| M-nisoldipine is 1, 4-dihydropyridine calcium ion antagonist, to be presented as a couple of enantiomers, which were firstly composed in School of Pharmacy, Hebei Medical University. The main effect of dihydropyridine calcium ion antagonist was relaxing blood vessel, and they showed remarkable effect on hepertension, cardiac angina etc., clinically. Therefore, the exploitation of the drug of preventing and curing hypertensive is meaningful.In the present study, the ES-OVM column was used to establish a method to separate chirally m-nisoldipine enantiomers and determine their contents. Meanwhile the studies on pharmacokinetics of m-nisoldipine enantiomers in beagle dogs, the absorption kinetics of m-nisoldipine enantiomers in rats'intestines in situ, tissue distribution of R, S-m-nisoldipine in rats and the protein binding rates of m-nisoldipine enantiomers with different plasma, were significant in understanding the pharmacological action and toxicological action of R, S-m-nisoldipine, obtaining the pharmacokinetics parameters, rational administration and quality control. By the way, it is helpful to optimize administration route, elevate the quality and stability of the drugs, and also offerring the experiment foundation for the exploitation of single enantiomer.Part one Chiral separation of m-nisoldipine enantiomers and determination of their contents by RP-HPLCObjective: To establish a method to separate chirally m-nisoldipine enantiomers and determine their contents. Methods: The ES-OVM (150 mm×4.6 mm, 5μm) column was used with the mobile phase of methanol-acetonitrile-0.1% acetic acid (15:15:70, pH 4.5) at the detection wavelength of 237 nm. The flow rate was 0.8 mL·min-1 and the column temperature was 30℃.Results: The resolution between the peaks of m-nisoldipine enantiomers was good. The linear ranges of R, S-m-nisoldipine were 1.25 mg·L-1~25 mg·L-1 (r=0.9998, n=7) and 1.25 mg·L-1~25 mg·L-1 (r=0.9999, n=7) respectively. Both of the limits of detection were 5 ng.Conclusion: The method is specific, accurate, sensitive and can be used for the chiral separation and determination of m-nisoldipine enantiomers.Part two Studies on pharmacokinetics and relative bioavailabilities of m-nisoldipine enantiomers in beagle dogs by LC-MS-MSObjective: To establish a LC-MS-MS method for the measurement of m-nisoldipine enantiomers in the plasma of beagle dogs and to investigate the pharmacokinetics of m-nisoldipine enantiomers after an oral administration in Beagle dogs.Methods: After 6 beagle dogs were orally administrated m-nisoldipine at a dose of 5.0 mg·kg-1, blood samples were obtained from hindlimb's saphenous vein according to the specific schedule, 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24, 48 h and collected in heparinized centrifuge tube, respectively. Sample was pretreated by a single-step protein precipitation with acetonitrile and 50μL was injected into the ES-OVM column. The analysis was performed on a ULTRON ES-OVM column(4.6 mm×150 mm, 5μm) at 20℃with the mobile phase of methanol-acetonitrile-2 mmol·L-1 ammonium acetate solution(15:15:70, v/v) at a flow rate of 0.8 mL·min-1. The detect wavelength was at 237 nm. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using ElectroSpray ionization (ESI) source.Results: A selective and sensitive LC-MS-MS method was firstly established using MRM mode for quantification of m-nisoldipine enantiomers in plasma samples. Moreover, pharmacokinetic and relative bioavailabilities of m-nisoldipine enantiomers in beagle dogs were studied for the first time. The method was sensitive with a lower limit of quantification (LLOQ) of 0.25 ng·mL-1, with good linearity (rs=0.9958 and rr=0.9983) over the linear range 0.25 to 20 ng·mL-1. All the validation data, such as accuracy, precision, intra-day and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic and relative bioavailability studies of m-nisoldipine enantiomers in beagle dogs. Plasma concentrations of R, S-m-nisoldipine after oral administration of m-nisoldipine were determined. The parameters were calculated according to concentration-time curve. After oral administration of m-nisoldipine, the relevant parameters for R- and S-m-nisoldipine were Cmax 12.96, 14.83 ng·mL-1, Tmax 2.5, 2.7 h, t1/2 10.98, 8.57 h, AUC0-t 86.07, 91.56 ng·h·mL-1, AUC0-∞104.09, 93.21 ng·h·mL-1, respectively. The result of statistics analysis shows that there are no significant differences (p>0.05) in Cmax, Tmax, t1/2, AUC0-t and AUC0-∞, the AUC of S-m-nisoldipine were the same as those of R-m-nisoldipine and relative bioavailability was 106.4%.Conclusion: The method is sensitive, simple and suitable for pharmacokinetic parameters studies of m-nisoldipine enantiomers, this study provides necessary evidences for the research and new drug development of m-nisoldipine.Part three Studies on tissue distribution of R, S-m-nisoldipine in ratsObjective: A HPLC method was developed and validated for determination of R, S-m-nisoldipine in rat tissues.Methods: Rats were randomly assigned to four groups. After oral administration of 20 mg·kg-1 methanol solution of R, S-m-nisoldipine, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 10, 30, 90 and 150 min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution(0.3 g·mL-1). Samples of tissues were prepared based on a liquid-liquid extraction. The chromatograms of R, S-m-nisoldipine were achieved on a reversed-phase C18 column(250×4.6 mm, 5μm) with a mobile phase was consisted of acetonitrile and water(62:38, v/v) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 237 nm and the temperature of column was kept at 30℃. The method was applied to study tissues distribution of R, S-m-nisoldipine in rats after a single enantiomer administration of R, S-m-nisoldipine at a dose of 20 mg·kg-1.Results: After a single administration of R, S-m-nisoldipine at a dose of 20 mg·kg-1, by the Two-sample t-test, there were no significant differences between the two enantiomers in each tissue (P>0.05, n=5). It was also found that the concentrations of R and S-m-nisoldipine were high at 30 min and 150 min than that at 90 min in small intestine, lung, liver and spleen, and there was a small quantity of the prototype of R and S-m-nisoldipine in brain.Conclusion: The method is simple and shows high precision and good repeatability and it can be successfully utilized to study the tissues distribution of m-nisoldipine enantiomers in rats. This study shows that m-nisoldipine enantiomers have the phenomenon of hepatoenteral circulation and they can cross the blood brain barrier to arrive at the brain tissue, which would make it possible in vivo to carry out pharmacokinetics and pharmacodynamic studies of R and S-m-nisoldipine in humans.Part four Studies on the absorption kinetics of m-nisoldipine enantiomers in rat's intestines in situObjective: To investigate the absorption kinetics of m-nisoldipine enantiomers at different intestine segments in rats, and develop a high performance liquid chromatography (HPLC) to determine the contents of m-nisoldipine enantiomers and phenolsulfonphthalein at the same time.Methods: The intestine in rats was cannulated for in situ recirculation. HPLC was used to determine simultaneously the concentrations of phenolsulfonphthalein and m-nisoldipine enantiomers, respectively. The chromatograms of R, S-m-nisoldipine were achieved on a reversed-phase Symmetry C18 column(250×4.6 mm, 5μm) with a mobile phase was acetonitrile-0.1% phosphoric acid (62:38, v/v) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 353 nm at 25℃.Results: The absorption rate constants (Ka) of R, S-m-nisoldipine at duodenum, jejunum, ileum, and colon were 0.0338, 0.0346, 0.0370, 0.0308 h-1 and 0.1229, 0.0680, 0.0567, 0.0621h-1, respectively; Ka from intestine at R, S-m-nisoldipine concentrations of 5, 10, 15μg·mL-1 were 0.0664, 0.0551, 0.0402 h-1 and 0.0412, 0.0623, 0.0357 h-1, respectively; Ka at pH of 6.5, 7.4 and 7.9 were 0.0543, 0.0568, 0.0621 h-1 and 0.0443, 0.0513, 0.0547 h-1, respectively; Ka at the concentrations of 0.3%, 0.5%, 1.0% of tween-80 were 0.0581, 0.0586, 0.0553 h-1 and 0.0483, 0.0487, 0.0512 h-1, respectively.Conclusion: The concentrations of R, S-m-nisoldipine and Tween-80 and the pH of drug solution had no distinctive effect on the absorption kinetics. R, S-m-nisoldipine were well absorbed at all segments of intestine in rats. The method is simple and shows high precision and good repeatability and it can be successfully utilized to determine simultaneously the contents of m-nisoldipine enantiomers and phenolsulfonphthalein in rats.Part five Determination of the protein binding rates of m-nisoldipine enantiomers with different plasma by HPLCObjective: To develop a high performance liquid chromatography (HPLC) to determine the protein binding rates of m-nisoldipine enantiomers in human plasma, rat plasma, bovine serum albumin (BSA), and to calculate the correlate parameters of m-nisoldipine enantiomers to different genera plasma proteins.Methods: The binding rates of m-nisoldipine enantiomers with different genera plasma proteins were determined by equilibrium dialysis method. The concentrations of m-nisoldipine enantiomers were assayed by HPLC.Results: The binding rates of R, S-m-nisoldipine with rat, human plasma and BSA were (97.4±8.2)%, (93.0±13.4)%; (90.8±10.5)%, (89.2±9.9)% and (95.3±8.7)%, (91.0±5.7)%, respectively. The method was sensitive with a lower limit of quantification of 0.01 ng in different plasmas.Conclusion: The binding rates of m-nisoldipine enantiomers to human plasma protein, rat plasma protein and BSA were very high. The plasma protein binding rate,βp, Kp and N of R-m-nisoldipine is higher than S-m-nisoldipine. Moreover the binding rate is inversely proportional to plasma concentration of R-m-nisoldipine, while the S-m-nisoldipine is identical.
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