Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T】C, 1146C】T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.

Antihyperglycemic effects of the methanol leaf extract of Diaphananthe bidens in normoglycemic and streptozotocin—induced hyperglycemic rats

Objective:To evaluate the methanol leaf extract of Diaphonathe bidem(D.bidenx)(AFZEL EX SW) SCHLTR for antihyperglycemic activity in order to confirm it antidiabetic potential.Methods: D.bidenx was extracted by cold maceration for 48 h and concentrated in vacuo to yield D.bidenx extract(DBE).Hyperglycemia was induced by intraperitoneal administration of slreplozotocin (75 mg/kg).Oral glucose tolerance test was done with 2 g/kg glucose load in normal rats.DBE (150.300 and 600 mg/kg) was administered orally,while tolbutamide(100 mg/kg.p.o.) was used as the standard reference drug.Blood glucose levels determined using ACCUCHEK glucose autoanalyzer. The acute toxicity and phytochemical studies were also carried out.Results:DBF. (600 mg/kg) and tolbutamide(100 mg/kg) significantly(P【0.05,0.005) reduced blood glucose levels of rats between 120 and 480 nun post administration in normal rats.In the streptozotoeininduced hyperglycemic rats,DBE(150.300.600 mg/kg) caused significant(P【0.001)dose- and time- dependent reduction in the blood glucose levels by 1.7%.22.8%and 43.4%,respectively at 480 min compared to the negative control group.DBE(600 mg/kg) reduced the blood glucose level of rats by 1.2%in the oral glucose tolerance test when compared with the normal saline treated group.The acute toxicity test showed that DBE was safe at the doses used and the phytochemical screening revealed the presence of saponins,steroids,tannins and terpernoids.Conclusions:D. bidens extract possess antihyperglycemic activity which may be mediated through pancreatic and extra-pancreatic pathways,thereby justifying it folkloric use.

In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS

This study explored the effects of cucurbitacin E （CUE）, a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chro- matography-（tandem） mass spectrometry （LC-MS/MS） assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE （1.25-100μmol· L-1） competitively inhibited tolbutamide 4-hydroxylation （CYP2C11） activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1 in vitro. In whole animal studies, no signifi- cant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, mul- tiple pretreatments of CuE （200 μg kg-1 d-1, 3 d, i.p.） significantly decreased tolbutamide clearance （CL） by 25% and pro- longed plasma half-time （T1/2） by 37%. Moreover, CuE treatment （50-200 pg kg-l d-1, i.p.） for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or nat- ural products containing CuE to avoid unnecessary drug-drug interactions.

Xiao Chai Hu Tang for Liver Diseases:A Literature Review

The objective of this study is to summarize the pharmacological effects and the mechanisms of action of Xiao Chai Hu Tang(XCHT, Minor Bupleurum Decoction) on liver diseases, so as to give relevant researchers a valuable insight and benefit patients with hepatopathy. PubMed was used to search for and collect scientific publications related to XCHT and liver diseases from 1986 to 2016. The available scientific results or evidence were read, classified, and analyzed. XCHT showed clinical efficacy in patients with hepatic diseases including hepatitis,hepatic fibrosis, and hepatoma. The mechanisms involved the production of cytokines, the regulation of immune function, the suppression of lipid peroxidation, etc., XCHT might work on the metabolism of some medications such as tolbutamide by the regulation of gastric emptying and intragastric pH. XCHT exhibited a very low toxicity profile, such as interstitial pneumonia due to duration of medication,patients' age, and drug combination. XCHT has been a eutherapeutic supplemental remedy for liver diseases. However, many mechanisms of action and effects of XCHT on new types of liver diseases still remain unclear, so more and more animal experiments and human clinical trials are needed to obtain enough proofs for the clinical use of XCHT in new types of hepatosis such as nonalcoholic fatty liver disease and autoimmune liver disease.