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OBJECTIVE: To identify the specific cytochrome P450 (CYP) enzymes involved in the metabolism of dipfluzine hydrochloride (Dip) in the rat liver microsomes. METHODS: The rat liver microsomes were prepared and incubated with Dip. The Dip metabolites (M1, M2, M4 and M5) were identified by LC-MS/MS, and the CYP isoenzymes were identified by the combination of the selective CYP inhibitor study, correlation analysis and a panel of recombinant rat CYP expereiment, respectively. RESULTS: The results from the experiments of selective CYP inhibitors, correlation analysis and recombinant rat CYP isoenzymes indicated that CYP2A1, CYP3A and CYP2C11 contributed to the formation of M1 and M5 in the rat liver microsomes. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolized Dip to M2. CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation. And the recombinant rat CYP researches further indicated that CYP3A2 exhibited more activity than CYP3A1. CONCLUSION: CYP3A and CYP2A1 are the major CYP isoenzymes responsible for catalyzing Dip to the four metabolites formation in the rat liver microsomes.
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OBJECTIVE: To evaluate the effects of dipfluzine hydrochloride (Dip) on CYP450s activities in vitro and in vivo in rats. METHODS: Markers were incubated in the normal rat liver microsomes with Dip (0-200 μmol·L-1) and the concentration of metabolites of the markers were determined by LC-MS/MS, and then the ratios were calculated to evaluate the effects of Dip on the CYP450s activities. Dip was administered by orally to the male SD rats at doses of 30, 60 and 90 mg·kg-1 body weight and phenobarbital was administered at doses of 120 mg·kg-1 body weight for 14 d. At the fifteenth day, the rats were orally administered the Cocktail probe markers and blood samples were collected via medial angle of eye at different time. The concentrations of markers were determined by LC-MS/MS and the drug-time curve was plotted, by which the pharmacokinetic parameters were calculated. And then the rat liver microsomes were prepared and the probe markers were added into the incubation samples. The concentrations of the probe markers and its metabolites were determined and the metabolism ratios were calculated. The effects of Dip on CYP450s activities were evaluated by comparing the following outcomes between the experimental groups and the control group: the relative liver weight, the concentrations of protein, the contents of CYP450 enzymes, the drug concentration-time curves, the pharmacokinetic parameters and the metabolism ratios of the probes. RESULTS: In the normal rat liver microsomes, Dip had the inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, and the IC50 were 8.85, 20.93 and 69.45 μg·mL-1, respectively. Dip had no effect on the relative weight of livers, the protein concentrations and the CYP450 content for the rats after they were fed on Dip for 14 d, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. The results displayed that the low-dose Dip inhibited the activity of CYP2D1 or induced the activity of CYP2C11 in rats. Moderate-dose Dip showed the abilities to inhibit CYP2D1, but induce CYP2C11 and CYP3A. High-dose Dip had the certain inhibitive effects to CYP2C6 and CYP2D1, and had the inductive effects on CYP2C11 and CYP3A. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A were all induced after administration of phenobarbital. CONCLU SION: The results of liver microsomes incubation and blood plasma from the normal and Dip-induced rats all show that Dip inhibit the activities of CYP2C6 and CYP2D1, however, Dip inhibit CYP2C11 in vitro and induce it in vivo.
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AIM To investigate effect of dipfluzine on sodium current (INa+) in isolated single guinea-pig ventricular myocytes. METHODS INa+ was measured by whole cell patch-clamp technique in single isolated guinea-pig ventricular myocytes which were prepared by enzymatic dissociation method. RESULTSCardiac INa+ was elicited by 50-ms pulses to +50 mV from holding potential at -80 mV with a step of +10 mV, which could be blocked completely by tetrodotoxin 10 μmol·L-1. The peak INa+ occurred at about -20 mV and the reversal potential for INa+ was about +30 mV. Dipfluzine inhibited cardiac INa+ in a concentration-dependent manner. The blocking effect of dipfluzine on INa+ was reversible. Dipfluzine suppressed cardiac INa+, without modifying maximum activation potential and reversal potential. The peak of INa+ was decreased by about 46% at -20 mV and shape of I-V curve was not altered by dipfluzine 40 μmol·L-1. Dipfluzine shifted the steady-state inactivation curve of INa+ towards more negative without changing the slope factor and produced very little change in the steady-state activation curve towards more positive. The mean half activation voltage was (-34.9±5.1) mV and slope factor was (6.0±4.8) mV under control condition and (-33.7±3.6) mV and (5.6±2.4) mV following exposure to dipfluzine 40 μmol·L-1. The half inactivation voltage was (-73.0±4.6)mV and slope factor was (4.8±1.8)mV under control condition and (-82.8±7.2)mV and (4.8±1.8)mV following dipfluzine 40 μmol·L-1 treatment. Dipfluzine delayed recovery of cardiac INa+ from inactivation state. The time course of recovery was (36±11) ms in control group and (79±28) ms in dipfluzine 40 μmol·L-1 group. CONCLUSION Dipfluzine inhi- bits cardiac INa+ in a concentration-dependent manner and has higher affinity for the inactivated state than that for resting state of Na+ channels.
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AIM To investigate whether dipfluzine (Dip) possesses antiarrhythmic effect on experimental arrhythmias and effect on cytosolic calcium in ventricular myocytes of guinea-pig. METHODS Experimental arrhythmias were induced by strophanthin G infusion through jugular vein in guinea-pigs and by myocardial ischemia-reperfusion (I-R) in rats respectively. Cytosolic calcium concentration ([Ca2+]i) of isolated guinea-pig ventricular myocytes was examined with laser confocal scanning microscope. RESULTSIn guinea-pigs pretreatment with Dip 20 mg·kg-1 increased the dosages of strophanthin G required to induce ventricular premature contraction (VP), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA), pretreatment with Dip 10 mg·kg-1 increased the dosages of strophanthin G required to induce VP. In the I-R-induced arrhythmic model of rats, Dip 20 mg·kg-1 decreased the number of rats exhibiting VT, VF and CA, and the number of rats exhibiting VF and CA was decreased by Dip 10 mg·kg-1. Both Dip and verapamil (Ver) decreased [Ca2+]i of the ventricular myocytes in normal Tyrode′s solution. The Ca2+ overload evoked by high extracellular Ca2+ levels was inhibited by Dip and Ver, and the prophylactic effect of Dip was less than that of Ver, while the curative effect of Dip was more obvious than that of Ver. CONCLUSION Dip has antiarrhythmic effect, which is likely related to the modulation on the intracellular calcium homeostasis.
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Aim To explore the relations between anti-apoptotic role of dipfluzine (DIP) and the death signaling transduction pathway initiated by CD95 molecules, and the transcription factor involved in the transcription regulation of CD95 molecules in the hippocampal CA1 region after transient forebrain ischemia. Methods The rat forebrain transient ischemia model was established through 15 min ischemia followed by 3 days reperfusion by using the four-vessel method. The rats were divided randomly into five groups: sham control group, ischemia / reperfusion (I/R) group, DIP treated groups (20, 40 and 80 mg·kg-1 body weight, ig, separately). Western blotting and RT-PCR were performed to detect the expression changes of Fas, FasL, caspase 10 p20, caspase 8, I-κB-α, and p-I-κB-α molecules in protein and mRNA levels, separately, and immunohistochemistry for molecular localization of Fas and FasL in rat hippocampus. Results The expression of Fas, FasL, and caspase 10 p20 in protein and mRNA levels increased after I/R, which was inhibited significantly after treatment with 20 and 40 mg·kg-1 of DIP (P<0.01). In 80 mg·kg-1 of DIP group, the expression of Fas and FasL protein was not significantly different from that of I/R group (P>0.05). The expression of caspase 8 and I-κB-α showed no significant differences in all groups (P>0.05), and no gene expression was observed for p-I-κB-α protein in the study. DIP significantly affected molecular distribution of Fas and FasL protein in CA1 subregion of hippocampus. Conclusion DIP inhibits the death signaling transduction pathway initiated by CD95 molecules in rat hippocampal CA1 subregion, and NF-κB transcription factor may not be involved in the transcription regulation of CD95 molecules after transient forebrain ischemia.
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Aim To investigate the pharmacokinetics of dipfluzine hydrochloride,a novel piperazines calcium antagonist.Methods Eighteen Beagle dogs were randomly divided into three groups,which were administered with dipfluzine hydrochloride at iv single dose of 1.5,3.0 and 6.0 mg?kg-1,respectively.The blood was collected at different time.A RP-HPLC method was developed to determine the concentration of dipfluzine hydrochloride in plasma.The pharmacokinetic parameters were calculated by 3P97 software.Results The specificity,lowest limit of detection and quantification,extraction recoveries,the precision of intra-and inter-day and stability were qualified to the pharmacokinetic study.The concentration-time courses of dipfluzine hydrochloride were best fitted to a two-compartment open model at three doses.The main pharmacokinetic parameters at three doses were 24.7,24.2 and 29.6 h for T12?,0.44,1.12 and 2.86 g?min?L-1 for AUC,1.30,1.22 and 1.28 L?kg-1 for Vc,and 3.4?10-3,2.7?10-3 and 2.1?10-3 L?kg-1?min-1 for CL,respectively.Conclusions The developed RP-HPLC method for determination of dipfluzine hydrochloride in plasma can satisfy the requirement of pharmacokinetic study after iv dipfluzine hydrochloride.Analysis of plasma concentration-time curves indicates a biphasic decrease.There was a linear relationship between AUC and dosage.
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Aim To study the protective effects of dipfluzine against the whole cerebral ischemia and reperfusion injury and its mechanisms.Methods Four-vessel occlusion method was used to make the cerebral ischemia-reperfusion model.In early period of reperfusion,several including LDH,MDA,SOD and brain water content were tested.And in the late period of reperfusion,the delayed neuronal death and amnesia induced by reperfusion were studied.Results The contents of brain water and MDA were increased,and the activities of SOD and LDH were decreased after ischemia and reperfusion injury.The hippocampal structure and memory of rats were also destroyed in the delayed neuronal death.Dip reversed the changes obviously.It had antagonistic effect on brain edema and lipid oxidation,it also protected the neurons of hippocampal CA1 regions from ischemia injury.Conclusion Dip had protective effects on the early stage of reperfusion injury,and delayed neuronal death after the whole cerebral ischemia and reperfusion,which were possibly due to the antagonistic effect on lipid peroxidation.
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Aim To investigate the effect of dipfluzine on hERG potassium currents expressed heterologously in xenopus oocytes.Methods Using Xenopus oocytes expression system,the current amplitude and kinetic characteristics of hERG were measured with the two electrode voltage-clamp technique before and after dipfluzine application.Results Dipfluzine(8 nmol?L-1~5 ?mol?L-1)concentration-dependently inhibited hERG currents;the concentration for half maximal inhibition(IC50)was 98.0 nmol?L-1.Dipfluzine-induced inhibition of hERG currents was voltage dependent at membrane potentials between-10 and 40 mV.Dipfluzine at 1 ?mol?L-1 didn't statistically shifted V1/2 of hERG currents activation.Dipfluzine at 1 ?mol?L-1 significantly decreased the activating time constants and the deactivating time constants,and enhanced hERG currents activation and deactivation.Conclusion Dipfluzine concentration-dependently inhibits hERG currents and modifies kinetic characteristics of hERG activation and deactivation,which may be correlated with its antiarrhythmic effect.
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Aim To investigate the effects of dipfluzine(Dip) on proliferation and collagen synthesis in cultured neonatal rat cardiac fibroblasts(CFB)stimulated by angiotensin Ⅱ(AngⅡ),and to explore the action mechanisms of dipfluzine.Methods CFB were treated with AngⅡ to induce fibrosis model.The effect of Dip on proliferation of CFB was observed by MTT coloricmetric assay;synthesis of collagen was observed by the hydroxyproline concentration detemined;cell cycle distribution and PCNA proein were determined with flow cytometer(FCM);The expression of collagenⅠmRNA,collagen Ⅲ mRNA and TGF-?_1 mRNA was examined using semi-quantitative RT-PCR analysis;The protein expression of cPKC? and ERK_1 in CFB was observed by immunohistochemical staining.Results ① Within a concentration coverage,Dip inhibited CFB proliferation and collagen synthesis(P
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Aim To study the effects of dipfluzine (Dip) on cell apoptosis after focal cerebral ischemia-reperfusion injury in rats. Methods Endothelin-1 induced focal cerebral ischemia-reperfusion model in rat was used in experiment. The cell apoptosis, the expressions of Bcl-2 and Bax proteins were observed by flow cytometric analysis. Results The tissues from the solvent group showed remarkably high apoptotic percentages with(9.34?1.22)% in cortex and(10.58?1.44)% in striatum, respectively, in contrast with(1.26?0.15)% in cortex and(2.50?0.35)% in striatum in sham group. Dipfluzine could decrease the cell apoptosis in cortex and striatum and showed a close correlation with the dose increment, which were (7.92?0.76)% in 10mg?kg -1 group, (6.78?0.77)% in 20 mg?kg -1 group, and (6.00?0.71)% in 40 mg?kg -1 group in cortex (r=0.9559, P0.05). The determination of Bcl-2 and Bax by flow cytometric analysis indicated that sham group showed high expression of Bcl-2 both in cortex and striatum and the ratio of Bcl-2 and Bax were the highest,they were 1.30?0.08 in cortex and 1.64?0.10 in striatum, respectively. The expression of Bax in solvent group was increased and the the ratio of Bcl-2 and Bax were 1.03?0.12 in cortex and 1.00?0.04 in striatum, significantly lower than those in sham group (P