Metabolism Of Emodin In Liver And Intestine And Its Gender-Dependent Differences

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is one of the major active ingredients of rhubarb(rheuw officinale B.), aloe(aloe barbadensis M.), and leaf of senna(cassia angustifolia) plants. Decade ago, emodin was extensively studied for its traditional pharmacological activities. However, recent studies have put emodin back into limelight with its anti-cancer activities in several types of cancer cells with apoptosis as a possible mechanism of action.Emodin also has an inhibitory effect on cancer cell migration and invasion in in vitro studies and that is why emodin has good prospect and promising future in the market. Even though emodin has so many excellent application prospects as a chemical, little is known about its pharmacokinetics and metabolism in vivo. These studies are very important for development of emodin into a new anti-cancer pharmaceutical compound.In modern era, more and more people, no matter men or women aspire to be fit and healthy. Therefore, in addition to the above mentioned effects, emodin's health food and slimming product intake has increased due to its weight loss property through its traditionally known stimulant laxative activity. However, again, no study has been done to show the gender differences in oral bioavailability of emodin in rodents as well as humans. Hence, we propose to study gender-dependent difference in absorption and metabolism of emodin as the main aim of our study. Toxic effects of emodin such as renal and liver toxicity have received significant attention from all over the world. Due to renal failure issues, number of weight loss products containing emodin was recalled back from the market by FDA. Recently the toxicity of emodin was assessed by National Toxicology Program. The acute and chronic toxicity of emodin is investigated in liver and kidney of rodents. This proposed study investigates the gender-dependent differences in absorption and disposition of emodin which might help in understanding the pharmacological mechanism, guiding drug design, improving efficacy and safety and development of scientific standards of the quality of emodin.The proposed study delineates gender-dependent differences in absorption and metabolism of emodin in different animal species using in vitro, in situ as well as in vivo study models. Our intentions were to put forth experimental data in different animal models to make an informative decision about the emodin dosage regimen in men and women. Our research has been divided into several parts as follows:1) to study stability and solubility of emodin in various buffer solutions; 2) to determine gender differences in absorption and disposition of emodin in SD rats using in vivo single dose (8mg/kg) oral and intravenous (4mg/kg) PK model; 3) to study gender difference in absorption and metabolism of emodin by using in situ four site rat intestinal perfusion model; 4) to determine the species and gender differences in glucuronidation of emodin by using male and female dog, guinea pig, mouse, rat and human liver microsomes; 5) to study gender-dependent and regioselective intestinal emodin glucuronidation difference by using female and male SD rat intestinal and liver microsome model; 6) to determine the major human uridine diphospho-glucuronosyl transferase (UGT) isoforms (s) responsible for glucuronidation of emodin using various human UGT isoforms available in the market. 1. Stability and solubility of emodinBefore conducting in vivo and in situ studies to understand the gender-dependent differences in bioavailability as well as absorption and disposition of emodin in rats, we needed clear understanding of stability and solubility of emodin. We determined emodin's stability and solubility by using UPLC method. Emodin stock solution was prepared in HPβCD solution to improve the solubility and stability of poorly soluble emodin in HBSS. We used emodin-HPβCD-HBSS (pH 7.4) as the perfusate, which was stable for 24hr under room temperature. Therefore, the perfusate was prepared fresh every time right before the start of the experiment. Emodin was stable in pH7.4～8.87, so Ph 7.4 HBSS buffer was used in intestinal perfusion model. Furthermore we also found out that not acidic but alkaline treatment was good for the stability of emodin in aqueous media.2. Pharmacokinetics of emodin in female and male SD ratsTo determine the plasma concentrations of emodin and its metabolites from in vivo PK plasma samples, we used LC/MS instrument. Emodin and emodin glucuronide showed a good linear plasma standard curve in the range of 0.01～20μg/ml. Pharmacokinetics studies to determine bioavailability of emodin were done using a single dose oral and intravenous administration in female and male SD rats. The oral bioavailability of emodin is extremely low whether in male rats (5%) and female rats (7.5%).Following a single intravenous injection of 4 mg/kg emodin, emodin was distributed rapidly and eliminated slowly. The results showed a good two-compartment model fit for emodin plasma concentration-time data for male and female SD rats. The t1/2αwere 13.26±6.28min (male rats) and 13.52±7.28min (female rats). The t1/2βwere 187.38±0.16min (male rats) and 118.5033±83.09min (female rats). Emodin showed significant gender differences in iv PK profiles with higher AUC values in male (430±160 mg*μg/ml) than female (283±98mg*μg/ml) SD rats (n=6). A little amount of emodin was detected using LC/MS after the oral administration of dose of 8mg/kg emodin. However, there was no good fit for any PK compartmental model for the plasma concentration-time data for single dose intravenous administration of emodin (8mg/kg). Analyzing the intravenous PK data using non-compartmental model, Cmax, Tmax and AUC0-∞, p.o of emodin in male rats were:0.31±0.094 wereμg/ml,18.0±6.71min and 65.76±34.77 mg*μg/ml respectively; whereas Cmax, Tmax and AUC0-∞,P.o of emodin in female rats were:0.039±0.011μg/ml, 18.75±7.51min and 33.82±4.09 mg*μg/ml respectively.Emodin was metabolized very fast after the intravenous administration, suggesting a good fit for single compartmental model for the plasma emodin metabolite concentrations. t1/2Ke were 167.4±50.9min(male rats) and 251.3±114min (female rats), the area under the curve (AUC0-∞,i.v) were 2210±980 mg*μg/ml and 1540±290 mg*μg/ml (female rats) (n=6).After oral administration of emodin, the metabolite of emodin appeared in the plasma rapidly. Non-compartmental model analysis was done for emodin and its metabolite plasma concentrations after single dose oral administration of emodin (20mg/kg). The parameters of emodin glucuronide were significant different with emodin, the Cmax, Tmax and AUC0-∞, p.o of emodin glucuronide in male rats were 6.69±1.058μg/ml,240min and 2261.89±655.87 mg*μg/ml respectively, in female rats, the Cmax, Tmax and AUC0-∞, P.o were 1.807±0.578μg/ml,60min and 458.5±373.39 mg*μg/ml respectively. The oral absolute bioavailability of emodin was less than 7.5% and 5% in male and female SD rats respectively. Emodin glucuronidation michaelis menten kinetics parameters showed significant gender-dependent differences.The pharmacokinetics results of emodin suggested emodin glucuronide as a major metabolite in vivo. Rapid glucuronidation with slow metabolite elimination was one of the reasons of low bioavailability of emodin. Our research had enriched the pharmacokinetics aspects of emodin and provided the information for clinical use of emodin.3. Gender-differences in intestinal absorption and disposition of emodin in ratsTo confirm the reason of low bioavailability of emodin, gender-dependent intestinal absorption and disposition of emodin were investigated in rats by using the in situ intestinal perfusion model. The perfusate was analyzed for emodin and emodin glucuronide. Absorption and glucuronide excretion of emodin displayed regioselectivity (p<0.05) in male rats. The rank order for amounts of absorption of emodin in male rats was duodenum≥jejunum>ileum>colon (p<0.05). On the contrary, in female rats, the amounts of absorption showed no regioselectivity (p>0.05). Furthermore, gender-dependent higher intestinal emodin absorption was observed in female than in male SD rats in all 4 regions of intestine (p<0.01).The amount of emodin glucuronide in duodenum was significant higher (p<0.05) than in jejunum, followed (p<0.05) by ileum and colon in male rats. In female rats group, the rank order of amount of metabolite excreted was jejunum≈duodenum> ileum>colon (p<0.05). In contrast to intestinal emodin absorption, emodin glucuronide excreted lower in all four regions of the male rat intestine than the female rat intestine (p<0.01). No biliary excretion of emodin or emodin glucuronide was detected in both male and female rats. The results suggested that not liver metabolism, but intestine metabolism, played an important role in disposition of emodin in rats.4. Species and gender-dependent hepatic glucuronidation of emodin by using liver microsomes of different animal species.To find out the main reason of low bioavailability of emodin, ten kinds of liver microsomal (male mouse, rat, guinea pig, dog, human and female mouse, rat, guinea pig, dog, human) were used, UGT metabolite formation was determined by LC/MS method. The results showed that emodin was glucuronidated fast in all ten kinds of liver microsomes.1H-NMR spectra of the metabolite displayed that the metabolite was emodin 3-O-β-D-glucuronide. The results also showed species-dependent different in glucuronidation rates of emodin among the five species (p<0.05). Significant gender difference in glucuronidation rates was shown in all test species. The results also indicated that metabolism of emodin was saturable at higher concentrations. Among the five species of males, guinea pig and human followed simple Michaelis-Menten kinetics whereas mouse and dog followed a biaphasic kinetics, and only rat followed autoactivation kinetics. Among the five species, mouse, rat, guinea pig and human all followed simple Michaelis-Menten kinetics whereas dog followed Autoactivation kinetics. In liver microsomes, the glucuronidation rates were faster in the female rats, especially at lower concentration. In the liver of other 4 species, rates were faster in male mice, but were slower in female guinea pigs and dogs. There was very little difference in emodin glucuronidation rates between human male and female liver microsomes, although, hepatic rate of glucuronidation showed significant gender differences in certain species such as mice.Significant lower amount of hydroxyemodin was detected after phaseⅠmetabolism of emodin by LC/MS. There was no detectable PhaseⅠmetabolites but the glucuronide emodin in the mixed reaction system, which suggested that PhaseⅡmetabolism (glucuronidation) was major route of disposition of emodin in rats.The resulted indicated that glucuronidation was the major pathway for emodin metabolism which was significant gender and species dependent.5. Region and gender-dependent glucuronidation of emodin by intestinal microsomesWe measured glucuronidation rates of emodin in jejunal and ileal microsomes of male and female rats, using by UPLC-MS/MS method. The result showed that emodin was glucuronidated faster in rat jejunal microsomes than in ileal microsomes regardless of gender (p<0.05). Furthermore, emodin was metabolized faster in male than in female rats at all tested concentrations (p<0.01). Emodin glucuronidation in jejunal microsomes showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed autoactivation kinetics. In female rat intestine, glucuronidation in jejunal microsomes also showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed biaphasic kinetics.The results indicated that glucuronidation of emodin showed significant gender and regioselective intestinal difference.6. Fingerprinting of UGT glucuronidation of emodin by 12 expressed human UGTs isoforms.To identify the main UGT isoform responsible for metabolism of emodin, we used 12 expressed human UGTs isoforms. Except UGT1A4 and UGT2B4, the other 10 UGTs metabolized emodin at different rates. The average glucuronidation rate of emodin in UGT2A familiy was faster than UGT2B familiy. Amongst the 10 UGT enzymes isoforms, glucuronidation of emodin by UGT 1A9, UGT1A10 and UGT2B15 followed classical Michaelis-menten kinetics, while others followed autoactivation kinetics, and UGT1A3 didn't fit any pattern.In the UGTs study, the four major isoforms, responsible were UGT1A1,1A8, 1A9 and especially 1A10. Qualitative and quantitative expression of these UGT isoforms in two main metabolic organ, liver and intestine were quite different. UGT1A1 was expressed in all organs, although its expression was higher in the liver than in intestine. UGT1A9 was only expressed in liver whereas UGT1A8 and UGT1A10 were predominantly expressed in the small intestine.In summary, systemic metabolic characterization study suggested rapid metabolite (glucuronide) formation might be the major reason for emodin's low bioavailability in rats. The elimination of emodin and emodin glucuronide might follow UGT enzyme. In liver microsomes, the gender-dependent metabolism of emodin was also species-dependent. In intestinal microsomes, the gender-dependent metabolism of emodin also showed regioselectivity. Therefore, informative conclusion can be drawn from our results about glucuronidation of emodin in humans. Based on in vivo, in situ and invitro studies of emodin, we predicted that the main organ of metabolism for emodin to be intestine.