The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes.

The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CLint (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 microM (0.13% albumin) and 42.2 microM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CLone) was assessed. Virtually identical values were obtained for CLint and CLone. The free-drug hypothesis was tested by comparing ratios of relative CLint/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.

Human NADPH-cytochrome p450 reductase overexpression does not enhance the aerobic cytotoxicity of doxorubicin in human breast cancer cell lines.

Doxorubicin is a useful antineoplastic drug with multiple mechanisms of cytotoxicity. One such mechanism involves the reductive bioactivation of the quinone ring to a semiquinone radical, which can exert direct toxic effects and/or undergo redox cycling. We hypothesized that human NADPH-cytochrome p450 reductase (CYPRED) catalyzes doxorubicin reduction and that overexpression of this enzyme sensitizes human breast cancer cell lines to the aerobic cytotoxicity of doxorubicin. cDNA-expressed human CYPRED catalyzed doxorubicin reduction, measured as the rate of doxorubicin-stimulated NADPH consumption. Using a bank of 17 human liver microsomal samples, the rate of doxorubicin reduction correlated with CYPRED catalytic activity and CYPRED protein immunoreactivity. Diphenyliodonium chloride, a mechanism-based inactivator of CYPRED, inhibited CYPRED activity and doxorubicin reduction in human liver microsomes with similar concentration dependence. Stably transfected clones of MDA231 human breast cancer cells overexpressing human CYPRED immunoreactive protein and catalytic activity showed enhanced sensitivity to the aerobic cytotoxicity of tirapazamine, a bioreductive drug known to be activated by CYPRED; however, no sensitization to the cytotoxic effects of doxorubicin was observed. Although human CYPRED is an important catalyst of doxorubicin reduction, overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin.

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin, alpha-globulins, alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6.

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.

Identification of the novel splicing variants for the hPXR in human livers.

The human pregnane X receptor (hPXR) plays a key role in the regulation of both drug metabolism and efflux by inducing the expression of CYP3A4 and MDR1 gene. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we identified seven novel splicing variants of hPXR in tissue from a single human liver. The expression of hPXR-related transcripts in the liver samples of 15 Caucasian individuals was subsequently determined by RT-PCR assays. The pattern of expression levels of these transcripts varied among liver samples. These results suggest that the hPXR is expressed as several different transcripts in liver tissues, apparently due to alternative as well as defective gene splicing. Furthermore, because this study provides the possibility of interindividual differences in hPXR transcript profiles, these alternative splicings for hPXR may largely contribute to the interindividual variability in CYP3A4 and P-glycoprotein induction.

The interaction of human and bovine serum proteins with CYP3A in human liver microsomes: inhibition of testosterone 6beta-hydroxylation by albumin, alpha-globulins, alpha(1)-acid glycoprotein and gamma-globulins.

The effects of human and bovine serum proteins on CYP3A activity, using testosterone as the probe substrate, were investigated in human liver microsomes. Serum albumin, alpha-globulins, and alpha(1)-acid glycoprotein (alpha(1)-AGP) of both species significantly inhibited testosterone 6beta-hydroxylation. When the inhibitory effects of serum proteins were compared with serum protein binding data, human alpha-globulins, with a ratio (relative metabolic activity/unbound fraction) of 0.3, showed higher, and bovine alpha(1)-AGP, with the ratio of 1.4, showed lower inhibitory effects than those expected from protein binding of testosterone. The effects of the other serum proteins were close to those expected from protein binding, according to the free drug hypothesis. The K(i) values obtained from the Dixon plots were 0.32% (w/v, 48 microM) for human serum albumin (HSA), 0.48% for human alpha-globulins, and 0.23% (52 microM) for human alpha(1)-AGP. K(i) values of bovine serum albumin, bovine alpha-globulins and bovine alpha(1)-AGP were 3-5 times higher than those of the respective human proteins. The results suggest a direct interaction of some of these serum proteins with the active site of the CYP3A isoform. Since the bovine serum proteins showed weaker inhibitory effects than human serum proteins, the wide use of BSA, which is viewed as interchangeable with HSA, needs to be cautioned.

Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs, CYP3A5*3 and *6, in a Japanese population.

In this study, we established useful and reliable methods for the direct detection of the variants of CYP3A5 gene by polymerase chain reaction (PCR) and DdeI restriction analysis. The frequency of CYP3A5 related SNPs in 200 healthy Japanese male subjects was determined. The homozygous wild-type (*1/*1) frequency was 7.0% (14/200), the heterozygous (*1/*3) frequency was 32.5% (65/200) and the homozygous mutant-type (*3/*3) frequency was 60.5% (121/200). The *6 allele was not detected in any of the Japanese individuals. This result suggests that an estimated 40% of the Japanese express relatively high levels of metabolically active CYP3A5 protein. The proposed detection assays are useful for screening the CYP3A5 related SNPs in pharmacogenetic research.

Estudio preliminar sobre el fenotipo de acetilador de los amerindios Teribes / Preliminar study on the phenotype of acetylator of Teribe Amerindians

El estudio de la capacidad de acetilación en 62 voluntarios teribes, procedentes de Sieykin, Sieyic y Santa Rosa, demonstró que 48.4% eran acetiladores rápidos y 51.6%, acetiladores lentos. Al comprobar estos resultados con los que obtuvimos en una población cuna, en la cual encontramos que el 78% era de acetiladores rápidos, se observa que tanto la distribución de los sujetos estudiados, según el porcentaje de isoniacida que acetilaron, como el porcentaje de acetiladores lentos y rápidos indican que, en ambos grupos, existen diferencias en la actividad de la N-acetiltransferasa. Estos resultados sugieren que pueden existir diferencias fundamentales entre los diferentes grupos amerindios, en la habilidade de biotransformar los medicamentos

La biotransformación de la esparteína en amerindios cunas de Panamá / Biotransformation of sparteine in Amerindians cunas of Panamá

Reportamos, por vez primera, un estudio de oxidación metabólica en un grupo amerindio: los cunas de Panamá. Estudios genéticos indican un escaso mestizaje de menos del 1% de mezcla racial y establecen la identidad genética del grupo cuna con respecto a otros grupos amerindios que se remonta, a lo menos, a 5,000 años. Ciento nueve voluntarios recibieron 50 mg de esparteína y recogieron sus orinas por 12 horas, para determinar la cantidad que contenían de esparteína y de sus dos productos de oxidación. El porcentaje del medicamento intacto recuperado de la orina, así como de sus metabolitos, fue menor que el encontrado en otros grupos étnicos y está normalmente distribuido. La razón metabólica, que es un índice de eficiencia de la actividad de la isozima involucrada en la biotransformación de la esparteína, indica que no existen metabolizadores deficientes en este grupo y que la frecuencia de los metabolizadores extensos muestra una distribución unimodal. Por esas razones concluimos diciendo que en el grupo cuna, a diferencia de los grupos caucasoides, no existe polimorfismo en la oxidación de la esparteína. Además la ausencia de metabolizadores deficientes entre los cunas indica que este grupo, en comparación con los caucasoides, se encuentra en menor riesgo de desarollar reacciones tóxicas al usar medicamentos que siguen la ruta de la esparteína