A background subtraction approach for determination of endogenous cortisol and 6ß-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women.

Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6ß-hydroxycortisol/cortisol ratio (6ß-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6ß-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6ß-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquid-liquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC®BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1-1.000 ng/mL and 2-1.000 ng/mL for C and 6ß-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6ß-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6ß-OHF:C ranged from 2.57 to 51.69, with a mean ± standard deviation (SD) of 15.12 ± 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6ß-OHF:C ranging from 3.48 to 44.54 with a mean ± SD of 14.37 ± 5.73 (n = 7). Even though the 6ß-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability.

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.

This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from ten non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.

Cloning of cytochrome P450 3A137 complementary DNA in silver carp and expression induction by ionic liquid.

Cytochrome P450 (CYP) enzymes, especially CYP 3A, are responsible for metabolizing of various kinds of endogenous and exogenous compounds in animals. In the present study, a full-length sequence of CYP 3A137 cDNA in silver carp was cloned and sequenced, and then a phylogenetic tree of CYP 3A was structured. Additionally, the acute toxicity of the ionic liquid 1-octyl-3-methylimidazolium bromide ([C8mim]Br) on silver carp and transcription and microsome enzyme activity of CYP 3A137 in the liver of silver fish after rifampicin or [C8mim]Br exposure were also determined in this study. The results show that the full length of CYP 3A137 cDNA is 1810 base pair (bp) long and contains an open reading frame of 1539bp encoding a protein of 513 amino acids. Sequence analysis reveals that CYP 3A137 is highly conserved in fish. Moreover, the results of quantitative real-time polymerase chain reaction reveal that CYP 3A137 in silver carp is constitutively expressed in all tissues examined and the sequence of expression rate is liver>intestine>kidney>spleen>brain>heart>muscle. Finally, the results of acute toxicity tests indicate that both rifampicin and [C8mim]Br significantly up-regulate the expression of CYP 3A137 at mRNA level and increase CYP 3A137 enzyme activity in fish liver, suggesting that CYP 3A137 be involved in metabolism of [C8mim]Br in silver carp.

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury.

Lipid-lowering effects of statins: a comparative review.

The pharmacological regulation of lipid metabolism in patients with dyslipidaemia is unequivocally associated with significant reductions in risk for cardiovascular morbidity and mortality. There is strong clinical trial data to support of the use of statin therapies in the settings of both primary and secondary prevention. This paper addresses: i) the mechanisms of action of antilipidaemic medications; ii) dosing regimens and the pharmacokinetic differences among drugs of the same class; iii) risk for drug interactions; and iv) reviews the clinical trial evidence used to support the use of particular antilipidaemic medications in specific physiological settings.