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An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-14C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsilyl)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.

Following our recent finding that calmodulin antagonists can reduce cancer cell attachment to extracellular matrix proteins, we investigated the calmodulin antagonistic and anti-attachment properties of the non-steroidal anti-oestrogens tamoxifen and droloxifene. These drugs and four of their active metabolites were found to have calmodulin antagonist activity with IC50 values of 2-4 microM and to be capable of inhibiting attachment of murine B16 melanoma to extracellular matrix proteins in vitro. IC50 values for inhibition of attachment were 11 microM for tamoxifen and ranged from 5 to 40 microM for the other five compounds tested. (Poor reproducibility in drug potency between attachment experiments was almost certainly due to the low aqueous solubility of these drugs.) The effects of tamoxifen on cell/matrix adhesion were most evident between 15 min and 3 h of cell attachment. No effects of tamoxifen were evident in cells which had been allowed to attach for 6 h or more. Tamoxifen at concentrations between 0.1 and 30 microM was without effect on intracellular free calcium concentration. Tamoxifen also inhibited attachment of human ocular melanoma cells and human breast cancer (MCF7) cells to type I collagen. The concentration at which tamoxifen and its metabolites affect cell attachment in vitro (2-14 microM) is of the same order as the tissue concentrations of these drugs achieved clinically. The possibility exists that reduction of cell/matrix interactions may contribute to the clinical anti-metastatic efficacy of tamoxifen and some of its active metabolites.

Diazepam metabolism has been investigated in rat hepatocytes cultured for 3, 24, 48 and 72 hr under five different conditions. Although four of the treatments studied reduced markedly the spontaneous loss of cytochrome P450, they had different effects on the metabolism of diazepam (DZ) presumably by affecting the relative proportions of cytochrome P450 isozymes during the period of culture. Thus P450 medium or dimethyl sulphoxide-supplemented medium maintained the rate of disappearance of DZ from the culture medium and metabolite profile in 24 hr cultures at the initial levels found in 3 hr cultures, while culture at 30 degrees or in metyrapone-containing medium resulted in the production of oxazepam, a metabolite normally only produced by dog, monkey and human hepatocytes. These findings indicate that the well recognized phenotypic alteration of cytochrome P450-dependent mono-oxygenase activities that occurs when rat hepatocytes are cultured in different media can result in a range of metabolic options that are normally only available in other animal species.