This article is available online at http://dmd.aspetjournals.org ABSTRACT:Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC 50 values for CYP3A (0.03-0.24 M) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 (ϳ50-fold lower). The IC 50 value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC 50 values <10 M) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 M azamulin in the presence of NADPH for 10 min inhibited ϳ95% of testosterone 6␤-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 M, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 M. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.Along with other experimental approaches, enzyme-selective chemical inhibitors are commonly used in reaction phenotyping studies to determine cytochrome P450 isoform contribution to a metabolic reaction (Clarke, 1998). Chemical inhibitors provide a simpler and more cost-effective alternative to immunoinhibitory antibodies and can be used in cells. However, proper use of chemicals may require foreknowledge of the reaction kinetics under investigation. For example, competitive inhibitors should be used with a substrate concentration near or below the apparent K M . In addition, specificity is often lost when the inhibitor concentration is too high. The effect of microsomal protein concentration and incubation time may also need to be considered, if the inhibitor is also a substrate.Chemicals used as selective inhibitors of CYP3A include triacetyloleandomycin, gestodene, and ketoconazole. Ketoconazole is most widely used, probably because of advantages in potency, selectivity, commercial availability, and ease of use (e.g., preincubation steps are not required) (Maurice et al., 1992;Baldwin et al., 1995;Newton et al., 1995;Bourrie et al., 1996;Sai et al., 2000;Zhang et al., 2002). However, selectivity of ketoconazole for CYP3A is often less than ideal. For example, CYP1B1, CYP2B6, and CYP2C8...

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Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4 ؉ and CD8 ؉ T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1␤ (IL-1␤) produced by PBMC upon stimulation by OKT3 were increased (P < 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.Lymphocytes are among the most extensively studied cells of the hematopoietic system because of their central role in the generation of immune responses. Information provided from the study of T lymphocytes is important not only in understanding the basic concepts of immune function but also in enabling the development of lymphocyte-based adoptive immune therapies. Lymphocytes can be collected from the peripheral blood, lymphoid tissues, and certain internal organs. In most cases, lymphocytes are initially isolated from the peripheral blood compartment and purified by Ficoll density gradient centrifugation. However, regardless of the method used to isolate T cells or peripheral blood mononuclear cells (PBMC), there always exists a low level of contaminating red blood cells (RBC). In addition, when PBMC are isolated on a large scale, as with most ex vivo adoptive immunotherapy approaches, the level of contaminating RBC increases even further. It has been shown previously that lymphocytes in whole blood stimulated with mitogen produce more interleukin-2 (IL-2) than Ficoll-Hypaque-purified lymphocytes in culture (5). What remains unknown is the effect various levels of contaminating RBC have on the ability of well-characterized Tcell stimulants to activate lymphocytes under normal cell culture conditions.A unique form of outpatient adoptive immunotherapy referred to as autolymphocyte therapy (ALT) for the treatment of patients with metastatic renal cell carcinoma has been developed (6, 6a). Patients are infused monthly with ϳ10 9 T lymphocytes activated ex vivo in a conditioned medium containing a mixture of OKT3 (mouse monoclonal anti-CD3 antibody) and a broad panel of autologous cytokines. The cytokine mixture is generated by stimulation of patient PBMC ex vivo with 25 ng of OKT3/ml for 3 days during the first cycle of the therapy (8). During the secondary cycles, i.e., monthly, of therapy, patient PBMC are cultured with the autologous cytokine mixture from the first cycle of therapy for 5 days and then infused back into the patient.For this procedure, lymphoapheresis is performed for each cycle to collect large numbers of PBMC. The resulting apheresis cell products (ACP) are highly enriched in white blood cells and contain various amounts of RBC, platelets, ...

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An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Further validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.