The Pharmacokinetics And Metabolism Of Matrine, Oxymatrine And Higenamine

Background and Objectives:It is well know that pharmacokinetic characteristics of traditional Chinese medicine can affect its toxicity and efficacy. Metabolic characterizations of traditional Chinese medicine are the most important aspect which can influence their pharmacokinetic behavior. Based on this, this study mainly chose oxymatrine, matrine (active ingredients in Radix Sophorae tonkinensis) and higenamine (active ingredient both in Aconite root and Asarum) as the main research objects.Radix Sophorae tonkinensis (S. tonkinensis) is the processed lateral root of Sophora subprostrata (Leguminosae) and is widely distributed over the southwest provinces of China. Radix S. tonkinensis has been used as a famous Chinese medicinal herb to treat jaundice, inflammation, and aches. Oxymatrine and matrine, the main effective and toxic ingredients that are well-known target markers of radix S. tonkinensis, have been found to have anti-inflammatory, antitumor and antipyretic functions as well as hepatoprotective effects. Radix S. tonkinensis extract has pharmacological activity, but it also causes side effects and liver toxicity. The therapeutic potential versus the toxic potential of OMT and MT has been extensively discussed in the literature for many years. Scientific research has demonstrated that OMT and MT are the foremost toxic alkaloids and the nervous system is the main target organ of the MT toxicity. The transformation of OMT to MT is the main reason for the low OMT concentration and high MT concentration in plasma. This transformation can affect the toxicity and efficacy of both OMT and MT. Therefore, determining the safe application of S. tonkinensis extract is urgent and essential. The first stage requires an in-depth understanding of its pharmacokinetic (PK) properties.Further more, oxymatrine metabolism was also studied to explain the different PK properties of S. tonkinensis and oxymatrine.Higenamine, an active ingredient of Aconite root and Asarum, has been traditionally used as cardiac inotropic and anti-inflammatory agents. Higenamine is now in the phase II clinical trial in China. However, there are significant challenges associated with the drug safety evaluation of higenamine. One of the most important reason was, there lacks the information of the higenamine metabolic characteristics, which could directly affect the pharmacokinetics and pharmacodynamics of higenamine. It was reported that urinary excretion of conjugated metabolites of higenamine was about24%～33%after IV bolus administration in rabbits. It may be the reason resulting in low bioavailability in rabbits. So we focus on the glucuronidation of higenamine in our study.In general, the aims of this thesis are (1) to investigate the PK behaviors of OMT and MT following oral administrations of pure OMT and raw S. tonkinensis extracts to compare the PK characteristics between pure OMT and S. tonkinensis extracts;(2) to look for the metabolic mechanism of OMT which affect the PK behaviors of OMT and raw S. tonkinensis extracts (3) to elucidate the metabolic mechanism of higenamine, looking for the reason of low bioavailability of higenamine and the effect of metabolic differences on pharmacokinetics(PK) in various species, which is helpful to the evaluations of drug therapy and clinical drug safety.Methods:The metabolic mechanism of oxymatrine was studied using CYP isoforms and chemical inhibitors in vitro and pharmacokinetics was evaluated in rats by UPLC-MS/MS. The metabolic mechanism of higenamine was studied using UGT isoforms and five kinds of liver microsomes in vitro. Statistical Analysis:All the data expressed as mean±SD. SPSS13.0for Windows was used as statistical software. One-way ANOVA with or without Tukey-Kramer multiple comparison and Student’s t tests were used to evaluate statistical difference.Results:1. Determination of oxymatrine and matrine in Radix Sophorae tonkinensis extractsA sensitive and reliable HPLC method was developed to detect the concentration of oxymatrine and matrine in Radix Sophorae tonkinensis extracts. The OMT and MT contents in the radix S. tonkinensis extract samples were0.60%and0.40%, respectively.2. Comparation of the PK behaviors of OMT and MT following oral administrations of pure OMT and raw extracts of S. tonkinensisA fast, sensitive and reliable UPLC-MS/MS method was set up to detect the concentration of oxymatrine and matrine in rats. Blood samples were taken from eyes. Compared with that in a single dose of pure OMT, t1/2had no significant variation, indicating fast absorption. Cmax, AUC0→0, and MRT0→t of OMT in rats after receiving doses of radix S. tonkinensis extract were significantly lower than those in the pure OMT group (t-test, p<0.05). MT was observed after administration of OMT. The absolute bioavailability values of OMT in rats after the oral administration of pure OMT and radix S. tonkinensis extract were6.79%and1.87%, respectively. However, the bioavailability of total alkaloids (OMT+MT) of pure OMT was81.14±8.83%and the bioavailability of total alkaloids (OMT+MT) of radix S. tonkinensis extract was69.36±17.37%:AUCCOMT+AUCMT following oral administration of radix S. tonkinensis extract3. Plasma protein binding of OMT and MTThe vitro plasma protein binding of OMT and MT was performed by ultrafiltration technique which was simple, low interference, time-saving. Protein binding of OMT and MT in plasma was very low (4.80%-8.95%for OMT,5.10～10.55%for MT), which is consistent with our pharmacokinetic study of OMT and MT that both of the two alkaloids were rapidly eliminated (MRTi.v,136.94±39.98min for OMT,260.32±45.88min for MT). 4. Comparation of oxymatrine metabolism in human liver and intestine microsomeThe kinetic characteristics of oxymatrine in human liver and intestine microsome were investigated. The glucuronidation rate and the clearance rate in liver are higher than that in intestine.5. Chemical inhibition experiments in human liver microsome (HLM)Chemical inhibitor for CYP was assayed for the metabolism of oxymatrine in human liver microsome. It showed that the inhibitor of CYP3A had a strong inhibitory effect; the inhibitors of CYP2C19had modest inhibitory effects; whereas inhibitors of CYP1A2, CYP2C8, CYP2E1, CYP2D6and CYP2C9had no obvious inhibitory effects on oxymatrine metabolism.6. Metabolism of oxymatrine by recombinant human cytochrome P450isoformsRecombinant human cytochrome P450isoforms were used to certify the major CYP isoform participating the metabolism of oxymatrine. The primary contributing CYP isoforms were CYP3A4, while CYP1A2,3A5,2C8,2C19,2B6,2E1,1BA,2D6,2A6and1Al played a minor role in the formation of matrine metabolites.7. The UGT-mediated metabolism of higenamine and the characterization of glucuronidation in UGT1A8and UGT1A9The commercial recombinant UGT isoforms1A1,1A3,1A4,1A6,1A7,1A8,1A9,1A10,2B4,2B7,2B15, and2B17were assayed for higenamine glucuronidation activity, in the presence of20,40and200μM concentration. The primary human UGTs responsible for the glucuronidation of higenamine are UGT1A6, UGT1A8, and UGT1A9. The order of glucuronidation rates:UGT1A9> UGT1A8> UGT1A6. UGT1A9is well expressed in the liver but not expressed or poorly expressed in the intestine. In contrast, UGT1A8are well expressed in the intestinal but not in the liver. However, expression of UGT1A6has been demonstrated in both human liver and intestine with large interindividual differences.We regarded liver and intestine as the main metabolic organs.We further investigated the kinetic characteristics of higenamine in UGT1A9and UGT1A6incubation systems. The kinetic profiles of UGT1A9followed autoactivation kinetics, while the kinetic profiles of UGT1A8followed Michaelis-Menten equation. The clearance rate in UGT1A9was higher than UGT1A8.8. Chemical inhibition experiments in recombinant UGT enzymes and HLMWe chose UGT1A9and UGT1A6as the targeted isoforms for the forward investigation of glucuronidation mechanism in human liver. To better understand the roles of UGT enzymes in hepatic higenamine glucuronidation, chemical inhibition studies with phenylbutazone and carvacrol were performed. The inhibition effects of phenylbutazone (UGT1A6inhibiter) and carvacrol (UGT1A9inhibiter) on higenamine glucuronidation in pooled HLMs and recombinant UGT enzymes were evaluated. A similar tendency was obtained in HLMs and the UGT1A9system using carvacrol to inhibit the activity of UGT1A9. However, phenylbutazone can not inhibited the metabolism of higenamine in HLM, which is opposite from the situation occurred in UGT1A6system.9. Comparation of higenamine glucuronidation in liver and intestine microsomeThe kinetic characteristics of higenamine in liver and intestine microsome were investigated. The glucuronidation rate in liver (1.26±0.30nmol/min/mg) is higher than that in the intestine (6.81±1.73nmol/min/mg).10. Metabolic kinetics Analyse for male and female human, rat, mouse, dog and guinea pig liver microsomeIt was reports that pharmacokinetics characteristics in rabbit, dog and human were differences. We assume that it may be the different metabolism in different species. It turned out that glucuronidation for five different species liver microsome showed different kinetic profiles.The kinetic profiles of rat and mouse followed substration inhibition kinetics and the kinetic profiles of human, guinea pig, dog followed autoactivation kinetics. The kinetic profiles of dog and human are similar that we believed dog could be a better animal for higenamine than other three animals. Based on the various Km of each species, we chose three substrate concentrations to compare the gender differences. Besides rat and dog (only at middle and high substrate concentrations), there were no significant difference in others.Conclusion:1. Compared with that in a single dose of pure OMT, t1/2had no significant variation, indicating fast absorption. Cmax, AUC0→t, and MRT0→t of OMT in rats after receiving doses of radix S. tonkinensis extract were significantly lower than those in the pure OMT group (t-test, p<0.05). MT was observed after administration of OMT. The absolute bioavailability values of OMT in rats after the oral administration of pure OMT and radix S. tonkinensis extract were6.79%and1.87%, respectively:We presume that other ingredients in radix S. tonkinensis could affect the transform of the two alkaloids. The formation rates and clearance rates of matrine in human liver microsome are both higher than that in intestine microsome. It indicated that the main metabolic organ was liver. At the same time, we certified that CYP3A4could metabolize oxymatrine which could affect the pharmacokinetic characterizations of ingredients in radix S. tonkinensis.2. The primary human UGTs responsible for the glucuronidation of higenamine are UGT1A6, UGT1A8, and UGT1A9, indicating liver and intestine were the main metabolic organs.The glucuronidation rates in liver are higher than that in the intestine. It turned out that glucuronidation for five different species liver microsome showed different kinetic profiles which lead to various PK behaviours in different species. The kinetic profiles of dog and human are exactly similar that prompting us dog could be a better animal for higenamine than other three animals.