per minute) was 5.2+/-1.4 (liver; n = 5) and 10.5+/-1.2 (kidney; n = 5; P = 0.0005). The MPA glucuronidation rates (mean +/- SD; nmol/min/mg) were 3.3+/-0.9 (liver; n = 10) and 7.8+/-1.5 (kidney; n = 10; P = 0.0002). The rate of MPA glucuronidation ranged between 2.0 and 5.1 nmol/ mg per minute with a 2.5-fold variation (liver) and between 5.7 and 9.8 nmol/mg per minute with a 1.7-fold variation (kidney). The inhibition study was performed in liver and revealed that the percentage of control ranged from 8%+/-3% (niflumic acid) to 119%+/-16% (Ketoralac). The inhibition curves for MPA glucuronidation rate were determined with the four most effective inhibitors: niflumic acid, flufenamic acid, mefenamic acid and diflunisal. Their IC50 estimates (microM) were 8+/-1, 19+/-9, 63+/-8 and 109+/-15, respectively (liver), and 8+/-2, 13+/-2, 49+/-4 and 122+/-18, respectively (kidney). The IC50 estimate for niflumic acid was eightfold lower than the peak plasma levels after a single oral dose of 250 mg of this drug. CONCLUSION: The human liver and kidney are important sites of MPA glucuronidation. MPA glucuronidation was inhibited to various extents by different NSAIDs and the four most effective inhibitors were niflumic acid, flufenamic acid, mefenamic acid and diflunisal. These drugs have similar molecular structures consisting of two aromatic rings bearing a carboxylic group.