Study Of Metabolic Activation Of Rhein And Interaction With Organic Anion Transporters

Rhein,a major bioactive compound of many medicinal herbs and the prodrug of diacerein,is often used with low dose of methotrexate as drug combination to treat rheumatoid arthritis.Despite wide use,intoxication cases associated with rhein-containing herbs are often reported.The present work aimed to investigate if rhein was subject to metabolic activation leading to toxicity and potential drug-drug interaction between methotrexate and rhein based on organic anion transporters（OATs）.Upon incubations with different species of liver microsomes,three monoglucuronides（RG1,RG2 and RG3）were identified,corresponding to two hydroxyl glucuronides and one acyl glucuronide via the carboxyl group,respectively.Further study revealed that rhein acyl glucuronide（RG3）was chemically reactive,and showed cytotoxicity toward hepatocarcinoma cells.In addition,significant species differences in glucuronidation of rhein were observed between humans and laboratory animals.All of RG1,RG2 and RG3formations by human liver microsomes（HLMs）and uridine 5’-diphospho-glucuronosyltransferases（UGTs）followed typical Michaelis-Menten kinetics.HLMs exhibited the highest V_max for the formation of RG3 among the three glucuronides,but similar K_m for the formation of RG1,RG2,and RG3（77.37±9.98,52.20±5.96,and 49.68±3.85μM for RG1,RG2,and RG3,respectively）.As such,the apparent intrinsic clearance of rhein in HLMs through RG3 was much higher than that through RG1 and RG2.Reaction phenotyping experiments demonstrated that RG3 was catalyzed predominantly by UGT1A1,1A9,and 2B7.UGT1A1,1A7,1A8,and 1A9 showed activity toward RG2formation,and UGT1A1,1A7,1A8,1A9 and 1A10 catalyzed RG1 formation.UGT1A1displayed the highest capacity in converting rhein to RG3 with the lowest K_m（14.81±2.29μM）and the highest V_max,followed by 1A9（K_m 20.86±2.10μM）and 2B7（K_m 66.77±8.73μM）.Further study demonstrated that RG3,the major metabolite of rhein in the human blood circulation,significantly inhibited the uptake of p-aminohippurate in hOAT1transfected cells with IC₅₀ value of 691.40±1.08 nM and estrone sulfate uptake in hOAT3transfected cells with IC₅₀ value of 78.46±1.30 nM.As the substrate of both hOAT1 and hOAT3,the methotrexate transport was significantly inhibited by RG3 in hOAT1transfected cells at 50μM and hOAT3 transfected cells at 1μM by 69%and 87%,respectively.Further in vivo study showed that after co-administrated with RG3 in rats the AUC_0-24 values of methotrexate increased from 3108.9±304.5 to 5370.6±723.7 ng/mL*h and the t_1/2 was prolonged by 40.5%（from 7.4±2.1 h to 10.4±6.9 h）,demonstrating the inhibitory effect of RG3 on methotrexate excretion.Taken together,the present study confirmed that rhein could be metabolically activated via the formation of acyl glucuronide,especially in human.And rhein acyl glucuronide could significantly decrease the transport of methotrexate by both hOAT1 and hOAT3.The combination use of rhein,diacerein or other rhein-containing herbs with methotrexate may cause obvious drug-drug interaction and require close monitoring for potential drug interaction in clinical practice.