| Objective:1. To determinate the dissolution in vitro of crystal type I and crystal type IV nitrendipine tablets and evaluate the similarity of dissolution between pharmaceutics for evaluating the effect of crystal polymorphism on the dissolution and providing theoretical basis for the clinical research.2. To establish a reliable high performance liquid chromatography tandem mass spectrometry (HPLC-MS) for determining the plasma concentration of nitrendipine in study of human bioequivalence.3. To study the the pharmacokinetics and bioequivalence of crystal type I and crystal type IV nitrendipine tablets in healthy volunteers, and to evaluate characteristics of absorption, distribution, metabolism and excretion and differences in bioavailability for providing theoretical basis for further clinical application and approval of new drug.4. To study the effect of CYP3A5 gene polymorphism on the pharmacokinetics of nitrendipine for searching the cause of the differences between individuals.Methods:1. The dissolution conditions are as follows:the 900 ml of 0.5% sodium dodecyl sulfate (SDS) solution as the dissolution medium, the temperature as 37±0.5 ℃, and the speed as 100 r/min. According to the paddle method of dissolution determination, six tablets of the new crystal type IV nitrendipine tablets and two kinds of crystal type I nitrendipine tablets were determinied, respectively. The 5 ml solution was removed at 5, 15,30,45,60,90,120,180,120,360 min, respectively, and then filtered by 0.45 μm microfiltration membrane. The subsequent filtrate was analyzed by the new established and validated HPLC method for determinating the dissolution in vitro of crystal type IV and two kinds of crystal type I nitrendipine tablets. The similarity factor f2 method was used to evaluate the similarity of dissolution among the three kinds of nitrendipine tablets.2. A new HPLC-MS method was established for determining the concentration of nitrendipine in human plasma.The chromatographic column was Inertsil(?)ODS-3 (4.6 x150mm,5μm), and the mobile phase was acetonitrile:5mM ammonium acetate buffer (70:30, V/V). The flow rate was 0.8 mL/min with the column temperature as 25℃, and 10μL of the extracting solution was analyzed. The ESI ion source was used in the mass spectrometry by negative ion mode. The mass to charge ratio (m/z) of 359.1 and 345.1 were monitored for nitrendipine and nifedipine as internal standard by the selective ion detection(SIM) mode, with the fragment voltage as 100 v and 80 v respectively. The capillary voltage wad 3500 v and the velocity of dry gas (N2) was 11.0 L/min, with the drying temperature as 350 ℃. The methodology validation was carried out after the nitrendipine plasma samples were dealed by the liquid-liquid extraction method.3. A 3-period crossover, double 3x3 Latin square design was employed with the new crystal type IV nitrendipine tablet as the test preparation (T) and two kinds of commercially available crystal type I nitrendipine tablets as reference preparations (R1, R2). using of experiment. The 24 healthy male subjects were randomly divided into six groups after paired according to the weight, and were orally administrated 20 mg the new crystal type IV nitrendipine tablets or crystal type I nitrendipine tablets. Blood samples were collected before dosing and at 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0,6.0, 8.0,12.0 and 24.0 h after dosing. The sensitive liquid chromatography mass spectrometry (HPLC-MS) method was used to determine nitrendipine in plasma. Drug And Statistics 2.1.1 was used to calculate the pharmacokinetic parameters, and the relative bioavailability was calculateted by the ratio of AUC0-24h of the test preparation with reference preparation. The variance analysis, two one-side t test and 90% confidence interval of the main pharmacokinetic parameters were adopted to evaluate the bioequivalence of test preparation and reference preparations.4. The blood (2ml) was collected from peripheral venous, and the DNA was extracted by DNA extraction kit. The fluorescence in situ hybridization (FISH) method was used to detect the CYP3A5 genotype of the 24 subjects, after the eluent of DNA was added to the CYP3A5 gene detection kits. According to the results of CYP3A5 genotype, the subjects were divided into different groups. On the basis of the human bioequivalence test, the average pharmacokinetic parameters of each group were calculated. The statistical analysis of two groups was counted using SPSS 17.0 software by the independent-sample t test. In consider of the effect of crystal polymorphism on bioavailability, the effect of CYP3A5 gene polymorphism on the pharmacokinetics of crystal type I and crystal type IV nitrendipine tablets was studied separately.Results:1. The f2 value of crystal type IV nitrendipine tablet (A) relative to the two kinds of crystal type I nitrendipine tablets (B and C) were 21.30 and 19.64, respectively. The f2 values were lower than 50 indicating that the dissolutiont of the former was not similar to the two latter. Relative to the crystal type I nitrendipine tablet, the dissolutiont of the crystal type Ⅳ nitrendipine tablet was increased significantly. The f2 value of the two kinds of crystal type I nitrendipine tablets was 72.85 which was higher than 50 indicating that the dissolutiont between the two kinds of crystal type I nitrendipine tablets was similar.2. The endogenous impurity in plasma did not interference the determination of nitrendipine and nifedipine as internal standard, indicating that the new established HPLC-MS method was of high specificity. Linearity of nitrendipine was achieved over a plasma concentration range from 0.2 to 40 ng/mL (r2>0.99), and the lower limit of quantification (LLOQ) was 0.2 ng/mL. The results of the intra- and inter-day accuracy and precision investigated by analyzing the low, middle and high (0.5,5,35ng/mL) QC samples were within ± 15%, meeting the determination requirements. The extraction recovery of nitrendipine was about 70%, and the matrix effect was 96.89-101.38%, indicating that the plasma matrix did not influence the plasma concentration of nitrendipine. Stability studies including post-preparative (16h), freeze-thaw (one and two cycles), long term freeze (56d) stability, and the results of them were stable. The HPLC-MS method was sensitive, accurate and suitable for analyzing the nitrendipine plasma samples for human bioequivalence study.3. The main pharmacokinetic parameters of the crystal type IV nitrendipine tablet (T) and the two kinds of crystal type I nitrendipine tablets (R1、R2) were as follows:t1/2 (5.075±2.430) h, (3.031±2.264)h and (3.468±1.916)h, respectively; Tmax (2.625±0.516) h, (2.188±0.640) h and (2.083±0.761) h; Cmax (10.759±7.809) ng-mL-1, (3.291±2.300) ng-mL-1 and (3.437±2.240) ng-mL-1; AUC0～24h (46.354±39.409) ng·mL-1·h, (13.316±13.271) ng·mL-1·h and (13.326±9.994) ng·mL-1 h; AUC0～∞ (49.394±41.909) ng·mL-1·h, (14.802±14.249) ng·mL-1·h and (14.807±11.195) ng·mL-1·h. The ratio of AUCo-24h between the two kinds of crystal type I nitrendipine tablets (R1、R2) was 100.08%. The bioavailability of crystal type IV nitrendipine tablet (T) relative to the two kinds of crystal type I nitrendipine tablets (R1、R2) were 348.11% and 347.84%, respectively. The 90% confidence interval of ln(Cmax) and ln(AUCo-24h) of crystal type IV nitrendipine tablet (T) relative to the two kinds of crystal type I nitrendipine tablets (R1、R2) were not within 80%-125%, indicating that the former was not bioequivalent to the two lattter. Compared with the crystal type I nitrendipine tablet, the bioavailability of crystal type IV nitrendipine tablet increased significantly.4. The results of the CYP3A5 genotype of 24 subjects were as follows: CYP3A5*1/*1 (wild type) as 10 subjects, CYP3A5*1/*3 (heterozygous mutation type) as 10 subjects and CYP3A5*3/*3 (homozygous mutations type) as 4 subjects.For the crystal type IV nitrendipine tablet, the average pharmacokinetic parameters of the CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3 groups were as follows:t1/2 4.909 h,4.796 h and 6.1905 h; Tmax 2.500 h,2.650 h and 2.875 h; Cmax 8.250 ng·mL-1, 11.337 ng·mL-1 and 15.587 ng·mL-1;AUC0～24h 35.835 ng·mL-1·h,48.202 ng·mL-1·h and 68.033 ng·mL-1·h AUC0～∞,37.957 ng·mL-1·h,51.132 ng·mL-1·h and 73.639 ng·mL-1·h. Compared with CYP3A5*1/*1 and CYP3A5*1/*3, the Cmax and AUC0-24h of CYP3A5*3/*3 were increased, but there were no significant differences among them (P=0.139,0.449 for Cmax; P=0.152,0.511 for AUC0～24h). There were no significant differences of AUC0～∞, t1/2 and Tmax (P>0.05).For the crystal type I nitrendipine tablet, the average pharmacokinetic parameters of the CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3 groups were as follows:t1/2 3.126 h,3.648 h and 2.561 h; Tmax 2.100 h,2.150 h and 2.188 h; Cmax 3.399 ng·mL-1,3.452 ng·mL-1 and 3.057 ng·mL-1; AUC0～24h 11.925 ng·mL-1,15.999 ng·mL-1 and 10.116 ng·mL-1·h; AUC0～∞,13.066 ng·mL-1,18.036 ng·mL-1 and 11.075 ng·mL-1·h. It was different from the crystal type IV nitrendipine tablet, because the Cmax and AUCo-24h of CYP3A5*3/*3 group were not increased compared with the CYP3A5*1/*1 and CYP3A5*1/*3 groups. There were no significant differences of all the pharmacokinetic parameters (P>0.05).Conclusion:1. Relative to the two kinds of crystal type I nitrendipine tablets, the dissolutiont in vitro of the crystal type IV nitrendipine tablet was increased significantly. The crystal polymorphism had a significant impact on the dissolutiont of nitrendipine.2. The crystal type IV nitrendipine tablet was not bioequivalent to the two kinds of crystal type I nitrendipine tablets, and there were significant differences for the pharmacokinetics between them. Compared with the crystal type I nitrendipine tablets, the bioavailability of crystal type IV nitrendipine tablet was increased significantly. The absorption, distribution and metabolism of different crystal types of nitrendipine were different, and the crystal polymorphism had a significant impact on the bioavailability of nitrendipine tablets.3.The CYP3A5 gene polymorphism had no significant effect on the pharmacokinetics of crystal type IV and crystal type I nitrendipine tablets, which was not the main reason for the pharmacokinetic differences between individuals. |
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