| Herba Leonuri (HL), which is generally referred to as’Yi-Mu-Cao’, has been used since ancient times for the treatment of gynecological and obstetric disorders in China. In the past thirty years, studies have indicated that HL has versatile bioactivity, especially showing excellent therapeutic effect on cardiovascular diseases. Evidences from animal experiment and clinical trial indicated that extracts of HL have protective effect both on myocardial ischemia and cerebral ischemia. Leonurine (4-guanidino-n-butyl syringate), as the major bioactive ingredient of HL, is a plant alkaloid exclusively isolated from effective extracts of HL. It was reported to show uterotonic action, anti-platelet aggregation, inhibition of creatine kinase and relaxation of vascular contractile response to vasoconstrictor. Nowadays in our laboratory, leonurine is available by chemically synthesis and named after SCM-198, which is being studied in preclinical phase as type I new drug for cardiovascular disease. Recently, it has been reported from in vitro experiments that leonurine could attenuated apoptosis in H9c2rat myocardial cells induced by hypoxia and hydrogen peroxide. Evidence from animal studies demonstrated that leonurine exhibits cardioprotective effects both on the acute and chronic myocardial ischemia rats. Moreover, it has proved that leonurine has neuroprotective activity against ischemic stroke and cerebral ischemia/reperfusion model in rats. The pharmacological effects of leonurine may be related to its antioxidation, anti-apoptotic effect and regulation of mitochondrial function. These findings have shown that leonurine has become a novel promising cardiovascular drug candidate. In research and develpoment of new drugs, preclinical pharmacokinetic (PK) study is obligatory to carry out prior to accessing into clinical study. However, the PK profile and metabolism of leonurine is still almost unknown so far. To develop leonurine and its derivants as new cardiovascular drugs, a systematic preclinical PK study of leonurine was performed in the present study as follows.A high performance liquid chromatography (HPLC) method for determination of leonurine in rat plasma and intestinal perfusate was established on the basis of existing literature. The PK parameters of leonurine after intravenous (i.v.) and intraperitoneal (i.p.) injection at a single dose of15mg/kg and the absorption properties of leonurine in in situ incubation with intestinal tract were measured by this HPLC method. The results showed that the bioavailability of i.p injection of leonurine is58.42%and the remaining percentage of leonurine incubated in intestinal tract over3h is27.13%. The level of leonurine in rat plasma after intragastric (i.g.) administration was very low and not detected by this HPLC method. For the first time, we successfully established a high performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS) method for quantitative analysis of leonurine in biological samples including plasma, tissues, urine, feces and bile and rigorously conducted systematic method validation procedure for determination of plasma samples. This HPLC/MS/MS method was applied to the study of PK properties, plasma protein-binding ratio, tissues distribution and excretion after oral dosing of leonurine. Results showed that this established HPLC/MS/MS method is simple, rapid, robust and reliable and achieves high sensitivity and specificity, which was successfully applied to the quantitative analysis of leonurine in biological matrix following oral administration of leonurine. The elimination of leonurine in rat after i.g administration at dose of15,30,60mg/kg fits the linear kinetics feature which demonstrated similar half life and mean residence time (MRT), and the absorption rate is generally coincident based the fact that the peak time (Tmax) and mean absorption time (MAT) has no obvious difference. From the dose15mg/kg to30mg/kg, the area under curve (AUC) showed a linear correlation with dose. However this linear correlation did not appear in the range of30-60mg/kg which may be explained by the fact that the drug absorption in intestinal tract reach saturation after i.g large dose of leonurine. The absolute oral bioavailability of leonurine is very low, which is probably related to the high extracted ratio of intestinal tract.The96-well equilibrium dialysis device was applied to the measurement of plasma protein binding ratio, and the results showed that the plasma protein binding ratios of rat, dog and human were all not more than80%, and showed significant differences in different species, which suggested that the risk of adverse drug reaction induced by the change of plasma protein binding rate was not high. Tissues distribution results showed that leonurine could distribute to many tissues after administration, and the peak time was about lh. Major part of the leonurine distributed to stomach, liver and kidney, and minor part was detected in lung, heart, spleen and pancrease. In brain tissue, level of leonurine was very low which suggested that leonurine was not easy to penetrate the blood brain barrier (BBB). The excretion test showed that sum of cumulative excretion percent of parent drug into urine and feces was less than18%, which increased to about30%after beta-glucuronidase treatment. The results suggested that leonurine suffered from extensive metabolism and glucuronidation may involved in the biotransformation.A total of seven metabolites (M1-M7) of leonurine in rat in vivo samples were identified by HPLC/Qtrap-MSn method. In these metabolites, M1(glucuronidation), M2(sulfation) and M7(methylation) are phase Ⅱ metabolites of leonurine, and M3(demethylation) is the unique phase I metabolite which can be further metabolized to M4(demethylation with sulfation), M5(demethylation with glucuronidation) and M6(demethylation with bis-glucuronidation). The leading metabolite in rat after oral dosing is M1, so we isolated and prepared Ml from urine and its structure was characterized as leonurine-10-O-β-glucuronide (L-O-G) by NMR spectroscopy. The bioactivity analysis showed L-O-G can increase the viability of cardiac myocytes injured by hypoxia, reduce the leakage of lactate dehydrogenase (LDH) and increase the vitality of antioxidant enzymes which predicted its potential cardioprotective effect. The potency of L-O-G was similar or slight lower than parent drug. The preliminary test showed the UDP-glucuronyl transferase (UGT) isoform involved in glucuronidation of leonurine is UGT1A1. The phase I metabolite of leonurine in rat liver microsome incubation is M3(demethylation), and the P450isoforms CYP2D6, CYP1A2, and CYP3A4may involved in this reaction. We utilized cocktail technique to investigate the inhibitory effect of leonurine on the P450isoforms. Results showed that leonurine has inhibitory effect on CYP1A2, and the value of IC50is about at80μM, however it has no inhibition on other P450isoforms, which also suggested that drug-drug interactions may occur when leonurine was in combination with the substrate of CYP1A2in clinical therapy.In summary, the systematic preclinical PK study of leonurine including absorption, distribution, metabolism, excretion (ADME) and the influence on the activity of P450enzymes were clarified. Above results provide pharmacokinetic information for the preclinical evalution of leonurine. |
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