Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.

High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS.

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Abscopal depression of G-CSF mobilized peripheral stem cells.

Transient suppression of hematopoietic colony formation from cytokine mobilized PBSC was observed in a patient following local radiation therapy for lymphoma. This effect was seen 2 weeks after completion of radiation and reversed by 6 weeks. The patient successfully engrafted after myeloablative chemotherapy with harvested PBSC. This report documents the first reported case of suppression of PBSC colony formation following radiation and we speculate that this finding may be due to an abscopal effect.

T-cell receptor variable beta genes show differential expression in CD4 and CD8 T cells.

Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) beta-gene products as part of the alpha beta heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR V beta-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain V beta genes were consistently overrepresented in the CD4 population (V beta-5.1, -6.7a, and -18); some were skewed to the CD8 population (V beta-14) while others showed variable patterns (V beta-12 and -17). Deletion of entire V beta gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

Monoclonal antibody 9.3 and anti-CD11 antibodies define reciprocal subsets of lymphocytes.

We have previously described antibody 9.3 which recognizes a 44-kDa polypeptide designated Tp44 expressed on 70-80% of peripheral blood T cells, including nearly all CD4+ cells and some CD8+ cells. Whereas the 9.3+ population contains helper cells, cytotoxic T cell precursors and cytotoxic T cell effectors, the 9.3- population has been reported in several models to contain precursors for suppressor cells. In this report, we demonstrate that 9.3- lymphocytes express CD11, an antigen which is also present on monocytes and granulocytes. Among lymphoid cells, Tp44 and CD11 represent markers that identify reciprocal, nonoverlapping subsets, each of which contains both CD8+ cells and CD4+ cells. With Tp44 and CD11, and CD4 and CD8, the development and function of T cells can thus be examined within the framework of two distinct systems of reciprocally expressed antigens.

Identification and functional characterization of two distinct epitopes on the human T cell surface protein Tp50.

The structural and functional domains of Tp50, the human T lymphocyte surface protein associated with the E rosette receptor, were probed with the use of two murine monoclonal antibodies. Lysostripping, immune precipitation, and competitive binding experiments demonstrated that antibodies 9.6 and 35.1 bind to Tp50 epitopes in close proximity. In functional studies, both antibodies caused a similar degree of antigenic modulation, inhibited T cell proliferative responses, and inhibited cytotoxic T lymphocyte function without affecting cells that mediate antibody-dependent cell-mediated cytotoxicity. The antibodies were strikingly different, however, in that antibody 9.6 inhibited E rosette formation and natural killer cell-mediated lysis, whereas antibody 35.1 did not. These results could not be ascribed to differences in antibody class or binding characteristics, because Scatchard analysis demonstrated that these two IgG2a antibodies have comparable avidity and that T cells bind each antibody in equivalent amounts. The differential binding of antibodies 9.6 and 35.1 to T cells from nonhuman primates further supports the interpretation that the differences between the antibodies in their effects on E rosette formation and natural killer function stem from the fact that they bind to distinct epitopes of Tp50. The implications of these findings for understanding the functions of Tp50 molecules are discussed.

Granulocytes and cultured human fibroblasts express common acute lymphoblastic leukemia-associated antigens.

We have investigated the expression of the common acute lymphoblastic leukemia antigen (cALLA) by nonlymphoid cells, using a new murine monoclonal antibody (designated 24.1), specific for cALLA. This antibody completely blocks the binding of monoclonal anti-cALLA antibody J-5. Furthermore, antibody 24.1 binds to cALLA with greater affinity and induces a greater degree of antigenic modulation than antibody J-5. With antibody 24.1, we have demonstrated that normal cultured marrow and skin fibroblasts and mature granulocytes express cALLA. By immune precipitation and polyacrylamide gel electrophoresis, cALLA from fibroblasts has a slightly lower molecular weight and cALLA from granulocytes a slightly higher molecular weight than cALLA from cultured human leukemic cell lines. Quantitative studies indicate, however, that cALLA expression is approximately 3-fold and 20-fold lower, respectively, on normal granulocytes and skin fibroblasts than on NALM-6 cells. cALLA may be expressed by cells with widespread distribution in multiple organ systems. These findings emphasize that differentiation markers that appear to be tumor or tissue specific may be found on cells of diverse origin.