Study On In-vivo Kinetics Of Leonurine And Its Metabolic Pathway

Leonurine (Leo), one of the major phytochemical components of Herba Leonuri, can be synthesized chemically nowadays. In previous studies, Leo has been proven to have protective effects against myocardial ischemia and cerebral ischemia both in vitro and in vivo. It has been recognized as a promising new drug candidate in strokeand cardiovascularprevention.Previous study indicated that Leo was mainly metabolized to leonurine-O-glucuronide(L-O-G) in vivo. At present, there are no reports about the pharmacokinetics of Leo in dogs, the toxicokinetics of Leo in dogs and rats, and the metabolism of Leo in vitro. The preclinical kinetics in animals and the metabolic pathway are important before a new investigational drug can be used in humans, which may in turn guide extrapolation to the appropriate dosing in clinical trials.ObjectiveFirst, this study aimed to acquire the pharmacokinetics data of Leo in dogs, which provides the change process of Leo in dogs. Second, we studied the toxicokinetics of Leo in dogs and rats, which provides the toxicokinetic characteristics of Leo and the relationship between doses and accumulation in animals. Finally, we studied the metabolic pathway of Leo in vitro with liver microsomal and recombinant enzymes. All the results may turn in guide extrapolation to the clinical design and drug combination therapy.MethodDog and rat plasma concentrations of Leo and its major metabolite, leonurine-O-glucuronide (L-O-G), were measured by LC-MS/MS methods. Quantification was performed in multiple reaction monitoring modes using m/z312.2�'181.3for Leo, m/z488.3�'312.2for L-O-G and m/z304.3�'156.3for scopolamine(IS). Separation of Leo and L-O-G from plasma was achieved by using Agilent Zorbax SB-C18column (150mm×2.1mm,5μm). The column temperature was maintained at27℃. The flow rate of0.2ml/min was used and the mobile phase was composed of acetonitrile-ammonium acetate buffer (lOmM; PH3.6):35:65(v/v) for dog plasma.The flow rate of0.3ml/min was used and the mobile phase was composed of acetonitrile-ammonium acetate buffer (10mM;PH3.6):20:80(v/v) for rat plasma.The method validation included the specificity and sensitivity, matrix effects and extraction recovery, stability, accuracy and precision. The kinetics in vivo involved the pharmacokinetics of Leo in beagle dogs (including the absolute bioavailability and accumulation studies) and the toxicokinetics of Leo in beagle dogs and SD rats (single and28consecutive doses of high, middle and low doses). Leo was incubated with liver microsomal of different species (rat, dog, human) and human recombinant enzymes to calculate the Kmax and Vmax of liver microsomal and search for the main metabolic enzyme.ResultLeo and L-O-G concentrations in dog and rat plasma were determined with a LC-MS/MS method. Protein was precipitated from the samples by mixing a50uL aliquot of plasma,10μL of IS (scopolamine,6μM, dissolved in deionized water), and30μL of7%perchloric acid. The mobile phase was composed of acetonitrile-ammonium acetate buffer (10mM; PH3.6):35:65(v/v) and the flow rate was0.2ml/min for dog plasma. The mobile phase was composed of acetonitrile-ammonium acetate buffer (10mM; PH3.6):20:80(v/v) and the flow rate was0.3ml/min for rat plasma. Separation of Leo and L-O-G from plasma was achieved by using Agilent Zorbax SB-C18column (150mm×2.1mm,5μm). A turbo ion spray interface was used as the ion source operating in positive ion mode. Leo and L-O-G were linear from6.2-624.0μg/L、48.8-4880μg/L, respectively for dog plasma. Leo and L-O-G were linear from31.2-3120μg/L.488-48800μg/L, respectively for rat plasma. All the results were complied with the Guidelines for Bioanalytical Method Validation of the Food and Drug Administration.Leo was conformed to be a two-compartment model after a4mg/kg intravenous administration in beagle dogs. The elimination half-life (t1/2) was1.3±0.4h, clearance(CL) was4.4±1.2L/kg/h, and the volume of distribution(Vd, area) was7.8±1.9L/kg after intravenous administration. After a single-oral dose of20mg/kg administration, Leo was conformed to be a two-compartment, with t1/2of1.9±0.4h, CLof71.8±39.4L/kg/h, Vd. area of193.7±07.9L/kg. The absolute bioavailability was7.5%. The accumulation ratios (AR) of the AUC for Leo and L-O-G were2.0and0.9after7consecutive doses of20mg/kg/dose every8hours. The AR of Cmax for Leo and L-O-G were1.6and0.7, respectively. Results indicated that there was no obvious-accumulation in beagle dogs. In the dog toxicokinetics study, three dose group of oral administration were80,240,800mg/kg, dose ratio of1:3:10. The increase ratio of AUC was1:1.3:3.0for Leo and1:1.7:3.0for L-O-G. Following the oral administration, the increase in plasma AUC for Leo and L-O-G was linear from dose but non-proportional. After multiple administrations, the AR of AUC was2.6、3.6、4.3for Leo and1.3、3.5、3.6for L-O-G, indicating that there was accumulation in beagle dogs after multiple administrations. In the rat toxicokinetics study, three dose group of oral administration were200,600,2000mg/kg, dose ratio of1:3:10. The increase ratio of AUC was1:3.5:13.6for Leo and1:3.2:8.4for L-O-G. Following the oral administration, the increase in plasma AUC for Leo and L-O-G was approximately dose proportional. After multiple administrations, the AR of AUC was1.5、1.3、0.8for Leo and1.8、1.1、0.8for L-O-G, indicating that there was no obvious accumulation in SD rats after multiple administrations.The kinetic parameters of Leo in the liver microsomal followed as:Km=88.3±21.6μmol/L, Vmax=19.3±1.5μmol·L-1·min-1·mg-1for rat; Km=Km=405.6±53.9μmol/L, Vmax=9.8±0.6μmol·L-1·min-1·mg-1for dog; Km=279.6±33.4μmol/L, Vmax=2.2±0.1μmol·L-1·min-1·mg-1for human. The results of incubations with recombinant enzymes indicated that the metabolic enzymes of Leo involved UGT1A1、UGT1A9、UGT1A10、UGT1A8、UGT1A3、UGT1A7, and the most important enzyme was UGT1A1.ConclusionThe LC-MS/MS method was simple, sensitive and rapid, which successfully applied to determine biological samples both in vivo and in vitro. The absolute bioavailability was7.5%in beagle dogs. There was no obvious accumulation after7consecutive doses of20mg/kg. Both intravenous(4mg/kg) and single-oral (20mg/kg) administration complied with two-compartment model in beagle dogs. After single-oral dose of Leo (80,240,800mg/kg) in beagle dogs, the AUC of Leo and L-O-G was linear from dose but non-proportional. The AR was increased with dose and there was accumulation in beagle dogs after multiple administrations. After single-oral dose of Leo (200,600,2000mg/kg) in SD rats, the AUC of Leo and L-O-G was approximately dose proportional. There was no obvious accumulation after multiple administrations. There was no obvious toxic reaction during all the toxicokinetic experimental. The main metabolic enzyme was UGT1A1. Combination therapy in the clinical trials should be noted to avoid drug-drug interactions.