P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99m Tc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 ϳ20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp ϩ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.

No abstract

We studied the inhibitory effects on colony formation by granulocyte-macrophage colony forming units (cfu-gm) of eight azole antifungal agents in vitro. All agents, except fluconazole, inhibited colony formation dose-dependently with 50% inhibitory concentrations (IC50) in the range of 0.78-49 micromol/L in cultures of murine and human bone marrow. For human cells, the IC50 values were 0.553 mg/L for itraconazole, 1.24 mg/L for saperconazole, 2.58 mg/L for clotrimazole, 5.33 mg/L for miconazole, 6.17 mg/L for econazole, 6.27 mg/L for ketoconazole and 8.38 mg/L for oxiconazole. The IC50 of itraconazole for human cfu-gm in vitro was similar to the plasma level of this drug recommended for systemic antifungal therapy (>0.5 mg/L) thus indicating the potential clinical relevance of our data. The IC50 of ketoconazole for human cfu-gm in vitro may be exceeded by plasma levels produced in vivo by high (> or =400 mg) doses, whereas fluconazole failed to reduce colony formation by 50% even at 100 mg/L, a concentration not reached in vivo even after extremely high doses (2000 mg/day). To most of the drugs studied, murine progenitor cells seemed to be less sensitive than the human ones. There was, however, a close correlation between the murine and human log IC50 values of the drugs (r2 = 0.964, P< 0.001), suggesting that cultures of murine bone marrow may be suitable to predict the in-vitro toxicity of azole antifungals to human cfu-gm.

Background:The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections. Methods: Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections. Results: The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of