Studies On Chiral Pharmacokinetics And Metabolism Of Tetrahydroprotoberbcrines Analogues

In the past few years, researches on chiral drugs have drawn global attention. Theevaluation of stereoselective characteristics of enantiomers in vivo is of paramountsignificance for developing effective and safe chirality drugs. Stereoselectivity plays animportant role in pharmacokinetics involving the absorption, distribution, metabolism, andexcretion of chiral drugs. It is necessary to evaluate the pharmacokinetic behavior, effectson the main drug-metabolism enzymes and the metabolic pathway of each enantiomerrather than those of the racemate.In this paper we study on tetrahydroberberine (THB) and tetrahydropalmatine (THP),which are two biologically active components of Chinese medicinal herb named Corydalisyanhusuo W.T. Wang. An enantioselective HPLC method was developed for thequantification of tetrahydroberberine (THB) enantiomers in rat plasma and the chiralpharmacokinetics of THB was investigated in rats. Transmembrane transport mechanismof tetrahydroberberine (THB) enantiomers was studied by a MDCK-MDR1cellmonolayers. The effect of THP enantiomers on the activity of cytochrome P450isozymesin rats was evaluated using a cocktail probe drugs method. The metabolites were identifiedby UPLC and the major metabolic pathways of THP enantiomers in rat urine wereproposed after oral administration of THP enantiomers by ultra performance liquidchromatography coupled to time-of-flight mass spectrometry. These studies aimed toclarify the stereoselective characteristics of tetrahydroprotoberberines (THPBs) analoguesin pharmacokinetics and to investigate the metabolites and effects of THPBs on the activityof cytochrome P450isozymes. The detailed results were presented as follows.1. Studies on chiral pharmacokinetics of tetrahydroberberine in ratsTo develop an enantioselective HPLC method for the quantification oftetrahydroberberine (THB) enantiomers in rat plasma，Supelclean LC-18solid phaseextraction (SPE) cartridges were used to extract the enantiomers from samples and thechiral separation was carried on a Chiral AD column (250mm×4.6mm,5μm) withmethanol-ethanol (80:20, v/v) as the mobile phases at a flow rate of0.4ml/min and acolumn temperature of25℃.The detection wavelength was230nm. The linear range ford-THB was0.010～2.5μg/ml and0.010～5.0μg/ml for l-THB. The absolute recoverisfor each enantiomer were greater than70%.The intra-and inter-day variations were less than15%. The method was applied to determination the concentrations of THBenantiomers in rats after oral administration and intravenous injection at different doses ofrac-THB (50mg/kg,10mg/kg), and the pharmacokinetics parameters were then calculated,The mean Cmaxand AUC0-∞values in rats after oral administration were1.09μg/ml,1.91μg·h/ml for d-THB, and2.01μg/ml,4.15μg·h/ml for l-THB. After intravenous injectionthe mean AUC0-∞values were1.95μg·h/ml for d-THB and2.79μg·h/ml for l-THB. Therewere significant differences between the main pharmacokinetic parameters of the twoenantiomers. The results herein showed the stereoselective pharmacokinetics of THBenantiomers in rats.2. Study of transmembrane transport of tetrahydroberberine enantiomers inMDCK-MDR1cell monolayersTo study transmembrane transport mechanisms of tetrahydroberberine (THB)enantiomers by MDCK-MDR1cell monolayers. The bi-directional transport characteristicsof l-THB and d-THB were discussed. The concentration of THB enantiomers wasdetermined by HPLC and the apparent permeability coefficient (Papp) was thus calculated.The Pappof l-THB from A to B was1.27×10-5nm/s, while the Pappfrom B to A was5.65×10-5nm/s, and the efflux rate of l-THB was4.45; while the Pappof A side to B side ford-THB was1.27×10-5nm/s and the Pappof B side to A side for d-THB was4.67×10-5nm/s,the efflux rate of d-THB was3.68. The result indicated that the transmembrane transportmechanisms of l-THB and d-THB in MDCK-MDR1cell monolayer might involved activetransport.3. Evaluation of THP enantiomers on the activity of cytochrome P450isozymes inrats using a cocktail probe drugs methodA liquid chromatography-mass spectrometry method（HPLC-MS）has been developedfor simultaneous determination of five major cytochrome P450probe drugs, metoprolol(CYP2D6), caffeine(CYP1A2), dapsone(CYP3A4), chlorzoxazone(CYP2E1) andtolbutamide(CYP2C9) in rats urine. SD rats were randomly divided into groups (each n=6).Three groups of the rats was separately given d-THP, l-THP and rac-THP once daily for7days while the control group received orally saline once daily. Seven days later, a mixtureof five cocktail probe drugs including metoprolol, caffeine, dapsone, chlorzoxazone andtolbutamide were given to rats by intravenous injection. The concentrations of the five probe drugs were determined by HPLC-MS and their corresponding pharmacokineticparameters were calculated to evaluate the possible effect of THP enantiomers on CYP450isozymes activity.The metabolism of caffeine and metoprolol slowed down after receiving d-THP, withshorter t1/2. The metabolism of caffeine and dapsone slowed down after receiving l-THP,with longer t1/2,but the metabolism of tolbutamide speeded up, with shorter t1/2.Themetabolism of caffeine slowed down after receiving dl-THP, with longer t1/2,while dapsone,chlorzoxazone and tolbutamide speeded up, with shorter t1/2. There were significantlydifferent of these t1/2values from that in blank control group (P<0.05). The resultsindicated that d-THP tended to be the inhibitor of CYP2D6and CYP1A2, l-THP tended tobe the inhibitor of CYP1A2, CYP2E1and inducer of CYP2C9. The effects of dl-THP onthe activity of CYP450isozymes caused by both enantiomers.4. Identification of metabolites of THP enantiomers in rat urine and study onpharmacokinetics of THP enantiomers and their metabolitesAn ultra-performance liquid chromatography coupled to quadropole time-of-flight massspectrometry (UPLC-Q-TOF/MS) method was applied to identify the metabolites in urineafter a single oral administration of each THP enantiomers to rats. The identification wasdetermined by accurate mass measurements and fragment ions under high energy CIDmass spectra. Thus, besides the parent drug, a total of32metabolites for d-THP and30metabolites for l-THP were detected in urine after oral administration of THP enantiomersto rats. The major components in urine were demethlyzed, didesmethlyzed, hydrolyzedmetabolites, glucuronide and sulfation conjugates. Compared with l-THP, the metabolitesof tridesmethlyzed metabolites and didesmethlyzed coupled with hydrolyzed metabolitesonly were found in d-THP treated rats. The results indicated that there may existstereoselective metabolism of THP enantiomers.A sensitive and rapid LC-MS/MS method has been developed for the quantification ofTHP enantiomers in rat urine. Urine sample were collected after a single intravenous THPenantiomers at a dose of60mg/kg within72h. And the concentrations of THP enantiomersin urine at different time periods were determination by the validated LC-MS/MS method.The concentrations of the possible metabolites were calculated by the semi-quantificationwith the equations of THP enantiomers, which described as molar concentration (μmol/ml)instead of mass concentration (μg/ml). In addition, according to the accurate molecular formulae provided by UPLC-Q-TOF/MS and urine excretion volume, the amounts of themetabolites were calculated. The results showed that the cumulative excretion amounts ford-THP and l-THP within72h were53.23±27.33μg and101.55±43.31μg, respectively. Thecumulative excretion rates for d-THP and l-THP were0.37%and0.72%, respectively. Thel/d ratios of cumulative excretion were2.23. The cumulative excretion amounts of mainphase I metabolites including demethylation (M7-2and M7-3) and didesmethylation(M3-2) were more than that of phase II metabolites including demethylation and sulfation(M9-1and M9-2).Furthermore, the cumulative excretion amounts of l-THP were higherthan those of d-THP at different time periods. The results indicated that only a smallamount of THP enantiomers was excreted in urine and there existed a stereoselectiveexcretion of THP enantiomers in rat urine.