| Dipfluzine, a novel diphenylpiperazine calcium channel blocker, was first synthetized by Hebei Medical University. The previous studies have demonstrated that Dip is a high selective cerebral vasodilator. Experimental studies have shown that Dip exerts the protective effects against focal or whole cerebral ischemic injury via multiple mechanisms. Dip also possesses the effects of anti-aggregation of platelet in vitro and prevention of thrombus formation in vivo. In vivo and in vitro evidence from those studies have revealed that pharmacological effects of Dip are more potent than its analogues, cinnarizine or flunarizine, marketed calcium channel blockers. Dip, therefore, is a promising candidate drug to treat cerebral vascular diseases. However, Dip is poorly water soluble. Considered of parenteral administration of the drug in ischemic stroke patients, we recently developed a preparation of water-soluble Dip, dipfluzine hydrochloride. In this study, a RP-HPLC method was developed to determine the concentration of dipfluzine hydrochloride in plasma and tissues. We studied the pharmacokinetics of dipfluzine hydrochloride in Beagle dogs by using this method, the acute toxicity and toxicokinetics of dipfluzine hydrochloride in rats and the toxicokinetics of dipfluzine hydrochloride in Beagle dogs. Metabolites of dipfluzine hydrochloride in rat and dog liver microsomes were determined. The study would provide nonclinical information to the further development of dipfluzine hydrochloride as a new drug.Part1 The pharmacokinetics of dipfluzine hydrochloride in Beagle dogs. Aim: To study the pharmacokinetics character of dipfluzine hydrochloride in vivo by determining the plasma concentration after iv administration in Beagle dog.Methods: 18 Beagle dogs were distributed into three groups with 6 animals in each group (3 female and 3 male). Dogs were respectively given a single intravenous injection of 1.5, 3.0 and 6.0 mg·kg-1 dipfluzine hydrochloride for each group. Blood samples were collected via the femoral vein at 0, 1, 3, 5, 10, 15, 30 min, 1, 2, 4, 8, 12 and 24h after administration of dipfluzine hydrochloride. Plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. A Zorbax C8 reversed-phase column was used as the analytic column. The mobile phase was developed by using 0.2% formic acid as aqueous phase and methanol/acetonitrile/ formic acid (60:40:0.2, v/v/v) as organic phase. The manner of gradient elution was achieved by a gradual increase of organic phase from 0% to 65% within 36 minutes. The flow-rate was maintained at 1 ml/min, and the detection was performed at a wavelength of 254 nm under constant column temperature of 40℃. Flunarizine was choosen as the inner standard. The pharmacokinetic parameters were calculated by 3P97 software.Results: Under the RP-HPLC method, the retention time of dipfluzine hydrochloride and flunarizine was 32 min and 34 min, respectively. Calibration curves was Y=0.2225X-0.0037 (r=0.9999). Results for the method were linear over the calibration range of 0.2-25 mg·L-1. The specificity, lowest limit of detection and quantification, extraction recoveries, the precision of intra- and inter-day were qualified to the pharmacokinetic study. Dipfluzine hydrochloride was found to be stable after three cycles of freeze and thaw ( room temperature), and no signs of degradation were found under the freeze condition. After intravenous administration of three doses of dipfluzine hydrochloride, the concentration-time courses of dipfluzine hydrochloride were best fitted to a two-compartment open model. The main pharmacokinetic parameters at three d?oses were ?24.7, 24.2 and 29.6 h for T1/2 , 0.44, 1.12 and 2.86 mg·min·ml-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.Conclusion: The developed RP-HPLC method for determination of dipfluzine hydrochloride in plasma can satisfy the requirement of pharmacokinetic study after intravenous administration of dipfluzine hydrochloride. Analysis of plasma concentration-time curves indicated a biphasic decrease. There was a linear relationship between AUC and dose. Part2 The acute toxicity and toxicokinetics of dipfluzine hydrochloride.Aim: To find the potential toxic target-organs by observing the symptom of acute toxicity and its expiration after a single intravenous administration of dipfluzine hydrochloride, and simultaneously study the toxicokinetics of dipfluzine hydrochloride to provide explanation for the toxicity.Methods: Dipfluzine hydrochloride was administered by intravenous injection to groups of rats (3 males and 3 females for each group) at doses of 5, 6, 10, 15, 25, 30, 35, and 40 mg·kg-1 body weight. The starting dose of 5 mg·kg-1 was a safety dose based on a preliminary study. The control group received the vehicle only. The general demeanor, clinical signs, and mortality of rats were continuously observed for 2 h after injection, and then once every 4 h for 24 h and thereafter once a day for 14 d. The body weights of all animals were measured before dosing and on days 2, 4, 8, and 15. All dead rats were immediately subjected necropsy and surviving animals were euthanized after the final observation (day 15) by exsanguination from the abdominal aorta under intraperitoneal pentobarbital and subjected to gross necropsy. The abnormal organ detected was preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue sections were microscopically examined.Another 6 rats (3 males and 3 females) were used for blood chemistry. Blood samples were collected via angular vein at 1 and 24 h after intravenous dosing at 30 mg·kg-1, the maximal tolerance dose. Clinical chemistry parameters were determined by automatic biomedical detector with assay kits and included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirebin (T-BIL), urea nitrogen (BUN), creatinine (CRE), total cholesterol (TCHO), glucose (GLU), albumin (ALB), total protein (TP), triglyceride (TG), gamma-glutamyl transferase (GGT) and creatinkinase (CK).A total 144 rats were used for toxicokinetic study, which were distributed into three groups with 48 animals in each group. Rats were respectively given a single intravenous injection of 5, 15 and 30 mg·kg-1 dipfluzine hydrochloride for each group. Six rats (three males and three females) at each time-point were sacrificed under intraperitoneal pentobarbital at 0.08, 0.16, 0.25, 0.5, 1.0, 2.0, 8.0, and 24 h after administration of dipfluzine hydrochloride. Blood samples were collected via the posterior vena cava and then the following organs were taken: heart, liver, spleen, lung, kidney, brain, pancreas and reproductive tissues. Each tissue sample was rapidly weighed and rinsed with 0.9% NaCl to remove the blood or content, and then homogenized in methanol/water (1:1, v/v) solution to obtain the concentration of 1.0 g·ml-1. The obtained tissue homogenates were centrifuged at approximately 9000×g for 10 min and the supernatants were stored at -20℃until analyzed. Blood and tissue samples were subjected to the determination of dipfluzine hydrochloride concentration by RP-HPLC.Results: The no-observed-adverse-effect level (NOAEL) was 5 mg·kg-1, whereas the lowest-observed-adverse-effect level (LOAEL) was 6 mg·kg-1. The clinical signs of toxicity were immediately evident after the intravenous administration of dipfluzine hydrochloride and included hair-upright, whole body tremor or convulsion, eye congestion, frothy sputum, dyspnea. The toxic symptoms were gradually reduced and disappeared within 20-60 min post-dose from 6 to 30 mg·kg-1. At 35 mg·kg-1, rats showed the severe symptoms, and two of six test rats died within 30 min and the activity of others was fully recovered within 1 h. Thus, maximal tolerance dose (MTD) was 30 mg·kg-1, and minimal lethal dose (MLD) was around 35 mg·kg-1. At 40 mg·kg-1, three of six test rats died within 20 min. All surviving rats showed no any abnormal signs during the following test period. The body weight of rats was not significantly changed during the period of the test.At 1 h after dosing, AST, ALT, ALP, GLU and CK levels in 30 mg·kg-1 dose group were significant higher compared to the control group. There were no difference in other parameters including T-BIL, BUN, CRE, TCHO, ALB, TP, TG, and GGT. By 24 h all of clinical chemistry parameters in rats administrated with dipfluzine hydrochloride were not different from the control levels. From the autopsies of test mice, obvious lung congestion was found in dead mice, but the organs of surviving rat lacked any other abnormal indications. The histological analysis further demonstrated the pulmonary congestion, which characterized by an infiltration of lots of blood cells into alveolar wall.Following i.v. administration, analysis of plasma concentration-time curves indicated a biphasic decrease. A good fit of the observed data for a two-compartment model was obtained. The results showed that AUC in plasma were respectively 2.9, 10.9, 29.4μg?h·ml-1 at 5, 15 and 30 mg·kg-1. A good linear correlation was obtained in correlation and regression analysis of AUC-dosage plots (r=0.9939). The T1/2αwere respectively 14.5, 36.0, and 23.8 min, and the T1/2βwere respectively 11.2, 11.6 and 23.3 h. The T1/2βwas significantly increased at 30 mg·kg-1. The apparent volumes of distribution were respectively 3.3, 3.5 and 2.0 L·kg-1, suggesting that Dip easily penetrated all tissues. A good linear correlation was obtained in correlation and regression analysis of AUC-dosage plots in all of detected tissues. The highest AUC level was found in lung and it was respectively 8.9, 26.2 and 52.9μg?h/ml after iv administration of three doses of dipfluzine hydrochloride, which were significantly higher than those in plasma (p<0.01). AUCs in brain, kidney, and pancreas were similar to those in plasma, whereas in heart, liver, spleen, and reproductive tissues were smaller than those in plasma. The T1/2βwas much longer in lung, kidney and brain than that in plasma at all of three doses. The longer T1/2βwas also found in liver and pancreas at doses of 15 and 30 mg·kg-1.Conclusion: After intravenous administration of dipfluzine hydrochloride, the acute toxicity study demonstrated that NOAEL, LOAEL and MTD were 5, 6, 30 mg·kg-1, respectively. The toxicokinetic result indicated that there was a linear relationship between the symptom of toxicity and the exposure of dipfluzine hydrochloride in rat. Congestion was found in lung which was also the tissue with the highest dipfluzine hydrochloride concentration. The longer T1/2βin kidney, brain, liver and pancreas revealed that dipfluzine hydrochloride probably accumulated in these tissues after repeated administration. The clinical chemistry results revealed that there were temporary impairment in liver and heart. These results provide evidence for long-term toxicity study of dipfluzine hydrochloride. Part3 The toxicokinetics of dipfluzine hydrochloride in Beagle dogs.Aim: To study the relationship between doses and exposure and the possible accumulation of dipfluzine hydrochloride in dogs after repeated intravenous infusion by investigating the toxicokinetics of dipfluzine hydrochloride in Beagle dogs in the long-term toxicity study.Methods: 24 Beagle dogs were randomly distributed into three groups with 8 animals in each group (4 male and 4 female per group) and were respectively given intravenous infusion of 2.5, 5.0 and 10 mg·kg-1 dipfluzine hydrochloride (2ml·min-1 ) for each group once a day for a 4-week period. The blood was collected at 0, 5, 15, 30 min and 1, 2, 4, 8 and 24 h on the first day and the 27th day immediately after intravenous administration. Plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. The plasma samples were subjected to the concentration determination of dipfluzine hydrochloride by HPLC.Results: A good fit of the observed data for a two-compartment model was obtained for intravenous infusion of dipfluzine hydrochloride at three doses of 2.5, 5.0 and 10 mg·kg-1. After a single (on the 1st day) intravenous infusion administration, the CL for three doses was 5.6, 4.0 and 5.3 mL·kg-1·min-1 and the Vc was 0.79, 1.14 and 1.15 L·kg-1, respectively. The CL and Vc were not significantly different among three doses. The T1/2βfor three doses were 7.8, 12.8 and 18.2 h, The T1/2βincreased with the dose. The AUC for three doses were respectively 0.47, 1.06 and 2.38 mg·min·ml-1, and there was linear relationship between AUC and dose. After 4 weeks (on the 27th day) of intravenous infusion administration, the CL for three doses was 4.5, 4.0 and 2.9 mL·kg-1·min-1. CL in high group was a little lower than that in low and medium group, but there was no statistic difference. The Vc was 0.97, 0.85 and 0.88 L·kg-1 respectively. Vc were not significantly different among three doses. The T1/2βfor three doses was 11.7, 14.2 and 18.0 h, The T1/2βincreased with dose. The AUC for three doses were respectively 0.61, 1.42 and 3.74 mg·min·mL-1, and there was nonlinear relationship between AUC and dose. When compared with those in the first single administration, the repeated administration resulted in an increase of Vc in low and medium group and AUC in the high group, and reduction in Vc and CL in high group. There was no difference in T1/2βin the three groups.Conclusion: After a single (on the 1st day) intravenous infusion administration of 2.5, 5.0 and 10 mg·kg-1, there was linear relationship between AUC and dose. However, after 4 weeks (on the 27th day) of repeated administration, AUC was not varied with dose in a linear relationship. This non-linear kinetics in vivo may result from the metabolism saturation in the high dose and thus the increment in AUC was not proportional to the dose. Our study suggested that repeated-administration may result in accumulation of dipfluzine hydrochloride in the body.Part4 The metabolic research of dipfluzine hydrochloride in rat and dog. Aim: To investigate the metabolites of dipfluzine hydrochloride in vitro and in vivo in rat and dog.Methods: The liver microsomes was prepared by differencial centrifugation method, the rat and dog microsomal protein contents were determined by Bradford assay method, with BSA as the standard protein. Dipfluzine hydrochloride stock solution in methanol was added to rat or dog liver microsomes respectively. The mixture was shaken for 3 min for equilibration in a shaking water bath at 37℃. The incubation was then initiated by addingβ-NADPH solution. The final concentrations of dipfluzine hydrochloride, NADPH and the microsomal protein were 1 mmol·L-1, 1 mmol·L-1 and 1 mg·ml-1 , respectively in a typical incubation mixture (1 ml) for metabolite identification study. The percentage of methanol in the incubation mixture was kept less than 1% (v/v). For metabolite identification study, samples were incubated for 30 min. The reaction was terminated with a ice bath. Negative controls were prepared with methanol added substituting the dipfluzine hydrochloride. All experiments were carried out in triplicate.Beagle dogs were intravenously given of 2.5 mg/kg dipfluzine hydrochloride. The blood was collected at 30 min and 1 h after administration of dipfluzine hydrochloride and plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. The urine was collected for 0-24 h after administration of dipfluzine hydrochloride. After centrifugation, the supernatant was stored at -20℃until analyzed. The metabolites of dipfluzine hydrochloride were analyzed by LC-MS/MS.Results: After incubation for 30 min, metabolite profiles in females were found similar as their corresponding males for the same species. There were eight metabolites of dipfluzine hydrochloride generated in rat microsomes, which were respectively 1-(4-fluoro-benzene)-4- piperazine-butanone (M1), 4-OH-benzophenone (M2), 4-fluoro-γ-OH- phenylbutyric acid (M3), benzhydrol (M4), benzophenone (M5), 1-OH- diphenylmethyl-4-[3-(4-fluoro-benzene)]-piperazine (M6), 1-OH- diphenylmethyl-4-[3-(4-OH-fluoro-benzene)]-piperazine (M7) and 1- diphenylmethyl -4-[3-(4- fluoro-benzene-methyl)]- piperazine (M8). A new metabolite 1-OH-diphenylmethyl -4-[3-(4- fluoro-benzene-methyl)]- piperazine (M9) was found in microsomes in dog in addition to M1, M2, M3, M4, M5, M6 and M8. While the M7 existed in rat microsomes was not found in dog microsomes.In the dog plasma at 30 min and 1 h after administration of dipfluzine hydrochloride, the metabolites were respectively M1, M2, M3, M4, M5 and M6, and the main metabolites were M1, M4 and M6. The metabolites detected in dog urine were almost the same as that in plasma, with the exception of M1, M2 and M4 as the main metabolites. Both of M8 and M9 were not found in plasma and urine.Conclusion: Based on the metabolite profiling, we proposed that the primary metabolic pathways of dipfluzine hydrochloride in rat and dog were 1,4-N- dealkylation, hydroxylation and/or methylation on benzene ring. The identical metabolites in rat and dog were M1, M2, M3, M4, M5, M6 and M8. M7 existed only in rat and M9 only in dog. The content of M6 in rat microsomes was much higher than that of in dog. The result indicated that the activity of hydroxylase in rat was higher than that in dog, which was also manifested by the different existence of M7 (only in rat and not in dog). The study suggested that there was species-difference for the metabolism of dipfluzine hydrochloride.
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