Cytochrome p450 inhibitory properties of common efflux transporter inhibitors.

Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.

Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition.

Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.

Influence of simvastatin on the thromboxane and prostacyclin pathways, in vitro and in vivo.

3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) are believed to exert beneficial effects against cardiovascular disease beyond correction of dyslipidemia. The aim of this combined in vitro and in vivo study was to investigate the influence of the commonly used simvastatin on prostacyclin and thromboxane A2, 2 prostaglandins with different cardiovascular effects, normally in homeostatic balance in the circulatory system. Single-dose administration of simvastatin significantly decreased urinary prostacyclin excretion of healthy volunteers (P < 0.01) and increased the ratio between thromboxane A2 and prostacyclin (2-fold increase, P < 0.01), as assessed by enzyme immunoassays of the corresponding metabolites in urine. Human vascular endothelial cells, exposed to corresponding concentrations of simvastatin and assayed in the same way, reduced the release of prostacyclin about 40% (P < 0.05), altered the transcriptional expression of cyclooxygenase and prostacyclin synthase as analyzed by real-time polymerase chain reaction, and reduced the prostacyclin synthase promoter activity by 50% (P < 0.05), evaluated in a luciferase reporter system. We speculate that simvastatin shifts the balance between thromboxane A2 and prostacyclin in favor of the thromboxane pathway in vivo, and after exposure to clinically relevant concentrations in vitro. This may have pathophysiological implications by promoting a prothrombotic state in the blood vessels.

Statins inhibit expression of thioredoxin reductase 1 in rat and human liver and reduce tumour development.

BACKGROUND: Statins have been reported to have anti-carcinogenic properties in addition to their cholesterol-lowering effects, but the mechanism is unknown. Thioredoxin reductases (TrxR) are selenium-containing enzymes of great importance for carcinogenesis and their levels are increased in neoplastic cells. The aim of the present study was to investigate if statin treatment is associated with alterations in the hepatic expression of TrxR. METHODS: Human liver biopsies from a study where patients had been randomised to statin treatment or placebo were analysed. In addition we used liver tissue from a human liver bank where statin treated subjects were compared with non-treated. We also used tissue from a rat liver cancer model in which we have previously shown anti-carcinogenic effects of statins. Real-time PCR and activity assay were used to determine TrxR-levels and activity in tissue extracts. RESULTS: In humans 80 mg atorvastatin treatment for 4 weeks (n = 6) was associated with 85% lower levels of TrxR1 and TrxR2 compared to placebo-treated patients (n = 8) (p = 0.03). In liver biopsies from a human donor liver bank 3 statin treated subjects had 90% lower expression of TrxR1 than 15 non-treated subjects (p = 0.04). Statin treatment was associated with 45% lower expression and activity of TrxR1 in a rat model for liver cancer (p = 0.03). There was a clear correlation between inhibition of carcinogenesis and decreased TrxR1-levels (p = 0.003). CONCLUSION: Statin treatment decreases the hepatic expression of TrxR1 in humans and rats. Suppression of TrxR1 expression could explain possible anti-carcinogenic effects of statins. In addition, decreased levels of TrxR1 during statin treatment may shed light on the mechanism of other side-effects of statins.

Beneficial vasoactive endothelial effects of fluvastatin: focus on prostacyclin and nitric oxide.

Statins are believed to exert beneficial effects against cardiovascular disease beyond correction of dyslipidemia. There are however still very sparse data on how individual statins interact with the production of vasoactive eicosanoids and nitric oxide (NO) in human vascular endothelial cells. Here we have determined how fluvastatin affects the mRNA expression of genes associated with vascular reactivity as well as the formation of two major vasodilators, prostacyclin (PGI2) and NO, in human endothelial cells. Also, the influence of fluvastatin on arterial resistance was assessed in isolated small arteries. We show that the promoter activity of prostacyclin synthase (PTGIS), the mRNA expression of PTGIS and endothelial nitric oxide synthase (eNOS), and the production of PGI2 and NO are significantly induced by fluvastatin. Also, strong rapid dilatation ex vivo was observed, with the equal contribution of PGI2 and NO. Our findings in cell culture experiments and in isolated human arteries indicate that fluvastatin-evoked endothelium-derived vasodilator production may confer protection of the endothelial cells via both acute and long-term effects of fluvastatin treatment. If these effects take place in vivo, we suggest a protective pleiotropic role of fluvastatin on the cardiovascular system, particularly at the level of the vascular endothelium, to ameliorate the process of atherogenesis and in the acute manner to reduce vascular tone.

Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.

BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

A supraphysiological dose of testosterone induces nitric oxide production and oxidative stress.

BACKGROUND: Accumulating evidence indicates that abuse of anabolic androgenic steroids may cause cardiovascular adverse side-effects, including endothelial dysfunction. The aim of the present study was to investigate the effects of supra-physiological doses of testosterone on the endothelial production of nitric oxide (NO) and oxidative stress in vitro and in vivo. METHODS: Testosterone enanthate was administrated as of a single 500 mg dose to healthy volunteers (N = 27). Gene expression was studied in human vascular endothelial cells exposed to testosterone. RESULTS: The in vivo results show that the urinary NO level and the antioxidative capacity were significantly decreased two days after testosterone administration. In agreement, our in vitro studies show that testosterone inhibits the gene expression of endothelial NO synthase (eNOS) after 48 hours. When the antioxidant seleno-L-methionine was added, the down-regulation of mRNA specific eNOS was partly abrogated. The mRNA expression of antioxidizing enzyme genes was significantly inhibited after eight hours and recovered 48 hours after testosterone treatment of endothelial cells. CONCLUSION: These results show that a supraphysiological dose of testosterone decreases the expression of eNOS and consequently the formation of NO, which could partly be explained by oxidative stress. These results indicate that supraphysiological doses of testosterone may induce endothelial dysfunction, which is of interest in relation to the cardiovascular adverse side-effects observed in anabolic androgenic steroid abusers.

Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA reductase.

BACKGROUND: Cholesterol is mainly synthesised in liver and the rate-limiting step is the reduction of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) to mevalonate, a reaction catalysed by HMG-CoA reductase (HMGCR). There is a comprehensive body of evidence documenting that anabolic-androgenic steroids are associated with deleterious alterations of lipid profile. In this study we investigated whether a single dose of testosterone enanthate affects the cholesterol biosynthesis and the expression of HMGCR. METHODS: 39 healthy male volunteers were given 500 mg testosterone enanthate as single intramuscular dose of Testoviron®--Depot. The total cholesterol levels prior to and two days after testosterone administration were analysed. Protein expression of HMGCR in whole blood was investigated by Western blotting. In order to study whether testosterone regulates the mRNA expression of HMGCR, in vitro studies were performed in a human liver cell-line (HepG2). RESULTS: The total cholesterol level was significantly increased 15% two days after the testosterone injection (p = 0.007). This is the first time a perturbation in the lipoprotein profile is observed after only a single dose of testosterone. Moreover, the HMGCR mRNA and protein expression was induced by testosterone in vitro and in vivo, respectively. CONCLUSION: Here we provide a molecular explanation how anabolic androgenic steroids may impact on the cholesterol homeostasis, i.e. via an increase of the HMGCR expression. Increasing knowledge and understanding of AAS induced side-effects is important in order to find measures for treatment and care of these abusers.

Daunorubicin metabolism in leukemic cells isolated from patients with acute myeloid leukemia.

BACKGROUND: Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of anthracyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. AIMS: To study C-13 reduction of DNR to daunorubicinol (DOL) in leukemic cells isolated from patients with AML and to determine the most important enzyme involved. METHODS: Mononuclear blood cells from 25 AML patients were isolated at diagnosis and used in a metabolic assay to determine the % DOL formed. mRNA and western blot analysis were performed on the 2 most likely candidates for anthracycline metabolism; carbonyl reductase 1 (CR1) and aldoketoreductase 1A1 (AKR1A1). DNR and DOL concentrations were determined by HPLC. RESULTS: We found a large interindividual variation (up to 47-fold) in leukemic cell DNR metabolism. The specific CR1 inhibitor zeraleone analogue 5 significantly inhibited DNR metabolism with a mean inhibitory effect of 68 %. No correlation between mRNA levels of the enzymes and metabolism were found. Cellular DNR metabolism correlated significantly with CR1 protein expression, determined by western blot, (p < 0.05, R2 = 0,229) while no significant correlation was found with AKR1A1 protein expression. CONCLUSIONS: DNR metabolism in AML cells shows a pronounced interindividual variability. Our results support that CR1 is the most important enzyme for conversion of DNR to DOL in AML cells. This information could in the future be used to genotype CR1 and possibly help to individualise dosing.

Regulation and expression of human CYP7B1 in prostate: overexpression of CYP7B1 during progression of prostatic adenocarcinoma.

BACKGROUND: Cytochrome P450 (CYP) 7B1 is involved in many metabolic processes including androgen metabolism. Cytochrome P450 (CYP) 7B1 is expressed within the prostate and may determine the levels of the natural estrogen receptor beta (ERbeta) ligand 5alpha-androstane-3beta,17beta-diol (3betaAdiol) available and hence affect the regulation of prostate proliferation. We hypothesized that CYP7B1 expression is increased in prostate tumors and that promoter methylation contributes to the regulation of CYP7B1 expression in human prostate tissue. METHODS: Expression of the CYP7B1 gene and protein in clinical prostate tissues and prostate cancer cell lines were investigated using real-time PCR and immunohistochemistry. The methylation status of the CYP7B1 gene was analyzed using methylation-specific PCR (MSP). RESULTS: The immunohistochemical results demonstrate that high expression of CYP7B1 protein occurs in high-grade prostatic intraepithelial neoplasia (PIN) and adenocarcinomas. The ERbeta/CYP7B1 mRNA ratio was significantly lower in tumor compared to the non-tumor area. The MSP analysis indicate that local methylation of CYP7B1 promoter region is an important mechanism involved in down-regulation of CYP7B1 in human prostate tissue. CONCLUSIONS: This is the first report showing that CYP7B1 is overexpressed in high-grade PIN and in prostate cancer and that local methylation of CYP7B1 promoter region may have significant effect on gene transcription.