Animals ; Hepatocytes ; Ketoconazole/pharmacology* ; Liver/pathology* ; Male ; Mice ; Testis/pathology*

OBJECTIVETo evaluate the effect of a 10-day course of triptolide (TP) on rat cytochrome (CY) P3A4 activity, and on the pharmacokinetics of cyclophosphamide (CPA).

METHODSIn the pharmacokinetics experiment, rats were orally given 0.9% NaCl solution (n=5) and TP [1.2 (mg/kg·d)] for 10 days and a single dose of CPA was administered intravenously (100 mg/kg) to rats on day 11. Blood samples were collected up to 4 h at predetermined time intervals, the plasma concentration of CPA was determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were determined. In the in vitro CYP3A4 activity inhibition research, rat blank liver microsomes were divided into 3 groups: a control group, a TS (5 μL, 200 μmol/L) with TP (5 μL, 12.5 μmol/L) group, a TS with ketoconazole (5 μL, 1 μmol/L) group. Concentration of 6β-hydroxylated testosterone (6β-OHT) in liver microsomes was measured by HPLC and the activity of CYP 3A4 was calculated through the following formula: Einhibitor/Econtrol × 100%=Cinhibitor/Ccontrol × 100%.

RESULTSCompared with the control group, the area under the plasma concentration-time curve (AUC0-∞) of CPA was significantly increased by 229.05% pretreated with TP (P<0.01). Peak plasma concentrations (Cmax) of CPA was significantly increased and plasma half-life was correspondingly extended. The CYP3A4 activity was significantly inhibited by ketoconazole 93.5%±0.2% and TP 84.6%±0.3% compared with the control group (P<0.01 and P<0.05, respectively).

CONCLUSIONOur results strongly suggested that long-term oral intake of TP can distinctly inhibit the CYP3A4 activity and this inhibition evidently decrease the formation of toxic metabolites of CPA.

Animals ; Cyclophosphamide ; pharmacokinetics ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; pharmacology ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Herb-Drug Interactions ; Hydroxytestosterones ; metabolism ; Immunosuppressive Agents ; pharmacokinetics ; Injections, Intravenous ; Ketoconazole ; pharmacology ; Male ; Microsomes, Liver ; drug effects ; metabolism ; Phenanthrenes ; pharmacology ; Rats, Sprague-Dawley

Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol L-1 Tris-HCI buffer (pH 7.4) containing 0.5 gL-1 microsomal protein and 50 micro molL-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 micro L. The incubation mixture was pre-incubated at 37 degrees C for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 degrees C, 200 micro L of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including alpha-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes.
Aconitine ; analogs & derivatives ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Enzyme Inhibitors ; pharmacology ; Ketoconazole ; pharmacology ; Male ; Metabolic Networks and Pathways ; Microsomes, Liver ; enzymology ; metabolism ; Quinine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfaphenazole ; pharmacology ; Tandem Mass Spectrometry

Limonin existed in citrus fruits has been shown to have anti-bacterial, anti-viral, anti-feedant, anti-nociceptive, anti-inflammatory activities and anti-carcinogenic activities. But the clinical use is limited by its low bioavailability. The aim of this study is to observe the absorption and secretion transport mechanisms of limonin in intestine which can pave the way for the further study and clinical use. The transport characteristics and mechanisms of limonin in rat were studied by in situ intestine perfusion and in vitro Caco-2 cells method. The intestinal absorption of limonin was probably via a facilitated diffusion pathway which was poor and without segment-selection. Verapamil and ketoconazole improved the absorption remarkably according to the result of in vitro Caco-2 cells study; however, probenecid had no significant effect on the absorption. The P-gp efflux and CYP3A4 metabolism were involved in the poor intestinal absorption and low bioavailability of limonin. The exploration of the intestinal absorption mechanism is crucial to the design of dosage form and clinical use of limonin.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Biological Availability ; Biological Transport ; drug effects ; Caco-2 Cells ; Cytochrome P-450 CYP3A ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Intestinal Absorption ; drug effects ; Ketoconazole ; pharmacology ; Limonins ; administration & dosage ; pharmacokinetics ; Male ; Perfusion ; Probenecid ; pharmacology ; Rats ; Verapamil ; pharmacology

The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.
Anti-HIV Agents ; metabolism ; pharmacokinetics ; Biological Availability ; Bridged Bicyclo Compounds, Heterocyclic ; metabolism ; pharmacokinetics ; Coumarins ; metabolism ; pharmacokinetics ; Cytochrome P-450 CYP3A ; Cytochrome P-450 CYP3A Inhibitors ; Humans ; Intestines ; metabolism ; Ketoconazole ; pharmacology ; Metabolic Clearance Rate ; Microsomes ; metabolism ; Ritonavir ; pharmacology ; Troleandomycin ; pharmacology

To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.
4-Butyrolactone ; analogs & derivatives ; metabolism ; Animals ; Benzoflavones ; pharmacology ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; Cytochromes ; antagonists & inhibitors ; Ketoconazole ; pharmacology ; Kinetics ; Male ; Microsomes, Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trimethoprim ; pharmacology

OBJECTIVETo observe the effect of ketoconazole on the activity of cytochrome P450 (CYP) 1A2 and 3A4 in hepatic microsomes of healthy adults.

METHODSHuman hepatic microsomes obtained from healthy adults were randomly divided into control group and ketoconazole-treatment groups at different concentrations. After 15 min of culture, the substrates (testosterone for CYP3A4 and phenacetin for CYP1A2) were added and incubated for another 20 min. The metabolites (6-testosterone and acetaminophen) were then measured with high-performance liquid chromatography (HPLC) to assess the activities of CYP3A4 and 1A2.

RESULTSSignificant difference was found between the groups in the quantity of 6-testosterone and the relative activity of CYP3A4 (P<0.05). The IC(50) of ketoconazole for CYP3A4 was 0. 16 mg/L. Both the quantity of 6-testosterone and the relative activity of CYP3A4 were reduced gradually with the increment of ketoconazole concentration. Significant differences were found between the ketoconazole groups and the control group in both the quantity of acetaminophen and the relative activity of CYP1A2 (P<0.05). Ketoconazole at low doses reduced CYP1A2 activity and but increased the activities at high doses (P<0.05).

CONCLUSIONIn the range of maximum clinical blood concentration, ketoconazole can inhibit the activity of CYP3A4, but not that of CYP1A2, in the hepatic microsomes in healthy adults.

Adult ; Antifungal Agents ; pharmacology ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Dose-Response Relationship, Drug ; Female ; Humans ; Ketoconazole ; pharmacology ; Male ; Microsomes, Liver ; drug effects ; enzymology

The present study was to evaluate feasibility of a limited sampling strategy (LSS) in the prediction of inhibited hepatic CYP3A activity with systemic clearance of midazolam (MDZ), a hepatic CYP3A activity phenotyping probe. Rats were pretreated with a serial doses of ketoconazole, a selective inhibitor on CYP3A. Blood samples were collected and detected for MDZ at specified time points after intravenous injection of MDZ. Stepwise regression analysis and a Jack-knife validation procedures were performed in one group of rats as training set to establish the most informative LSS model for accurately estimating the clearance of MDZ. Another group of rats with same treatment was used as validation set to estimate the individual clearance based on predictive equations derived from the training set. Bland-Altman plots showed a good agreement between the systemic clearance calculated from DAS (CLobs) and corresponding parameter that was derived from three LSS models (CLest). LSS models derived from two or three sampling time points, including 60, 90 min, 30, 60, 90 min and 30, 60, 120 min, exhibited a good accuracy and acceptable error for estimating the CLobs of MDZ to evaluate hepatic CYP3A activity, especially the 60, 90 min LSS model is most accurate and convenient. The results supported that limited plasma sampling to predict the systemic clearance of MDZ is easier than the usual method for estimating CYP3A phenotyping when the hepatic activity of CYP3A is reduced in the rat. The present study provided theoretical basis and laboratory evidence for LSS to clinically evaluate metabolizing function of liver and
Animals ; Area Under Curve ; Aryl Hydrocarbon Hydroxylases ; antagonists & inhibitors ; genetics ; metabolism ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Injections, Intravenous ; Ketoconazole ; administration & dosage ; pharmacology ; Male ; Metabolic Clearance Rate ; Midazolam ; blood ; pharmacokinetics ; Phenotype ; Random Allocation ; Rats ; Rats, Wistar

To evaluate the effects of p-octyl polyethylene glycol phenyl ether (Triton X-100), polyoxyl 35 caster oil (EL35) and polyoxyl 40 hydrogenated caster oil (RH40) on the activity of Cytochrome P450 3A (CYP3 As) in vivo. Rats were administered with saline, ketoconazole (75 mg x kg(-1) x d(-1)), Triton X-100 (30 mg x kg(-1) x d(-1)), EL35 (150 mg x kg(-1) x d(-1)) and RH40 (150 mg x kg(-1) x d(-1)) intragastrically for 5 consecutive days, and then given midazolam 10 mg x kg(-1) 20 min after the last treatment of ketoconazole or three surfactants with the same dose through duodenal administration. Pharmacokinetics parameters for midazolam and its metabolite 1'-hydroxymidazolam were estimated from the plasma concentration-time data by a noncompartmental approach. The results showed that multiple dose administration of Triton X-100, EL35 and RH40 decreased the ratios of 1'-hydroxymidazolam and midazolam AUC0-infinity from 1.14 to 0.90, 1.03 and 0.64, respectively. In contrast, multiple dose administration of ketoconazole caused the ratios of 1'-hydroxymidazolam and midazolam a significant decrease to 0.50. This study indicated that Triton X-100 and EL35 would have no inhibition on CYP3A, while RH40 had significant inhibition on CYP3A. Therefore, RH40 might be used to prepare drug formulations in pharmaceutical industry and would increase the bioavailability of some drugs transformed by CYP3As and further lead to significant clinical pharmacologic effects.
Animals ; Area Under Curve ; Biological Availability ; Biotransformation ; Cytochrome P-450 CYP3A ; metabolism ; Ketoconazole ; pharmacology ; Male ; Midazolam ; analogs & derivatives ; pharmacokinetics ; Octoxynol ; pharmacology ; Polyethylene Glycols ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Surface-Active Agents ; pharmacology

INTRODUCTIONThe advent of prostate specific antigen (PSA) has resulted in an increased incidence of early detection of prostate cancer recurrence. Patients treated with androgen deprivation therapy (ADT) become hormone-resistant after 18 to 24 months. In patients with biochemical failure, where there is a rise in PSA but no objective evidence of metastases, or in whom there are small volume metastases but who are asymptomatic, there is no standard of care after ADT. Ketoconazole, an antimycotic which affects the synthesis of androgens and other steroids, has shown direct cytotoxic effects in prostate cancer cell lines in in-vitro studies. This study describes our experience with ketoconazole treatment for hormone refractory prostate cancer (HRPC).

MATERIALS AND METHODSA retrospective study of HRPC patients given ketoconazole at the National Cancer Centre and The Cancer Institute from 2004 to 2005 was performed. All eligible patients had histologically proven adenocarcinoma of the prostate and a rising PSA level despite ADT with orchidectomy or luteinising hormone-releasing hormone (LHRH) agonist therapy. All patients received 200 mg of ketoconazole thrice daily. Response was defined as a decline in PSA of at least 50% from the pre-treatment level and confirmed by a second PSA value 4 or more weeks later. The endpoints evaluated were the presence and duration of a response and the toxicity profile of the treatment.

RESULTSA total of 32 patients with HRPC were treated with ketoconazole. Twelve (38%) of the 32 patients had a greater than 50% decrease in their PSA values. The median duration of response was 6.75 months. The median time to reach PSA nadir was 3.5 months. Five patients continue to exhibit progression-free response at the time of writing. Ketoconazole was generally well tolerated. Eighteen (56%) patients recorded mild toxicities related to ketoconazole. There were no grade 3 or 4 toxicities.

CONCLUSIONSLow-dose ketoconazole bridges the gap in the continuum of treatment for patients who have failed ADT and in whom cytotoxic chemotherapy would have a significant impact on the quality of life. Its good toxicity profile, low cost and ease of administration makes it a viable option for this group of patients.

Adenocarcinoma ; Aged ; Aged, 80 and over ; Androgen Antagonists ; administration & dosage ; therapeutic use ; Androgens ; biosynthesis ; Humans ; Ketoconazole ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Prostate-Specific Antigen ; Prostatic Neoplasms ; drug therapy ; Retrospective Studies ; Singapore