| The extracts, derivatives and the active ingredients derived from natural plants play an important role in preventing and curing diseases in traditional Chinese medicine. In the past decades, as more and more difficult in developing chemical synthetic drugs, natural medicines have gained more popularity in many countries and regions worldwide such as Japan, India, the United States, Canada and North America countries. It was reported that approximately20%patients in the United States receiving conventional pharmacotherapy also take herbs or other natural supplements, but only one-third tell their physician about this case. And in these populations, about40%of adverse effects are due to the interactions between the herbal and chemical drugs. Many consumers believe that herbal medicines are natural and they are relatively safer than synthetic drugs, but this is a dangerous oversimplification. This understanding produce risks to clinical application because it is no doubt that we exaggerated the role of herbal medicine, ignoring their potential side effects and even toxicity. While, the facts prove that many natural drugs have side effects or even toxicity. Adverse reactions and drug-drug interactions related to natural medicines increase when natural medicines were co-administered with other herbs or drugs was mainly due to their complexity material foundation. In addition to, the biotransformation of most natural medicines in vivo and their effect on body are still unclear. With the strengthening of the adverse drug reaction (ADR) monitoring, the reports of natural medicines adverse effects showed a rising tread. This adverse reaction not only has a serious impact on the clinic treatment, but also is a huge obstacle on the way of natural medicine entry into the international market. Chinese medicines, which were under the guidance of traditional Chinese medicine, is the main source of natural medicines. Therefore, the researches related to metabolic pathway and herb-drug interactions on natural medicines, particularly for their effective ingredients, have important significances for the rationality and safety of clinical therapy.Aconite medicines (Wutou and Fuzi) are the basic crude medicines in tranditinal Chinese medicine and they were used extensively in clinic. However, the toxicity and frequent poisoning of aconite medicines produce risk to clinic application. Aconite medicines were frequently used combined with other herbal medicines in order to alleviate toxicity and increase efficacy according to traditional Chinese medicine theory. While, the mechanisms of their active components which produce efficacy are unclear, even though there are numerous studies on aconite medicines. Meanwhile, the mechanism of aconite medicines co-administered with other herbal medicines is also unclear. It is well know that the ingredients of Wutou and Fuzi are complex as well as other natural medicines, and this situation will become more complex and volatile when they are co-administered with other herbs or drugs. In recent years, with more and more drug-drug interactions (DDIs) related to metabolic among herbal medicines or between herbal medicines and chemical drugs reported, studies on the effect of herbs on drug metabolizing enzymes have received increasing concern. Aconitine (Aconitine, AC) is a one of the main diterpenoid alkaloids contained in plants of aconite medicines. It was found to possess a wide range of pharmacological effects, such as anti-inflammatory and analgesic. It was reported that the major metabolic pathways of aconitine are hydrolysis, demethylation, hydroxylation and dehydrogenation. And CYP3A is the main enzyme involved in the metabolic of aconitine However, the effect of aconitine on CYP450enzymes remained unknown. Does the toxicity of aconitine relate to the effect of aconitine on CYP450enzymes? Whether the DDIs will occur when aconite medicines are co-administered with other herbs or chemical drugs? Thus, aconitine was chosed as a representative effective/toxic ingredient which was derivated from aconite medicines and the effects of aconitine on major CYP450isozymes were elucidated by using probe drugs. The current study will provide scientific basis for the rational use and toxicity warning of aconite medicines in clinic. The main contents of this thesis are as following.1. Metabolism of buspirone hydrochloride in rat liver and intestinal microsomes and its pharmacokinetics in ratsMethods:A rapid and sensitive UPLC-MS/MS method of buspirone and it’s metabolites (1-PP and6’-OH-BP) was established. The metabolism of buspirone hydrochloride in rat liver and intestinal microsomes and its pharmacokinetics in rat were evaluated.Results:1-PP,6’-OH-BP and M3were the major metabolites of buspirone in rat liver and intestinal microsomes. The formation rates of its metabolites in decreasing order is6’-OH-BP>M3>1-PP in rat liver microsome, while this order is M3>6’-OH-BP>1-PP in rat intestinal microsome. Bioavailability of buspirone is linearly related to the doses of buspirone hydrochloride, suggesting that the first-pass metabolism of buspirone showd first order kinetic when the dose in the range of12.5to50mg/kg in rats. The mean systemic bioavailability of buspirone is about4.3%to6.6%. Plasma levels of1-PP and6’-OH-BP are higher than that of buspirone after oral administration of buspirone hydrochloride, while the concentrations of1-PP and6’-OH-BP much lower than buspirone after intravenous injection of buspirone hydrochloride (0.5mg/kg).2. The effects of aconitine on CYP3A activity in vitro and in vivoMethods:We adopted buspirone as a probe drug of CYP3A to elucidate the effect of aconitine on mRLM, mHLM and rCYP3A4in vitro.The effect of aconitine (single or7days, p.o.) on the pharmacokinetics of CYP3A probe drug buspirone (0.5mg/kg, i.v.) were evaluated.Rats were pretreated with aconitine for7days by oral administration. At the end of the pretreatment, the livers of rats were excised and the liver microsomes were prepared. The activity of rat liver microsomes were evaluated by CYP3A probe drugs buspirone and testosterone in vitro and its protein level were determined by Western blot.Three specific probe substrates/metabolites, phenacetin/acetaminophen (CYP1A2), desipramine/2-hydroxydesipramine (CYP2D6), chlorzoxazone/6-hydroxychlorzoxazone (CYP2E1), were used to determine the activities of rat liver microsomes which were pretreated with aconitine for7days.Results:The formation rates of1-PP showed substrate inhibition enzyme kinetic characteristic. Buspirone N-dealkylation rates were determined in mRLM (mHLM or rCYP3A4), in the presence of various concentration of aconitine (0,10,2040/μM). The maximum formation rates (Vmax) of1-PP from buspirone in rCYP3A4were207.70nmol/min-mg,1497.00nmol/min-mg,655.80nmol/min-mg and48.71nmol/min-mg, respectively, while Km values were1.74μM,25.18μM,14.96μM and0.79μM. The clearance rates of1-PP were decreased, as indicated by the VmaK/Km value was119.37ml/min/mg in the absence of aconitine, while there were59.45ml/min/mg,43.84ml/min/g and61.50ml/min/mg, respectively, in the presence of various concentrations aconitine (10,2040μM). The results demonstrated that the activity of rCYP3A4was inhibited by aocnitine in vitro, but this inhibition showed no obvious concentration dependence.The maximum formation rates (Vmax) of1-PP from buspirone in mHLM were136.30nmol/min-mg,316.80nmol/min-mg,253.30nmol/min-mg and473.80nmol/min-mg, respectively, while the Km were26.41μM,4.34μM,7.88μM and17.22μM. The clearance rates of1-PP were increased, as indicated by the Vmax/Km value was5.16ml/min/mg in the absence of aconitine, while there were72.96ml/min/mg,32.14ml/min/g and27.51ml/min/mg, respectively, in the presence of various concentrations aconitine (10,2040μM). The results indicated that aconitine induced the activity of rCYP3A4in vitro, but this induction showed no obvious concentration dependence. This results implied that the activity of mHLM was activated by aconitine in vitro, but this induction showed no apparent concentration dependence.The maximum formation rates (Fmax) of1-PP from buspirone in mRLM were425.6nmol/min-mg,343.3nmol/min-mg,179.5nmol/min-mg and276.9nmol/min-mg, respectively, while the Km were1.41μM,2.88μM,0.32μM and2.58μM. The values of Vmax/Km were300.99ml/min/mg,119.28ml/min/mg,566.42ml/min/g and107.20ml/min/mg, respectively. Aconitine did not significantly affect the rates of buspirone N-dealkylation in mRLM at the tested concentrations. The formation rates of6’-OH-BP showed Michaelis-Menten, Autoactivation and Biphasic enzyme kinetic characteristics respectively. Buspirone6’-hydroxylation rates were determined in mRLM (mHLM or rCYP3A4), in the presence of various concentration of aconitine (0,10,2040μM). The maximum formation rates (Vmax) of6’-OH-BP from buspirone in mRLM were1599.90nmol/min-mg,952.30nmol/min·mg,964.60nmol/min-mg and723.20nmol/min-mg respectively, while the Km were26.53μM,9.51μM,23.12μM and21.24μM. The clearance rates of6’-OH-BP were decreased, as indicated by the Vmax/Km value was60.31ml/min/mg in the absence of aconitine, while there were100.14ml/min/mg,41.72ml/min/g and34.05ml/min/mg, respectively, in the presence of various concentrations aconitine (10,2040μM). The results demonstrated that the activity of rCYP3A4was inhibited by aocnitine in vitro, but this inhibition showed no obvious concentration dependence. Aconitine did not significantly affect the rates of buspirone6’-hydroxylation in mHLM and mRLM at the tested concentrations.Single dose of aconitine (0.125mg/kg, p.o.) pretreatment had slightly but significantly changed the pharmacokinetics of buspirone (0.5mg/kg, i.v.) and its metabolites in rats. The AUC0-t of buspirone,1-PP and6’-OH-BP were increased by32%(P<0.05),23%and66%(P<0.05) respectively, in aconitine group, with the Cmax of buspirone,1-PP and6’-OH-BP were increased by57%(P<0.05),37%(P<0.05) and16%. Compared with control group, aconitine decreased the tm of buspirone,1-PP and6’-OH-BP as34%,54%(P<0.05) and28%, respectively, and the CL of buspirone,1-PP and6’-OH-BP reduced by21%(P<0.05),10%and45%. Compared with control group, the Vd of buspirone,1-PP and6’-OH-BP reduced to45%,57%(P<0.05) and40%respectively. However, the formation ratios of1-PP and6’-OH-BP from buspirone has not been significantly changed by aconitine, suggesting the activity of CYP3A in rats has not been affected by aconitine. Pretreated with aconitine (0.125mg/kg, p.o.) for7days did not significantly affect the pharmacokinetics of buspirone (0.5mg/kg, i.v.) and its metabolites in rats. This result indicated that CYP3A activity has not been changed by7days aconitine pretreatment.Rat liver microsomes were prepared after7days of aconitine (0.125mg/kg or0.25mg/kg, p.o.) pretreatment. CYP3A activities of rat liver microsomes were determined by using buspirone and testosterone as probe drugs. The activities of CYP3A had slightly but significantly increase in aconitine groups (P<0.05). But this increase does not exceed20%of the CYP3A activity level in control group, and this increase does not exceed40%of the CYP3A activity level in positive control group. These results indicated that7days of aconitine pretreatment did not affect the activityof CYP3A in rats. The increase of CYP3A activities in aconitine grou may be relate to the agonist of enzyme. The results of Western bolt showed that7days of aconitine pretreatments have no effect on the protein expression of CYP3A in rats.Pretreated with aconitine for7days did not affect the activities of CYP1A2, CYP2D6and CYP2E1in rats.3. The effects of aconitine on pharmacokinetics of oral buspirone hydrochloride in ratsMethods:The effect of aconitine (single dose or7days, p.o.) on the pharmacokinetics of the CYP3A probe drug buspirone hydrochloride (25mg/kg, p.o.) were evaluated.Results:Single dose of aconitine (0.125mg/kg or0.25mg/kg, p.o.) pretreatment had significantly changed the pharmacokinetics of buspirone and its metabolites in rats. Compared with control group, the A UC0-t of buspirone showed a different variation trend between0.125mg/kg aconitine group and0.25mg/kg group. For aconitine0.125mg/kg group, compared with control group, the AUCv.t of buspirone decreased by54%(P<0.05), and its Cmax and t1/2by84%(P<0.05) and26%(P<0.05) respectively. The Vd of buspirone had33%decrease compared with that of control group. The AUC0-t and Cmax of1-PP also decreased by32%and39%(P<0.05) respectively compared with those of control group. While the t1/2and Vd of1-PP increased by44%(P<0.05) and70%(P<0.05) respectively compared with those of control group. For6’-OH-BP, except its Cmax had40%decrease compared with that of control goup, other pharmacokinetic parameters of it remained unchanged. Aconitine increased the formation ratios of1-PP and6’-OH-BP from buspirone by49%and74%(P<0.05) respectively compared with those of control group.For aconitine0.25mg/kg group, compared to control group, the AUC0-t and Cmax of buspirone showed slightly but no significantly increases. The AUC0-t and Cmax of1-PP increased by32%(P<0.05) and47%respectively compared with those of control group, and the AUC0-t and Cmax of6’-OH-BP also increased by53%(P<0.05) and84%respectively compared with those of control group. The Vd of6’-OH-BP reduced by40%(P<0.05) compared with that of control group. The formation ratios of1-PP and6’-OH-BP from buspirone had slightly but no significantly increase compared with control group.Theses reaults suggested that there were different effects on the pharmacokinetics of oral buspirone hydrochloride between different doses of aconitine. Thus, We speculated that this interactions may be caused by aconitine affects the absorption, distribution and excretion of buspirone. The activity of CYP3A was not changed by aconitine as the formation ratios of1-PP and6’-OH-BP had no significantly affected by aconitine.7days of aconitine (0.125mg/kg, p.o.) pretreatment had greatly significantly changed the pharmacokinetics of buspirone and its metabolites in rats when buspirone hydrochloride at the dose of25mg/kg by oral administration. Aconitine increased the Cmax and AUC0-t of buspirone by more than1fold (P<0.05). Compared with control group, aconitine increased the AUCo-t of1-PP by more than5fold (P<0.05), and its Cmax by2.3fold (P<0.05). The t1/2and Vd of1-PP also significantly decreased compared with control group. In contrast, compared with control group, the Cmax of6’-OH-BP reduced by91%(P<0.05), accompanied by the AUC0-t reduced by88%(P<0.05), while the Vd value of6’-OH-BP increased more than7fold (P<0.05).We supposed that the7days of aconitine expose changed the pharmacokinetic of buspirone and its metabolites due to aconitine may affect the disposition and excretion of buspirone and its metabolites.In summary, our studies focus on the effects of aconitine on the major C YP450isozymes. The results showed that aconitine inhibited rCYP3A4activity but activated the activity of mHLM in vitro at the tested concentrations (10,2040μM). Single dose of aconitine (0.125mg/kg, p.o.) pretreatment had slightly but significantly affected the pharmacokinetics of CYP3A probe drug buspirone hydrochoride (0.5mg/kg, i.v.) and its metabolites (P<0.05), while7days aconitine (0.125mg/kg, p.o.) pretreatment did not affect the pharmacokinetics of buspirone hydrochloride (0.5mg/kg, i.v.) and its metabolites. CYP3A activity of rat liver microsomes which were pretreated with aconitine (0.125mg/kg or0.25mg/kg, p.o.) for7days had slightly but significant increase, and this increase may due to the activation of enzyme but not its expression level.7days of aconitine (0.125mg/kg or0.25mg/kg, p.o.) pretreatment did not affect the activities of CYP1A2, CYP2D6and CYP2E1in rats. Single dose or7day aconitine exposure significantly affected the pharmacokinetics of buspirone and its metabolites after oral buspirone hydrochloride (P<0.05). We speculated that this interactions may be caused by aconitine affects the absorption, distribution and excretion of buspirone. The results of this thesis suggested that there will not be drug-drug interaction related to CYP450in liver between aconitine and other drugs. |
