Studies On Quality Control Of M-Nisoldipine And It's Metablism In Vitro And In Vivo

m-Nisoldipine[3-iosbutyl-5methyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3, 5dicarboxylate] is a new kind of DHPs which was developed by School of Pharmacy of Hebei Medical University. m-Nisoldipine and nisoldipine which is widely used in clinic to treat coronary artery disease, hypertension, Chronic congestive heart failure and cardiac shock etc. with significant effect are isomerides. After structure modification, m-nisoldipine is not only kept the same pharmacological action of nisoldipine, but also has superior photostability to nisoldipine which is not stable when exposed to light. At present there are several assay methods for m-nisoldipine, such as cerium sulfate method, ultraviolet spectrophotometry, high performance liquid chromatography, and so on. Our present paper described the GC and GC-MS methods for the assay of m-nisoldipine with nimodipine as the internal standard for the first time.The understanding of drug metabolism plays an important role in the development of new drug entities. The studies on drug metabolism can predict the probalble metabolites of a new candidate drug and its potential toxicity, on the other hand, we can foresee whether the studied drug owns the permanent effect, as many candidate drugs have therapeutic effect in vitro but weak pharmacologic action or inefficacy in vivo. The main reasons are the problems about absorption and metabolism of the compounds. Therefore the work pertinent metabolism can provid significant clues for the metabolic process and toxicity evaluation of new drugs, which makes it possible that the much more safer and effective compounds are synthetized.Our present paper described the preliminary research of m-nisodipine's metabolism from two directions in vitro and in vivo and we found several metabolites. In addition, we got a pure product of m-nisoldipine by microbial transformation and confirmed its structure as 3-(2-methyl-2-hydroxyl)-propyl -5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3, 5-dicarboxylat(ewhich was represented by the letter of M in our paper). At last we got some massage about the metabolic pathway of m-nisoldipine in rat.Part one Studies on the assay of m-nisoldipine by GC and GC-MSObjective: To establish methods for the assay of m-nisoldipine by GC and GC-MS.Methods: 1. GC method: (1) Chromatographic conditions'definition: We selected capillary chromatographic column, optimized equipment parameters, such as injector temperature, column temperature and the detector temperature, and adjusted mobile phase flow rate in order to make sure the analytical conditions. (2) Validated the methodology and determinate the samples. 2. GC-MS method: (1) Chromatographic conditions'definition: such as different chromatographic column, injector temperature, column temperature, flow rate of carrier gas, sample injection mode and so on. (2) Mass spectra conditions'definition: such as interface temperature, ion source temperature, quadr-pole temperature, ionization modus, detection mode, and so on. (3) Validated the methodology and determinate the analytical conditions.Results: 1. GC method: (1) Chromatographic conditions were as follows: capillary column was HP-1 (50m×320μm×0.52μm) and the stationary phase was 100% dimethyl-siloxane. Column temperature and injector temperature were controlled at 265℃and 295℃, respectively. The detector was ECD and the temperature was 300℃. The carrier gas was high purity nitrogen with follow rate of 4.0mL·min-1. Split stream sampling and split ratio 20:1 was used. The injection volume was 1μL. (2) The reproducibility of this method was good with RSD of 0.1% and the average recovery was 99.5% with RSD of 0.4%(n=9). The content of three batches of samples were not less than 97%. 2. GC-MS method. (1) Chromatographic conditions were as follows: a DB-1ms capillary column (30m×250μm×0.25μm) was used. The carrier gas was high purity helium with follow rate of 0.8mL·min-1. The programmed column temperature was set as follows: the intial temperature was kept at 200℃, then was raised to 260℃at 20℃·min-1 followed by holding 5min and continually went up to 300℃at 10℃·min-1, and subsequently sustained for 3min, the whole running time was 15min. Injector temperature was 250℃and the sample injection form was spitless. (2) Mass spectra conditions: Interface temperature, ion source temperature and quadr-pole temperature were 250℃, 230℃and 150℃, respectively. Electro-ionization was EI with electron energy of 70ev. Analysis was carried out in SIM mode at m/z 266.0, 210.0 for m-nisoldipine and m/z 296.0,254.0 for internal standard nimoldipine. (3) The RSD for reproducibility of this method was 0.4% and the average recovery was 98.9% with RSD of 0.3%(n=9).Conclusion: The methods we established are proved to be accurate, sensitive and convenient and can be used for the determination of m-nisoldipine, which provided some guideline for qulity control.Part two Studies on metabolism of m-nisoldipine in vitroObjectives: To study the enzymatic reaction kinetics of m-nisoldipine; to find its metabolites by multiple means, such as incubation of liver microsome, microbial transformation, anaerobic culture of intestinal tract bacteria, and prepare pure metabolite as far as possible.Methods: 1. Studies on the enzymatic reaction kinetics of m-nisoldipine: (1) Chromatographic conditions and sample's disposal methods'definition. (2) To validate the methodology. (3) To inspect the effect factor of m-nisoldipine's metabolism rate, such as time, concentration of protein and the original concentration substrate. 2. Liver microsome experiment: m-nisoldipine was added to the incubation liquid which was prepared according to the pertinent literature, after some time, the incubation culture was treated and analyzed by HPLC-PDA and HPLC-MS. 3. Microbial transformation experiment: firstly, we selected a robust strain for the transformation of m-nisoldipine by screening seven kinds of strain, then we got a pure trasformation product and confirmed its structure by its MS and NMR data. 4. Culture solution of intestinal bacteria were produced from rat feces and then incubated with m-nisoldipine to find whether there were metabolites. 5. Fresh content in rat stomach and m-nisoldipine were incubated together, then the mixture was analyzed by HPLC-PDA.Results: 1. Studies on the enzymatic reaction kinetics of m-nisoldipine. (1) Chromatographic conditions: column was Welchrom C18 (4.6mm×250mm×5μm). Mobile phase was acetonitrile-water (62:38) with the flow rate of 1.0mL·min-1. Detect wavelength was set at 237nm and column temperature was controlled at 30℃. The injection volumn was 20μl. Sample treatment method: 200μL natrium hydroxydatum solution was added into 1.0mL incubation system and then the mixture was vortexed for 20s. After that 5mL ethylether-n-hexane was spiked with the mixture and vortexed for another 2min. The new mixture was centrifugated for 5min at 2850×g, from which 3mL organic phase was took out. After the organic phase being dried by N2 at 37℃, the residue was resolved by 40μL methanol and determined by HPLC. (2) The method had good precision with RSD of 6.1% and 1.6%(n=15) for intra-day and inter-day RSD. The accuracy (93.0%) for the method was high and the RSD was 3.8%(n=15). (3) The elimination rate of m-nisoldipine was different with the different time, protein concentration and original concentration of substrate. 2. Liver microsome experiment: we found several metabolites in the liver microsome for both rat and mouse, but there was difference between the two, which demonstrated the species difference of m-nisoldipine's metabolism. 3. Microbial transformation experiment: after screening, Cunninghamella elegans , AS 3.1207 was selected to transform m-nisoldipine for its potential metabolic capability and good reproducibility. We got a metabolite of m-nisoldipine from AS 3.1207 whose chemical name was 3-(2-methyl-2-hydroxyl)-propyl -5-methyl-1, 4-dihydro-2, 6-dimethyl-4-(3-nitrophenyl)-pyridine -3, 5-dicarboxylate. 4. We fonud 4 new products in both culture solution of intestinal bacteria and blank control experiment, which made it difficult to decide whether they were metabolites of m-nisoldipne or not. 5. We found no metabolites in the content of rat stomach.Conclusions: We got a pure product by microbial transformation and confirmed its structure. From liver microsome experiment we found there was species difference for m-nisoldipine's metabolism. Part three Studies on metabolism of m-nisoldipine in vivoObjective: To study the process of m-nisoldipine and its metabolites in rat in order to illuminate the metabolic pathway of m-nisoldipine.Methods: After taking orally m-nisoldipine, blood, urine, bile, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC-PDA. The peaks of m-nisoldipine and its metabolites were identified by comparing their UV spectra. The bile and feces were also analyzed by HPLC-MS.Result: Except hepatic tissue, blood and urine, metablites were found in gastrointestinal tract, bile and feces.Conclusion: m-Nisoldipine was transformed extensively in rat body, metabolites were multiple. The main metabolism site was liver and the principal excretion pathways for m-nisoldipine and its metabolites were bile and feces.