The Metabolism Characteristics Of Dipfluzine Hydrochloride In Rat Liver Microsomes

Metabolism is a important topic in drug research, and its main aim is to find lead compound which is characterized good DM and PK in the initial stage of drug development. Drug is metabolized in the liver through two general sets of reactions, called PhaseⅠand PhraseⅡ. The Phrase I reactions may increase or decrease the activity or toxicity of micromolecular compounds in body, and CYP450s are the critical enzymes to catalyze the reactions. So, it is meaningful to identify CYP450 isoenzymes which is invovled in the drug metabolism and evaluate the interaction between the drug and P450 isoenzymes. These studies can elucidate metabolism-mechanism, anticipate drug-drug interaction and metabolism-polymorphism. Therefore in Europe and America, identification of CYP450s and their isoenzymes has been used to screen new drugs or study metabolism, and it is one of content in applying for new drug.Dipfluzine Hydrochloride 1-diphenylmethyl-4-(3-(4-fluorobenzoyl))-piperazine hydrochloride, Dip), a novel diphenylpiperazine calcium channel blocker, was first synthetized by Hebei Medical University. Studies have demonstrated that Dip exerts protective effects against focal or whole cerebral ischemic injury via multiple mechanisms and its pharmacological effects are more potent than its analogs, cinnarizine (CZ) or flunarizine (FZ).In the present study, the LC-MS-MS method was established to determine four metabolites of Dip in rat liver micrsomes, and illuminate its metabolism-pathway. The conditions of rat liver microsomes incubation were optimized and enzymes kinetics was investigated at the initial stage. And then, inhibitory effects of known CYP450 isoform-selective inhibitors, correlation analysis and recombinant rat CYP450 isoforms were used to identify the isoform of CYP450s invovled in the formation of metabolites and explore the enzyme catalyzing mechanism of Dip. Isoform-specific reaction markers were used to evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats. These studies are useful to illuminate metabolism-pathway or enzyme catalyzing mechanism of Dip and to understand the interactions of drugs or interacting mechanism, thus drug-drug interaction and adverse effect can be decreased based on these results.Part one The metabolism of Dip in different sex rat liver microsomes and the establishment of LC-MS-MS methoad for the determination of Dip and its metabolites.Aim:To determine the metabolism of Dip in different sex rat liver microsomes and establish the LC-MS-MS method for Dip or its metabolites.Methods:20 Sprague-Dawley rats (10 Male and 10 female), weighing 230±20g were acrificed by decapitation and the livers were removed rapidly. Then the rat liver microsomes were prepared and the concentration of the protein or CYP450 was determined by the method of Lowry or Omura and Sato, respectively. Dip (100μM) was incubated in 500μl pooled rat liver microsomes deluted in potassium phosphate buffer (100 mM, pH 7.4) for 30min at 37℃. Reaction was terminated by placing tubes on ice and the reaction solution was extracted by ethyl acetate, and then 20μl of the organic layer was transferred into LC-MS-MS. Chromatographic separation was performed on a Diamonsil C18 column (250×4.6 mm,5μm, Dikma Technologies), at a flow rate of 0.8 ml/min using a linear gradient elution of A (0.5‰formic acid in methanol) and B (0.5‰formic acid in water). An 8-min linear gradient from 40% A to 95% A was applied, followed by a 5-min elution at 95% A and a 1-min wash to return to the starting condition. The column was maintained at 40℃. Mass spectra was recorded by electrospray ionization with a positive mode.Results:The contents of total protein and CYP450s in male or female rat liver microsomes were 11.26±2.10 versus 10.47±1.87 mg/ml and 0.627±0.10 versus 0.573±0.077 nmol/mg protein(means±S.D., n=10), respectively. Dip was rapidly metabolized and four metabolites were identified in male rat liver microsomes, which were 1-(4-fluoro-benzene)-4- piperazine-butanone (M1), 4- OH-benzophenone (M2), benzhydrol (M4) and benzophenone (M5), respectively. But three metabolites of Dip were generated in female rat liver microsomes, which were M1, M2 and M5. So metabolite profiles in male rat liver microsomes were different from those in females, indicating there is the gender difference in the metabolism of Dip in rat liver microsomes. The initial two metabolic reaction of Dip were the dealkylation of piperazine ring and the oxidation of diphenyl methylene, which formed M1 and M5, respectively. And then M5 could be further metabolized to form metabolites M2 and M4. Comparing with other metabolites, the content of Ml was the highest in male or female rat liver microsomes, suggesting that M1 is the main metabolite of Dip.The recovery of Dip and its metabolites was 78-86%, and the precisions of intra- and inter-day were 0.15-14.42% for the developed LC-MS-MS method. The calibration curve was linear in the range from 2.59 to 1295 ng/mL for Dip, from 25.92 to 12961 ng/ml for M1, from 0.065 to 32.38 ng/mL for M2, from 2.63 to 1315 ng/ml for M4, and from 2.46 to 1228 ng/ml for M5. Dip and its metabolites were found to be stable for short-term and long- term or after three cycles of freeze and thaw. These results showed this methoad is qualified to the pharmacokinetic study.Conclusion:Dip and its metabolites were determined and the metabolic pathway was identified. The developed LC-MS-MS method can satisfy the requirement of pharmacokinetic study and was suitable for simultaneous analysis of Dip and its four metabolites in vitro. There is the gender difference in the metabolism of Dip in rat liver microsomes and M1 is the main metabolite of Dip.Part two The optimization of incubation condition and the study of enzyme kinetic of Dip in rat liver microsomesAim:To optimize the conditions for microsomal incubation and measure kinetic parameters of the metabolites for Dip to provide the reference for the further research of rat liver microsomes.Methods:Time for microsomal incubation, protein concentration of rat liver microsomes and substrate concentration were used as investigating objects. When one of them was changed after the immobilization of the other, the conditions for microsomal incubation could be optimized by observing the concentrations of metabolites. The kinetic parameters (Km, Vmax and Clint) in rat liver microsomes or recombined CYP450s were calculated by Lineweaver-Burk plotResults:Incubation time of 60 min or 30min, protein concentration of 1 mg/ml and substrate concentration of 10μM or 25μM were found to be optimal for the metabolism of Dip in female or male rat liver microsomes, respectively. In male rat liver microsomes, Km and Vmax for M1, M2, M4 and M5 were 3.97,0.0064,4.14μM,3.69μM, and 650.26,0.98,108.66, 100.92 pmol/min/nmol P450, respectively. In female rat liver microsomes, Km and Vmax for M1, M2 and M5 were 3.89,0.0055,5.63μM and 330.17, 0.58,6.54 pmol/min/nmol P450, respectively. In the recombinant CYP450s incubation,3.52,0.0067,5.67,3.90μM and 683.15,1.14,136.40,115.41 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3Al,and 3.49,0.0081,5.03μM,3.95μM and 719.44,1.57,144.91,132.80 pmol/min/nmol P450 were Km and Vmax for M1, M2, M4, M5 in CYP3A2.Conclusion:The optimal incubation time in male or female rat liver microsomes is 30min or 60min, respectively, the optimal concentration of protein is all 1mg/mL, the optimal concentration of Dip in male or female rat microsomal incubation is 25μM or 10μM, respectively. Under these optimal conditions, the formation velocity for all metabolites is saturable, and measured enzyme kinetic parameters of rat liver microsomes and recombined CYP450s, such as Vmax, Km and Clint, were accdurate and reliable, which could satisfy the requests for Dip metabolism research. Thus these results can provide the reference for further research on rat liver microsomes or recombinant CYP450s.Part three Identification of rat liver microsomal CYP450 enzymes involved in the metabolism of DipAim:To study the CYP450 enzymes involved in the metabolism of Dip in different gender rat liver microsomes.Methods:The CYP450 isoforms involved in the metabolism of Dip were investigated by the experiments of selective CYP450 inhibitors, correlation analysis and recombinant CYP450 enzymes.The following P450 isoform-selective inhibitors were used:furafylline (CYP1A2 inhibitor used at 1～100μM), pilocarpine (CYP2A1 inhibitor used at 0.5～100μM), orphenadrine (CYP2B1 inhibitor used at 1～100μM), sulfaphenazole (CYP2C6 inhibitor used at 1～200μM), cimetidine (CYP2C11 or CYP2C12 inhibitor used at 0.5～100μM), quinidine (CYP2D1 inhibitor used at 1～100μM), diethyldithiocarbamate (CYP2E1 inhibitor used at 2.5～200μM) and ketoconazole (CYP3A inhibitor used at 0.5～200μM). Dip was added and incubated with inhibitors in male or female rat liver microsomes or recombinant CYP450 isoforms, and then the concentration of metabolites was determined. The ratio of metabolites in samples with and without inhibitor was calculatedand the effect of chemical inhibitors on the metabolism of Dip was evaluated.Dip was incubated with microsomes from 15 different rat livers to test the correlation of Dip metabolism and the activity of individual CYP450s. The following isoform-specific reaction markers were used to determine the activity of each rat CYP450 isoform: phenacetin O-deethylation (CYP1A2), testosterone 7a-hydroxylation (CYP2A1), testosterone 16β-hydroxylation (CYP2B1), diclofenac 4-hydroxylation (CYP2C6), testosterone 16a-hydroxylation (CYP2C11),5α-androstane-3α,17β-diol-3,17-disulphate 15β- hydroxylation (CYP2C12), dextromethorphan O-demethylation (CYP2D1), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1'-hydroxylation (CYP3A). The correlation coefficients between the formation rates of Dip metabolites and the activity of each CYP450 isoform in the different rat liver microsomes were calculated.Dip was incubated with a panel of recombinant rat CYP450 isoforms (Rat CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2) to assess the involvement of each individual recombinant rat CYP450 enzyme in metabolite formationResults:The results from experiments of selective CYP450 inhibitors, correlation analysis and recombinant rat CYP450 isoforms all confirmed that in male rat liver microsomes in order of contribution, CYP2A1, CYP3A, CYP1A2, CYP2E1 and CYP2C11 contributesto M1 generation. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolize Dip to M2, CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation, and CYP3A, CYP2Aland CYP2C6 involved in the M5 formation. So, CYP3A and CYP2A1 were the major CYP isoenzymes responsible for catalyzing Dip to four metabolites in male rat liver microsomes. For female rat liver microsomes, CYP1A2 and CYP3A metabolize Dip to M1, CYP3A, CYP1A2, CYP2C6, CYP2A1and CYP2E1 involved in M2 formation, CYP3A, CYP2A1, CYP2D1 and CYP1A2 have major roles to M5 formation. Thus, CYP3A and CYP1A2 were the major isoenzymes for all four metabolites in female rat liver microsomes. These results above suggest that CYP3A is the most important isoenzyme for the metabolism of Dip in rat liver microsomes for different sex, and CYP3A2 exhibited more activity than CYP3A1.Conclusion:CYP3A is the most important isoenzyme for the metabolism of Dip in two gender rat liver microsomes, and CYP3A2 exhibited more potent activity than CYP3A1. Both CYP1A2 in female rats and CYP2A1 in the male rats involve in the formation reactions of four metabolites of Dip, which reveals predominant noticeable gender difference.Part four Effects of Dip on CYP450s activities in ratsAim:To evaluate the effects of Dip on CYP450s activities in vitro and in vivo in rats.Methods:Markers were incubated in normal rat liver microsomes with Dip (0-200μM). After reactions were terminated and extracted by ethyl acetate, then the organic layer was transferred into LC-MS-MS and determined the concentration of metabolites for markers. Calculate ratio of content of metabolites in samples (added Dip) and in the control (no Dip), and evaluate the effect of Dip on the CYP450s.Male and female SD rats were divided into five groupes. Dip was administered by orally to rats at doses of 30,60 and 90 mg·kg-1 body weight for fourteen days. At the fifteen day, rats were orally administered Cocktail probe markers:phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and aminophenylsulfone. Blood samples were collected via medial angle of eye at 5,15,40,60,90,120,180,240,360,480 and 720 min after administration of Dip, put into heparinized centrifuge tube, respectively, and centrifuged. The separated plasma was extracted by ethyl acetate and 20μl of the supernatants were transferred to LC-MS-MS for determination. The concentrations of probe markers and their metabolites were determined in plasma samples at 360 min after administration of Dip andthe drug-time curve was plotted, by which pharmacokinetics parameteres were calculated.Rats were sacrificed by decapitation, the livers were weighed, removed rapidly and rat liver microsomes were prepared. Probe markers were added into incubation samples, and after reaction was completed, samples were extracted by ethyl acetate,20μL of the supernatants were transferred to LC-MS-MS for determining the concentration of probe markers and its metabolites. The metabolism ratio of probe in plasma and in liver microsome at 360 min after administration of Dip were calculated and the effects of Dip on CYP450s activities were evaluated by comparing the relative liver weight, concentration of protein and CYP450 content, the drug concentration-time cueves, pharmacokinetics parameteres and the metabolism ratio of probes between the experimental groups and control group..Results:In the normal male rat liver microsomes, Dip had inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, IC50 were 8.85,20.93 and 69.45μg/mL, respectively. However, for the female, Dip showed inhibitor on CYP2C12, CYP2C6, CYP2D1and CYP3A, IC50 were 19.3,35.01,36.69 and 138.53μg/mL, respectively.Dip had no effect on the relative weight of livers, protein concentration and CYP450 content for male and female rats after they were feeded on Dip for fourteen days, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. Results from pharmacokinetics parameteres and markers displayed that low-dose Dip inhibited the activities of CYP2D1 in female rats, or induced the activities of CYP2C11 in male rats and CYP2C6 in female rats. Moderate-dose Dip showed the ability to inhibit CYP2D1 in male rats and CYP2C12, CYP2D1and CYP3A in female rats or induce CYP2C11, CYP3A in male rats and CYP2C6 in female rats. High-dose Dip had certain inhibitive effect to CYP2C6, CYP2D1 in male rats and CYP2C12, CYP2D1, CYP3A in female rats and inductive effect to CYP2C11, CYP3A in male rats and CYP2C6 in female rats. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A in male or female rats were all induced after administration of phenobarbital.Conclusion:The results of normal rat liver microsomes incubation show that Dip inhibits the activity of CYP450s in either male or female rats, in whom there is a significant gender difference. The results of all Dip-induced rat liver microsomes incubation show that Dip had inhibitive effects on the activity of certain CYP450s, which is essentially consistent with those from rat liver microsomes incubation, but Dip can also induce the activity of some CYP450s. These results also exist the gender difference. Thus, co-administration of Dip and other drugs that were metabolized by the P450s must be avoided to decrease drug-drug interaction and adverse effect.