Potentiation of the anticoagulation effect of warfarin by the herbal remedy Shu-Jing-Hwo-Shiee-Tang in rats: The dosing regimen and pharmacokinetic interaction

The herbal remedy Shu-Jing-Hwo-Shiee-Tang (SJHST) has been used in traditional Chinese medical care for the treatment of osteoarthritis. This study aims to examine the influence of SJHST on the oxidation and anticoagulation effect of warfarin in male rats. In three SJHST preparations (S1–S3), hesperidin, gentiopicrin, and paeoniflorin were identified as chemical marker ingredients. The inhibition of liver microsomal warfarin 7-hydroxylation (WOH) activity by 50% methanolic extracts of SJHST was potentiated by β-glucosidase pretreatment, but not by NADPH-fortified microsomal preincubation. Among various ingredients and their β-glucosidase-hydrolyzed products, hesperetin caused the most potent inhibition of WOH. Oral administration of S2 to rats at 2 h after warfarin treatment (WS2_{2-h post}), but not co-treatment (WS2_co), decreased warfarin clearance and increased the maximal plasma concentration and the area under the curve (AUC_0-t, AUC_0-∞) of plasma concentration versus time of warfarin administration. S2 and S3 did not change the coagulation parameters. At 24 h after warfarin administration, the WS2_{2-h post} and WS3_{2-h post} groups had a prothrombin time longer than that of the warfarin group. These results demonstrate that a 2-h post-treatment of rats with SJHST caused pharmacokinetic interaction with warfarin, resulting in prothrombin time prolongation.     

Stimulation of synaptosomal tyrosine hydroxylation by phencyclidine in vitro

Phencyclidine (PCP), a potent psychoactive drug, produces some animal behavior that are belived to be mediated by dopaminergic and/or cholinergic neurons in the basal ganglia. In this study, we have monitored the effects of PCP in vitro on the synthesis, uptake, and release of dopamine (DA) in rat striatal synaptosomes. Using tyrosine hydroxylation as an index of DA synthesis, we observed a concentration-dependent stimulation of DA synthesis by PCP. The stimulatory effect was antagonized by reserpine (1 μM) and was observed only when synaptosomes were preincubated under conditions which prevented the spontaneous release of [³H]DA. Two hydroxylated metabolites of PCP were also tested and found to have little effect on tyrosine hydroxylation. Like PCP these metabolites are potent inhibitors of synaptosomal [³H]DA uptake, but they apparently lack PCP's ability to release synaptosomal DA. Taken together, these results support our hypothesis that PCP stimulates synaptosomal DA synthesis by releasing DA from an inhibitory pool.     

Benzo(a)pyrene metabolism in hepatic microsomes from female DA rats with a genetic sparteine oxidation deficiency

The impact of the genetic hydroxylation deficiency described for several drugs such as sparteine, debrisoquine, and phenformin, has been studied with respect to benzo(a)pyrene (BP) metabolite formation. 14C-BP (80 microM) was incubated with liver microsomes from female DA rats deficient in sparteine oxidation; microsomes from female Sprague-Dawley rats served as controls. BP metabolites were separated by high pressure liquid chromatography (HPLC). No significant differences were detected in the overall formation rate of 9,10-,4,5-, and 7,8-dihydrodiol-BP, of 4 quinones, or of 9-OH- and 3-OH-BP. Thus, the study suggested no association between genetic hydroxylation polymorphism (debrisoquine/sparteine type) and the formation of at least 6 BP metabolites.     

丹参酚酸类对羟自由基的捕捉作用

研究了丹参酚酸类对EDTA－H2O2－Fe^2 和Vit C-H2O2-Fe^2 系统中水杨酸的羟基化作用，在后一体系中，这类化合物剂量依赖性地抑制羟化作用，其作用强度和抗脂质过氧化基本一致，而在前一体系中，作用较复杂，大多数丹参酚酸类剂量依赖性地抑制水杨酸羟化作用，但丹参酚酸A作用例外－和Vit C作用相似，丹参酚酸A在低剂量时促进水杨酸羟基化作用，而在高剂量时则抑制，这类化合物能与铁离子结合或络合，但其络合常数较EDTA－铁络合常数低二个数量级以上，因而认为丹参酚酸类化合物可能具有羟自由基捕捉作用。     

Pharmacokinetics and chronic toxicity of cyclosporine A in genetic hydroxylation-deficient dark Agouti rats

Since oxidation plays a key role in the metabolism of cyclosporine A (CsA), the pharmacokinetics and the toxicity of CsA was investigated in female dark Agouti rats exhibiting a deficiency for debrisoquine hydroxylation and for dextromethorphan demethylation. When compared with Wistar rats (n=10), dark Agouti rats (n=10) had a higher mean clearance (4.8 ml/min per kg vs. 3.3 ml/min per kg) and a lower mean residence time (606 min vs. 1361 min) after intravenous dosing of CsA. The systemic availability of subcutaneous CsA was close to 100%. The steady state CsA concentrations assessed by HPLC in whole blood after subcutaneous dosing of 20 mg/kg per day for 23 days (n=10) were about 1000 ng/ml in dark Agouti rats. When compared with dark Agouti rats treated with cremophore (n=10) or not treated at all (n=12, dark Agouti rats on chronic subcutaneous CsA plus cremophore for 23 days (n=10) had no difference in kidney histology but had slightly increased liver fatty changes. Rats on CsA and/or cremophore had a decreased uric acid clearance and evidence of hypoaldosteronism. The urinary ratio of debrisoquine/4-hydroxydebrisoquine decreased in rats on CsA, whereas the O-demethylation and N-demethylation of liver obtained from rats on cremophore was impaired. Thus, dark Agouti rats show no difference in the metabolism of CsA and when given CsA for 23 days show drug-induced functional but no relevant structural light microscopic changes in the kidney, and functional and slight structural changes in the liver.Supported by the Swiss National Foundation for Scientific Research, Grant 32-9497-88. B. M. Frey was supported in part by the Cloetta Foundation.     

Polymorphic debrisoquine and mephenytoin hydroxylation in patients with pulmonary hypertension of vascular origin after aminorex fumarate

Summary During the period 1967 to 1971 an increase in the incidence of pulmonary hypertension of vascular origin (PHVO) was observed in Austria, Federal Republic of Germany, and Switzerland. Most patients had been given aminorex fumarate and a possible link was suspected. We therefore investigated the possibility of genetically — determined drug hydroxylation deficiencies (debrisoquine or mephenytoin type) in these patients as an explanation for the development of PHVO. Seventeen patients took 10 mg debrisoquine and 100 mg mephenytoin orally. Sixteen PHVO patients were classified as extensive metabolizers of debrisoquine with logarithmic metabolic ratios of –0.35±0.11 (mean±SEM), whereas one patient was a poor metabolizer with a logarithmic metabolic ratio of 1.82. For the mephenytoin hydroxylation sixteen patients with PHVO were extensive metabolizers, with logarithmic hydroxylation indices of 0.27±0.05. One poor metabolizer of mephenytoin had a logarithmic hydroxylation index of 1.59. Deficient hydroxylation of debrisoquine and mephenytoin was found in two different patients. The prevalence of poor metabolizers among patients with PHVO after aminorex fumarate was therefore approximately 9% for both debrisoquine and mephenytoin. This corresponds closely to the data of our reference population study where genetic debrisoquine and mephenytoin hydroxylation deficiencies occured independently, with a prevalence of 10% and 5% respectively. Thus, the normal prevalence of extensive drug hydroxylation phenotypes in patients with PHVO is not consistent with the hypothesis that the development of PHVO after aminorex fumarate might be related to a pharmacogenetically determined impairment of polymorphic drug oxidation.Supported by the Swiss National Science Foundation, Berne, Switzerland     

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1

1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes.

2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC–MS/MS and/or NMR analyses.

3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors.

4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible.

5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.     

Pharmacogenetics of mephenytoin: A new drug hydroxylation polymorphism in man

Summary Inherited deficiency in mephenytoin hydroxylation was observed in a family study. It is important that the propositus was of the extensive metabolizer phenotype for the genetically controlled hydroxylation of debrisoquine. Thus, a genetic polymorphism of drug hydroxylation was suspected for mephenytoin. A population study of mephenytoin hydroxylation, combined with identification of extensive and poor debrisoquine hydroxylation phenotypes, was carried out in 221 unrelated normal volunteers. Twelve of them (5%) exhibited defective aromatic hydroxylation of mephenytoin, and 23 (10%) could be identified as poor metabolizers of debrisoquine. Amongst these 35 subjects with a drug hydroxylation deficiency, 3 (or 0.5%; 1 female, 2 males) displayed both defects simultaneously. A panel study of 10 extensive and 10 poor metabolizers of mephenytoin showed that the ability to perform aromatic hydroxylation of the demethylated mephenytoin metabolite nirvanol (5-phenyl-5-ethylhydantoin) was co-inherited with the mephenytoin hydroxylation polymorphism. Family studies suggested that poor metabolizer phenotypes of nirvanol and mephenytoin were most likely to have the homozygous genotype for an autosomal recessive allele of deficient aromatic drug hydroxylation. Intra-subject comparison of the debrisoquine and mephenytoin hydroxylation phenotypes in these subjects indicated that deficiency in the two drug hydroxylations occurred independently. Consequently, the co-inheritance of extensive and poor hydroxylation of mephenytoin and nirvanol, respectively, represents a new drug hydroxylation polymorphism in man.     

Isolation and identification of phase I metabolites of butyrolactone I in rats

1.?Butyrolactone I (BL-I), one of the major secondary metabolites of fungus Aspergillus terreus, is a selective cdc2 kinase inhibitor. In the present study, the metabolism of BL-I in male Wistar rats was investigated by characterizing metabolites excreted into feces.

2.?Following an oral dose of 40?mg/kg BL-I, 10 phase I metabolites were isolated from the feces of rats, and their structures were identified on the basis of a range of spectroscopic data and ICD analysis. These metabolites were fully characterized as butyrolactone VI (M1), aspernolide E (M2), 7′′S-hydroxy-9′′-ene-butyrolactone I (M3), 7′′R-hydroxy-9′′-ene-butyrolactone I (M4), 7″S, 8″R-dihydroxy-aspernolide E (M5), 7″R, 8″S-dihydroxy-aspernolide E (M6), 7″R-acetyl-8″S-hydroxy-aspernolide E (M7), 7″S-acetyl-8″R-hydroxy-aspernolide E (M8), 7″R-methoxy-8″S-hydroxy-aspernolide E (M9), butyrolactone V (M10), respectively. It is the first time to describe the metabolites of BL-I in vivo, and metabolites M3 to M9 are new compounds.

3.?BL-I and metabolites M2 to M10 were evaluated for their antimicrobial activity and in vitro antiproliferative activities. Only M-3 and M-4 showed inhibitory effect against staphylococcus aureus both with MIC of 125?μg/ml. BL-I and metabolites M-4 and M-5 exhibited potent cancer cell growth inhibitory activities against HL-60 (human leukemia) cell lines with the IC₅₀ values of 13.2, 28.8 and 35.7?μM, respectively.

4.?On the basis of metabolites profile, a possible metabolism pathway for BL-I in rats has been proposed. This is the first systematic study on the phase I metabolites of BL-I.     

Microbial Models of Mammalian Metabolism: Stereoselective Metabolism of Warfarin in the Fungus Cunninghamella elegans

Biotransformation stereoselectivity of warfarin was studied in the fungus Cunninghamella elegans (ATCC 36112) as a model of mammalian metabolism. This organism was previously shown to produce all known phenolic mammalian metabolites of warfarin, including 6-, 7-, 8-, and 4-hydroxywarfarin, and the previously unreported 3-hydroxywarfarin, as well as the diastereomeric warfarin alcohols, warfarin diketone, and aliphatic hydroxywarfarins. Using S-warfarin and R-warfarin as substrates, and an HPLC assay with fluorescence detection to analyze metabolite profiles, the biotransformation of warfarin was found to be highly substrate and product stereoselective. Both aromatic hydroxylation and ketone reduction were found to be stereoselective for R-warfarin. Ketone reduction with the warfarin enantiomers exhibited a high level of product stereoselectivity in that R-warfarin was predominantly reduced to its S-alcohol, while S-warfarin was reduced primarily to the corresponding R-alcohol.