Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development for screening of potential tamoxifen-drug/herb interaction via in vitro cytochrome P450 inhibition assay.

Screening for potential drug-drug interaction (DDI) or herb-drug interaction (HDI) using in vitro cytochrome P450 inhibition (IVCI) assays requires robust analytical methods with high sensitivity and reproducibility. Utilization of liquid chromatography-mass spectrometry (LC-MS) for analyte quantification is often hampered by the presence of non-volatile IVCI sample buffer constituents that often results in ion suppression. In this study, to enable screening of drug interactions involving tamoxifen (TAM) metabolism using IVCI-LC-MS/MS, a liquid-liquid extraction (LLE) method was developed and optimized for sample clean-up. Utilization of chloroform as extraction solvent and adjustment of sample pH to 11 was found to result in satisfactory recovery (>70%) and low ion suppression (<19%). A LC-MS/MS method was subsequently developed and validated for simultaneous quantification of major TAM metabolites, such as N-desmethyltamoxifen (NDT), endoxifen (EDF) and 4-hydroxytamoxifen (HTF) to enable IVCI sample analysis. Satisfactory separation of E-/Z-isomers of endoxifen with peak resolution (Rs) of 1.9 was achieved. Accuracy and precision of the method was verified within the linear range of 0-50 ng/mL for NDT, 0-25 ng/mL for HTF and 0-25 ng/mL for EDF (E/Z isomers). Inhibitory potency (IC50, Ki and mode of inhibition) of known CYP inhibitors and Strobilanthes crispus extract was then evaluated using the validated method. In summary, the results demonstrated applicability of the developed LLE and validated LC-MS/MS method for in vitro screening of DDI and HDI involving TAM metabolism.

In vitro biocompatibility of chitosan porous skin regenerating templates (PSRTs) using primary human skin keratinocytes.

Biopolymer chitosan (beta-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-alpha and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-alpha and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.

The transcription of the peroxisome proliferator-activated receptor alpha gene is regulated by protein kinase C.

The transcriptional regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) by a variety of peroxisome proliferators was investigated. The treatment of primary cultures of rat hepatocytes with Wy14,643 or clofibrate increased mRNA steady state levels of both PPARalpha and acyl coenzyme A oxidase (ACOX). In contrast, fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARalpha. Inhibition of protein kinase C (PKC) activity using bisindolylmaleimide or calphostin C abolished the increased PPARalpha expression by the peroxisome proliferators whereas the expression of the ACOX gene remained unaffected. Phorbol-12-myristate-13-acetate increased PPARalpha mRNA levels without altering ACOX mRNA levels. It can thus be concluded that a number of peroxisome proliferators activate a PKC-dependent signalling pathway in addition to the PPARalpha pathway. The PKC signal transduction pathway contributes to the regulation of PPARalpha expression but does not influence the transcriptional activity of PPARalpha.

Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation.

The technique of quantitative competitive RT-PCR to determine the levels of mRNA expression of genes encoding peroxisome proliferator-activated receptor alpha (PPAR alpha), acyl coenzyme-A (ACOX) and cytochrome P450 4A1 (CYP4A1) in primary rat hepatocyte cultures is described. This technique is based on the co-amplification of an internal standard (PCR MIMIC) and target DNA sequence with one set of primers. Following total RNA extraction and reverse transcription, competitive PCR was carried out by mixing various dilutions of known concentrations of PCR MIMIC with constant amounts of cDNA. Densitometry was then carried out on the DNA bands obtained following gel electrophoresis and, after correcting for size differences between the target DNA and MIMIC, the concentration of target DNA was calculated and expressed as attomoles (10-18 moles) per microgram total RNA. Constitutive levels of PPAR alpha, ACOX and CYP4A1 obtained were 0.037 +/- 0.003, 1.858 +/- 0.470m and 0.035 +/- 0.007 attomoles/microgram RNA, respectively. Following 24 h culture of rat primary hepatocytes in the presence of sodium clofibrate (a peroxisome proliferator), the levels of PPAR alpha, ACOX and CYP4A1 were increased by 2.1-, 3.3- and 12.8-fold, respectively. Thus the technique described in this study has high sensitivity and can be used to accurately measure the mRNA steady state levels in cell cultures.