Exploring The Elimination Pathways(Metabolism And Excretion) Of Gossypol In Vivo And In Vitro

ObjectivesThe natural polyphenol gossypol possesses many therapeutic benefits. In recent years, the antitumor properties are widely researched by scholars. As further comprehensive in-depth of gossypol anti-tumor mechanism, gossypol might be a promising antitumor drug. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro(metabolism and excretion), which should facilitate drug development of this promising candidate for treating cancers. Methods and Results 1. Metabolite identification in rat plasma, urine, and fecesMetabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis(a data mining tool for detecting both expected and unexpected metabolites). On the basis of Metabolynx analysis, only one metabolite G3 was found in rat feces, whereas none was detectable in plasma and urine. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. 2. Pharmacokinetic studies in rats with bile duct cannulation.In bile duct cannulated rats, gossypol and three glucuronides were found significantly in bile under either low or high dose. At low dose, the mean values of biliary excretion within 8 h were 0.05 μmol/kg,0.03 μmol/kg,2.92 μmol/kg and 0.18 μmol/kg for G1, G2, G3 and gossypol, respectively. The percent of glucuronidated and excreted gossypol were 83.2 ± 29.1% and 5.0 ± 1.1%, respectively. At high dose, the mean values of biliary excretion within 8 h were 0.29 μmol/kg,3.32 μmol/kg,1.30 μmol/kg and 0.27 μmol/kg for G1, G2, G3 and gossypol respectively. The percent of glucuronidated and the excreted gossypol were 58.8 ± 17.4% and 20 ± 13% respectively. The results indicated that hepatic glucuronidation followed by biliary excretion of glucuronides was the predominant mechanism for gossypol clearance. 3. Transport and metabolism experiments in perfused rat intestine model.In the perfused rat intestine model, gossypol was well absorbed with a P*eff(the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides(G1, G2 and G3) were excreted into the gut lumen(2.5%) and into the bile(4.8%). Biliary excretion of unchanged gossypol(6.0%) was comparable to that of glucuronides,indicating that excretion of unchanged gossypol and glucuronides into bile were important clearance mechanism. 4. Microsomal glucuronidation of gossypol(RLM, RIM, HLM, HIM).In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes. The rat and human microsomes consistently generated three glucuronide isomers(G1, G2, and G3) from gossypol. The efficiency of glucuronide formation varied with the enzyme source for different isomers. The relative catalytic efficiency was 14:15:1(G3:G2:G1) for RLM, 7:11:1 for RIM, 1:1.1:1 for HLM and 1.5:2.9:1 for HIM. Of note, G2 or G3 was generated at a much higher efficiency(>seven-fold) than G1 in rat microsomes. However, this large discrepancy was not observed in human microsomes. Kinetic profiles were modeled using Michaelis-Menten equation(RLM, RIM) or substrate inhibition equation(HLM, HIM). The Km values for microsomal glucuronidation of gossypol ranged 0.64-5.6 μM, indicating that gossypol was a high affinity substrate for the UGT enzymes. 5. Reaction phenotyping of gossypol using recombinant human UGT enzymes.Reaction phenotyping using available UGT isoforms(UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17) were performed to identify the main UGT enzymes contributing to gossypol metabolism. Reaction phenotyping showed that multiple UGT1 A enzymes(including UGT1A1, 1A3, 1A7 and 1A8) were mainly responsible for gossypol metabolism. ConclusionIn conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism. Gossypol was well absorbed in the perfused rat intestine model and a high-affinity substrate for the UGT enzymes. Reaction phenotyping showed that multiple UGT1 A enzymes(including UGT1A1, 1A3, 1A7 and 1A8) were mainly responsible for gossypol metabolism.