| Research objective and background:Minipig was a good animal model for research on the cardiovascular deseases of human, because its heart and cardiovascular system were very similar to human. Minipig models of cardiovascular system diseases are closely resemble those of human. Thus, clinical application of cardiovascular drugs could be well guided using minipigs model to study pharmacokenetic. Additionally, minipig was also considered as a good model of human CYP 3A4 without induction. It has been reported there were similar enzymes as human CYP 3A4/5 in liver microsomes of minipigs. Sequences analysis indicated the CYP 3A of minipig had 60% homologous with human CYP 3A4. These studies suggested minipigs have a predicted prospect as experimental animals for evaluting drugs which were metabolized by human CYP 3A4. However, no systematic research was carried out.China has copious resource of minpigs. Bama miniature pigs were found in China with many advantages, such as high extent of inbreeding, white hair, bearing inbreeding, stable heredity and a consistent phenotype, and were proved to be a superior animal models. Additionally, there are much background information about anatomy, physiology, biochemistry and basic biological characteristics. In China, however, they have never been used in drug safety evaluation due to the scanty application research background on pharmacology and toxicology evaluation.In our previous study, lovastatin, a basic drug widely used in curing AS, was chosed as model drug to investigate its metabolism in Bama miniature pigs completely. In this study, we further used this drug to investigate the disposition (distribution and excretion) and long-term toxicity in vivo, metabolism of microsome (comparing activity and analysising metabolites) and main metabolic enzyme (cytochrome P450 3A) in miniature pigs. All data were compared with human and other animals.Contents and results of the research:(1) Distribution and excretion of lovastatin in Bama miniature pigs.6-month male Bama minipigs were intragastric administrated (45 mg/kg or 2.4 mg/kg). The concentration of lovastatin in tissues and body-fluid in 6-month old male Bama minipigs after oral administration were detected by RP-HPLC. The distribution-excretion process and transformation efficiency of LV-HA were investigated. Protein binding was determined by dialysis method. All results were compared with different experimental animals.Lovastatin was showed to be widely distributed in many tissues after i.g. dose of 45 mg/kg. The higher level of lovastatin was found in the stomach, intestine and liver. The concentrations in stomach, small intestine, liver, heart, adrenal and bladder at 1 h were higher than those of 4 h. The drug was found in blood-brain barrier of Bama miniature pig. 7.4% of the given dose was excreted in unchanged form in urine within 96 h. More than 80% was excreted in bile and feces within 96 h. The plasma protein binding ratio was higher than 95% and transformation efficiency of LV-HA was very similar between human and pig plasma.(2) Long-term toxicity test of lovastatin in Bama miniature pigs.6-month male Bama minipigs were randomly divided into two dose groups(12 mg·kg-1 and 135 mg·kg-1)and a blank group. The drug was given once a day for 42 successive days. Manifestation and behaviors, hematology, blood chemistry and histopathology were detected.The results revealed that 135 mg·kg-1 dose caused serious toxicity. The main toxic manifestations were piloerection and diarrhea, body weight loss and declined foraging quantity. Compared to control, total white cell count increased, however red blood count and hemoglobin and blood platelets count declined. ALT,AST,ALP and CK were increased 2-10 fold. Lovastatin has been shown to be highly effective in reducing plasma total-C, LDL, total-C/HDL ratio and LDL/HDL ratio. In addition, lovastatin modestly decreased plasma TG.The pathological histology analysis result showed that there were some morphological abnormalities and characteristic pathological change in the major target organ, liver and kidney.(3) Lovastatin metabolism of liver microsomes and essential metabolites in Bama miniature pigsTo evaluate the feasibility of Bama miniature pigs to be used as a model on pharmacokenetic properties of CYP 3A4, we compared the enzymatic kinetics of lovastatin and nifedipine in liver microsomes of Bama miniature pigs with that of human and rats. We evaluated the biotransformation activity in the minipig liver microsomes and compare with those of human and rat. The inhibitory and induction effects were determined by using chemical inhibitor and inductor specific to the CYP 3A in human. Metabolites of lovastatin were detected by UV, HPLC and MS.The enzymatic kinetics of minipig liver microsomes was more close to human than rats. The metabolism of lovastatin in three various genera was significantly inhibited by ketoconazole but the descendent rate of metabolism in pigs was similar to human. The average activity levels of nifedipine of Bama miniature pigs increased remarkablely when treated with rifampicin. Otherwise, the metabolites of lovastatin in vitro and in vivo of pigs were also close to that of human.(4) Expression of CYP 3A29 in Escherichia coliA P450 gene (3A29) from Bama miniature pigs, which was homologous to human CYP 3A4 gene, was successfully expressed in Escherichia coli. The expressed CYP 3A29 was confirmed by SDS-PAGE and western blot analysis. We investigated the effects of culture broth, temperature, IPTG concentration and additives on the expression level of CYP 3A29. The metabolism characteristics of CYP 3A29 expressed in E. coli were compared with those from human.Through constructed the truncated CYP 3A29 gene without 28 N-terminal amino acids (membrane-anchor signal peptide), we obtained active CYP 3A29 protein in cytoplasmic fraction of E. coli cells. We found the optimal conditions to produce active CYP 3A29 were to induce E. coli cells contained pET28b-3A29 with 10mM IPTG in TB broth for 10h at 25℃. 3A29 produced in E. coli has the oxidation activity of Nifedipine (NOD), which was the same as that in liver microsomes of pigs.Conclusions:(1) Our results showed the distribution-excretion, plasma protein binding ratio and transformation efficiency of LV-HA of lovastatin in the Bama minipigs were similar to those in humans. Thus, it could be concluded that Bama miniature pigs should be an ideal experimental animal of pharmacokinetics for cardiovascular drugs similar to lovastatin.(2) Bama miniature pigs could sensitively show toxic reactions which were observed in clinical therapy of lovastatin.(3) Compared with rats, Bama miniature pigs were more close to humans in the biotransformation activity of CYP 3A and the inhibitory and induction effects by chemical inhibitor and inductor specific to the CYP 3A. Therefore, Bama miniature pigs are suitable for the pharmacological studies relating to human CYP 3A.(4) The CYP 3A29 enzyme of Bama miniature pigs expressed in E. coli showed the oxidation activity of Nifedipine as human CYP 3A4. This indicated Bama minipig was a suitable animal model for human drugs which was metabolized by CYP 3A.Combined these results, we can collecte a complete data about metabolism of lovastatin in Bama miniature pigs. We evaluate the feasibility of Bama miniature pig to be used as a good model on safety evaluation of cardiovascular drugs which was metabolismed by human CYP 3A4.
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