Human metabolism of phenothiazines to sulfoxides determined by a new high performance liquid chromatography--electrochemical detection method.

The metabolism of phenothiazine drugs may contribute to both their therapeutic and toxic actions by production of active metabolites in vivo. Idiosyncratic reactions or treatment failure may be a consequence of differing patterns of metabolism in different patients. In this report, a modification of our method for the detection of metabolites of phenothiazines is described, which also permits the simultaneous determination of sulfoxide metabolites in human plasma. Application of this method to human plasma identifies marked individual differences in patterns of phenothiazine metabolism.

Measurement of blood levels of neuroleptics and metabolites by combined high performance liquid chromatography-radioreceptor assay for D2 and sigma sites.

The dopamine (D2) receptor blocking property of antipsychotic medications has been proposed as the mechanism of the therapeutic activity of this class of drugs. This property has also been exploited as a method to quantify therapeutic levels of these drugs in patients. However, the lack of correlation among dosage, blood levels and clinical response has resulted in a contradictory literature on both mechanism and quantification of these drugs. Bioactivity and chemical identity of the commonly prescribed neuroleptic drug fluphenazine and its metabolites in human plasma were determined by a new method which combines the selectivity of chemical methods with the sensitivity and bioassay of the radioreceptor assay (RRA) method. Fluphenazine and its metabolites were separated and identified in human plasma by an ion-pairing reverse phase high performance liquid chromatographic method with electrochemical detection. A volatile buffer system was employed which was compatible with facile sample preparation for post-column analyses, and which provided sharp, symmetrical chromatographic peaks of parent compound and metabolites. Post chromatography, HPLC fractions were assayed by RRA for D2, alpha 1 and sigma receptors. More than one pattern of metabolism of the drug was seen, including biosynthesis of drug metabolites with biological activities at these receptor types. The individual differences with which this occurs may contribute to the variabilities seen in clinical response to neuroleptics, and to difficulties in neuroleptic blood level determinations.

Comparison of cyclosporine and FK506 effects on glutathione levels in rat cochlea, brain, liver and kidney.

Cyclosporine treatment (50 mg/kg/day, p.o.) caused increases in rat renal reduced glutathione (GSH) levels of 205 and 673%, respectively, after 5 and 10 days. No changes were seen in liver GSH with either dose of cyclosporine. FK506 (2.5 mg/kg/day, p.o., for 7 days) caused an approximately 200% increase in kidney GSH, and an approximately 250% increase in hepatic GSH levels. Oxidized glutathione (GSSG) was never more than 1-2% of the level of the reduced form in any tissue from control animals. Small increases in the ratios of oxidized to reduced glutathione were seen in livers and kidneys from both cyclosporine- and FK506-treated animals. No changes in GSH or GSSG levels were seen in brains or cochleas from any animal.

Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause.

To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC. PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle. Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase. Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC. Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity. When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation. These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens. This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.

Solid-phase extraction and high-performance liquid chromatography for therapeutic monitoring of haloperidol levels.

This laboratory has developed a simple and efficient solid-phase extraction method that is combined with a high-performance liquid chromatographic method for rapid and precise therapeutic monitoring of haloperidol (Haldol) blood levels. The solid-phase extraction utilizes a mixed bed column. Sensitivity of the chromatographic method is 0.5 ng/ml (1.3 nM) of drug in serum, and separations can be performed in a 15-min chromatographic run. Advantages of this approach include enhanced speed, sensitivity, and efficiency. A high level of sensitivity may be achieved because of the absence of interferences from other drugs, metabolites, or serum components.

Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva.

Ketoconazole, cis-1-acetyl-4-[4[[2-(2,4-dichlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine, a clinically used antifungal agent, is also an inhibitor of steroid hormone biosynthesis. A high-performance liquid chromatographic method is described which resolves ketoconazole with selectivity and high sensitivity provided by the use of electrochemical detection. Ketoconazole can be detected in high-performance liquid chromatography by electrochemical oxidation at a glassy carbon electrode at a potential of +1.0 V. Electrochemical detection offers improved sensitivity and selectivity over ultraviolet absorbance or fluorescence detection after derivatization. The method utilizes a volatile buffer system compatible with postcolumn analyses and an internal standard which is electrochemically active. This technique provides a simple method to assay ketoconazole. Ketoconazole can be detected in human plasma and saliva after a single oral therapeutic dose.

Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract.

Viable tissue sections and isolated cell cultures from the human fallopian tube, uterus, cervix, and vaginal mucosa were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1). We examined infectivity by using the monocytotropic strain HIV-1(JR-FL) and several primary isolates of HIV-1 obtained from infected neonates. HIV-1 infection was measured by p24 production in short-term culture and by immunofluorescence detection of HIV-1 Nef and p24 proteins by laser scanning confocal microscopy. Three-color immunofluorescence was used to phenotype HIV-infected cells within tissue sections from each site. Our findings indicate that epithelial, stromal, and dendritic cells and cells with CD14+ CD4+, CD14-CD4-, and CD4+ CD14- phenotypes from the female reproductive tract are infectable with HIV-1. Of importance is the finding that tissues from the upper reproductive tract are susceptible to infection with HIV-1. Moreover, tissue samples from women in all stages of the menstrual cycle, including postmenopausal women (inactive), could be infected with HIV-1. Female reproductive tract cells required a minimum of 60 min of exposure to HIV-1 in order for infection to occur, in contrast to peripheral blood lymphocytes, which became infected after being exposed to HIV-1 for only 1 min. These findings demonstrate that HIV-1 can infect cells and tissues from different sites within the female reproductive tract and suggest that multiple cell types, including epithelial cells, may be targets for the initial infection by HIV-1.