Hunan Yi Ke Da Xue Xue Bao, 2002 Dec 28, 27(6), 522 - 4 {Expression of multidrug resistance P-170 and MRP and their correlation with clinical drug resistance in acute leukemia}; Lin XM et al.; OBJECTIVE: To investigate the relevance between the expression of P-170 and MRP and clinical drug resistance in acute leukemia . METHODS: The expression of P-170 and MRP in mononuclear cells of bone marrows was analyzed by the immunohistochemical technique in 72 acute leukemia patients . RESULTS: The expression of P-170 was positive in 46 and negative in 26 of the 72 post-chemotherapy acute leukemia patients . The therapeutic effect of the P-170 positive expression patients was significantly poorer than that of the negative expression patients (P < 0.01) . The expression of MRP was positive in 39 and negative in 33 of the 72 post-chemotherapy acute leukemia patients . The therapeutic effect of the MRP positive expression patients was significantly poorer than that in the negative expression patients (P < 0.01) . The expression of P-170 and MRP had a significant concordance (Kappa = 0.427, P < 0.01) . The sensitivity of P-170 and MRP which were analyzed simultaneously was 97.5%, which was higher than that of P-170 (90%) or MRP (77.5%) analyzed respectively in drug resistance patients . CONCLUSION: The expression of P-170 and/or MRP was significantly related with drug resistance in clinical chemotherapy . The therapeutic effect was significantly poorer in P-170 and/or MRP positive expression patients than that in negative expression patients . These data suggest that P-170 and MRP analyzed simultaneously can improve the value of diagnosis and prognosis in patients with drug resistance leukemia. Mol Cancer Ther, 2003 Mar, 2(3), 307 - 16 Cloning and functional characterization of the multidrug resistance-associated protein (MRP1/ABCC1) from the cynomolgus monkey; Godinot N et al.; The multidrug resistance-associated protein 1 (ABCC1) gene from human (hMRP1), dog (canMRP1), and mouse (muMRP1) all encode proteins that efficiently transport the endogenous MRP1 substrate glutathione-S-leukotriene C(4) and confer resistance to anticancer agents, including vincristine and etoposide . hMRP1 also confers resistance to anthracyclines, whereas this is not true of canMRP1 or muMRP1 . To determine whether MRP1 from another animal species used in toxicological studies would be more functionally similar to hMRP1, we cloned and characterized two alleles of the MRP1 homologue from the cynomolgus monkey Macaca fascicularis (monMRP1) . The monMRP1 cDNAs encode proteins of 1531 residues that are 98, 90, and 88% identical to hMRP1, canMRP1, and muMRP1, respectively . Stable overexpression of both monMRP1 alleles and hMRP1 in transformed human embryonic kidney cells was achieved using an episomal expression vector . Transporters encoded by both monMRP1 alleles were functionally very similar to hMRP1 . monMRP1 conferred an increased resistance to vincristine and etoposide and transported glutathione-S-leukotriene C(4) into membrane vesicles . In addition, MRP1-mediated drug resistance was effectively reversed in monMRP1 and hMRP1 transfectants by LY402913, a new MRP1-selective inhibitor in the class of tricyclic isoxazoles . However, monMRP1 transporters conferred a reduced level of resistance to the anthracyclines doxorubicin, daunorubicin, and epirubicin relative to hMRP1, although resistance levels were significant relative to vector control cells . These functional differences between human and monkey MRP1 transporters will need to be considered when designing pharmacokinetic and toxicological studies for the preclinical evaluation of MRP1 modulators. Infect Immun, 2003 Apr, 71(4), 2058 - 64 Recombinant gamma interferon stimulates signal transduction and gene expression in alveolar macrophages in vitro and in tuberculosis patients; Condos R et al.; Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year . Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-gamma) . We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-gamma (rIFN-gamma) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month . We hypothesized that rIFN-gamma would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM) . AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-gamma . STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection by Mycobacterium tuberculosis TN913 . In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 +/- 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity . After 4 weeks of treatment with rIFN-gamma aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved . Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens . rIFN-gamma aerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial. Endocr Relat Cancer, 2003 Mar, 10(1), 43 - 73 ATP binding cassette transporters and drug resistance in breast cancer; Leonessa F et al.; Resistance to chemotherapy is a critical issue in the management of breast cancer patients . The nature of clinical drug resistance is likely to be multifactorial . However, in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily, and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1) . Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins, due to both methodological and biological factors . Nonetheless, Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively, by immunohistochemistry), without a clear and consistent association with cancer stage . Exposure to chemotherapy increases the expression of both proteins . In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy . However, the correlation with clinical drug resistance is not as well defined . A stronger association of Pgp/MDR1 with response rates has been observed when expression or an increase in expression are detected immediately following chemotherapy . Correlations with prognosis appear more evident in studies using immunohistochemistry, in adjuvant and neoadjuvant settings . Evidence of clinical reversal of drug resistance by verapamil suggests a functional role of Pgp in drug resistance, although the significance of the evidence is generally weakened by poor trial designs . Future studies should take into account the multifactorial nature of drug resistance in breast cancer and use standardized approaches with adequate controls . Expression studies should be complemented by well-designed trials of drug-resistance reversal using target-specific chemosensitizing agents, and relating the results to the levels of expression of the target proteins. Biochemistry, 2003 Apr 1, 42(12), 3544 - 55 Purified human MDR 1 modulates membrane potential in reconstituted proteoliposomes; Howard EM et al.; Human multidrug resistance (hu MDR 1) cDNA was fused to a P . shermanii transcarboxylase biotin acceptor domain (TCBD), and the fusion protein was heterologously overexpressed at high yield in K(+)-uptake deficient Saccharomyces cerevisiae yeast strain 9.3, purified by avidin-biotin chromatography, and reconstituted into proteoliposomes (PLs) formed with Escherichia coli lipid . As measured by pH- dependent ATPase activity, purified, reconstituted, biotinylated MDR-TCBD protein is fully functional . Dodecyl maltoside proved to be the most effective detergent for the membrane solubilization of MDR-TCBD, and various salts were found to significantly affect reconstitution into PLs . After extensive analysis, we find that purified reconstituted MDR-TCBD protein does not catalyze measurable H(+) pumping in the presence of ATP . In the presence of physiologic {ATP}, K(+)/Na(+) diffusion potentials monitored by either anionic oxonol or cationic carbocyanine are easily established upon addition of valinomycin to either control or MDR-TCBD PLs . However, in the absence of ATP, although control PLs still maintain easily measurable K(+)/Na(+) diffusion potentials upon addition of valinomycin, MDR-TCBD PLs do not . Dissipation of potential by MDR-TCBD is clearly {ATP} dependent and also appears to be Cl(-) dependent, since replacing Cl(-) with equimolar glutamate restores the ability of MDR-TCBD PLs to form a membrane potential in the absence of physiologic {ATP} . The data are difficult to reconcile with models that might propose ATP-catalyzed "pumping" of the fluorescent probes we use and are more consistent with electrically passive anion transport via MDR-TCBD protein, but only at low {ATP} . These observations may help to resolve the confusing array of data related to putative ion transport by hu MDR 1 protein. Probl Tuberk, 2003, (1), 28 - 30 {Incidence of adverse reactions to chemotherapy and their types in adolescents with tuberculosis}; Panova LV et al.; During treatment, 65.7% of the patients developed adverse reactions to antituberculosis drugs . Allergic reactions were most commonly associated with the use of streptomycin . The simultaneous inclusion of isoniazid, rifampicin, and pyrazinamide into the treatment regimens irrespective of the total number of drugs was found to increase the incidence of hepatotoxic reactions as compared with the regimens which do not contain a combination of these 3 drugs . Cycloserin may be given to adolescents with multidrug resistant tuberculosis . Glutaminic acid prevents the toxic effect of the drug on the CNS, therefore cycloserin may be prescribed for a long period (for as long as 9 months). Leuk Res, 2003 Jun, 27(6), 509 - 16 Involvement of DNA-dependent protein kinase in regulation of the mitochondrial heat shock proteins; Um JH et al.; Since DNA-dependent protein kinase (DNA-PK) has been known to play a protective role against drug-induced apoptosis, the role of DNA-PK in the regulation of mitochondrial heat shock proteins by anticancer drugs was examined . The levels of basal and drug-induced mitochondrial heat shock proteins of drug-sensitive parental cells were higher than those of multidrug-resistant (MDR) cells . We also demonstrated that the development of MDR might be correlated with the increased expression of Ku-subunit of DNA-PK and concurrent down-regulation of mitochondrial heat shock proteins . The basal mtHsp70 and Hsp60 levels of Ku70(-/-) cells, which were known to be sensitive to anticancer drugs, were higher than those of parental MEF cells, but conversely these mitochondrial heat shock proteins of R7080-6 cells over-expressing both Ku70 and Ku80 were lower than those of parental Rat-1 cells . Also, the mtHsp70 and Hsp60 levels of DNA-PKcs-deficient SCID cells were higher than those of parental CB-17 cells . Our results suggest the possibility that mitochondrial heat shock protein may be one of determinants of drug sensitivity and could be regulated by DNA-PK activity. Expert Rev Anticancer Ther, 2002 Aug, 2(4), 449 - 59 Integrins and extracellular matrix: a novel mechanism of multidrug resistance; Elliott T et al.; Multidrug resistance remains the major hurdle to successful cancer treatment . Classical mechanisms of multidrug resistance include drug efflux pumps, glutathione-S-transferase upregulation and topoisomerase II-associated multidrug resistance . However, despite extensive research, the clinical relevance of these mechanisms remains unclear and no significant clinical benefit has materialized . Recently, a novel mechanism of multidrug resistance has been identified--extracellular matrix-mediated multidrug resistance: integrin-mediated adherence of cells to extracellular matrix proteins results in significant resistance to many anticancer agents that induce cell death via unrelated mechanisms . Verification of the mechanisms of action of this novel phenomenon will hopefully identify new therapeutic targets to aid in the fight against cancer. Cancer Chemother Pharmacol, 2003 Feb, 51(2), 161 - 6 Epub 2002 Dec 03. Modulation of multidrug resistance-associated protein 1 (MRP1) by p53 mutant in Saos-2 cells; Tsang WP et al.; The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancer and the mutation is correlated with a poor prognosis in cancer therapy . Upregulation of multidrug resistance-associated protein 1 (MRP1) and increase in drug resistance have been found to be induced by p53 mutation . Human osteosarcoma Saos-2 cells, a p53-null cell line, was transfected with p53 with mutations at codon 143 (V to A), 175 (R to H), 248 (R to W), 273 (R to H) and 281 (D to G) . Among the different transfectants, overexpression (about 42-fold) of MRP1 was detected in p53-R175H cells . Furthermore, the p53-R175H cells were 2.5-fold more resistant to doxorubicin (DOX) and had a 4-fold greater DOX efflux rate than the control cells 1 h after DOX treatment . Transfection with antisense MRP1 oligonucleotides demonstrated a DOX sensitization effect (about 2-fold) in p53-R175H transfectants but not in control cells . In addition, transfection with antisense p53 oligonucleotides greatly suppressed MRP1 expression and reversed DOX resistance in p53-R175H cells but had no effect in control cells . The results suggested that p53-R175H might induce MRP1 expression and DOX resistance in cells. J Biol Chem, 2003 Jun 6, 278(23), 21018 - 23 Epub 2003 Mar 19. A novel lytic peptide composed of DL-amino acids selectively kills cancer cells in culture and in mice; Papo N et al.; The high toxicity of most chemotherapeutic drugs and their inactivation by multidrug resistance phenotypes motivated extensive search for drugs with new modes of action . We designed a short cationic diastereomeric peptide composed of d- and l-leucines, lysines, and arginines that has selective toxicity toward cancer cells and significantly inhibits lung metastasis formation in mice (86%) with no detectable side effects . Its ability to depolarize the transmembrane potential of cancer cells at the same rate (within minutes) and concentration (3 micro m), at which it shows biological activity, suggests a killing mechanism that involves plasma membrane perturbation . Confocal microscopy experiments verified that the cells died as a result of acute injury, swelling, and bursting, suggesting necrosis . Biosensor binding experiments and attenuated total reflectance-Fourier transform infrared spectroscopy using model membranes have substantiated its high selectivity toward cancer cells . Although this is an initial study that looked at tumor formation rather than the ability of the peptides to reduce established tumors, the simple sequence of the peptide, its high solubility, substantial resistance to degradation, and inactivation by serum components might make it a good candidate for future anticancer treatment. AAPS PharmSci . 2002;4(4):E29. Detection of MDR1 single nucleotide polymorphisms C3435T and G2677T using real-time polymerase chain reaction: MDR1 single nucleotide polymorphism genotyping assay; Song P et al.; The objective of this study was to develop a real-time polymerase chain reaction (PCR) method to detect MDR1 (human multidrug resistance gene) single nucleotide polymorphisms (SNPs) C3435T and G2677T . C3435T and G2677T are linked to MDR1*2, which is associated with enhanced efflux activity in vitro . Using the Smart Cycler, an allele-specific real-time PCR-based genotyping method was developed to detect C3435T and G2677T . The MDR1 genotype of human genomic DNA templates was determined by direct DNA sequencing . PCR reactions for genotyping C3435T and G2677T by using allele-specific primers were conducted in separate tubes . An additional nucleotide mismatch at the third position from the 3' end of each allele-specific primer was used to abrogate nonspecific PCR amplification . The fluorescence emitted by SYBR Green I was monitored to detect formation of specific PCR products . PCR growth curves exceeding the threshold cycle were considered positive . Fluorescence melt-curve analysis was used to corroborate results from PCR growth curves . Using PCR growth curves, our assay accurately determined hetero- and homozygosity for C3435T and G2677T . Genotype assignments based on PCR growth curve, melt-curve analysis, agarose gel electrophoresis, and direct DNA sequencing results of PCR products were in perfect agreement . We have developed a rapid MDR1 genotyping method that can be used to assess the contribution of MDR1*2 to pharmacokinetic and pharmacodynamic variability of P-glycoprotein substrates. Br J Cancer, 2003 Mar 24, 88(6), 973 - 80 Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway; Lin HL et al.; Hepatoma cells are known to be highly resistant to chemotherapy . Previously, we have found differential Taxol resistance in human and murine hepatoma cells . The aim of this study was to examine the effect of a multidrug resistance inhibitor, cyclosporin A in combination with Taxol on hepatoma in vitro and in vivo, and to identify the possible mechanism involved in Taxol resistance . Simultaneous treatment of cyclosporin A (0-10 microM) and Taxol (0.1 microM) inhibited cell growth in vitro . Cyclosporin A interfered with Taxol (0.1 microM)-induced AKT activation and BAD phosphorylation . Cyclosporin A combined with Taxol treatment augments caspase-9, -3 activation and loss of mitochondrial membrane potential in HepG2 cells . PI3 kinase inhibitor, wortmannin, or a dominant-negative AKT1 expression vector treatment partially enhanced Taxol-induced apoptosis indicating that PI3 kinase-AKT pathway was involved in Taxol-resistance pathway . Moreover, combination treatment reduced tumour growth in SCID and C57BL/6 mice as compared to either Taxol or cyclosporin A treatment . Our results indicate that the combination of cyclosporin A and Taxol is effective in the reversal of Taxol resistance through the inhibition of PI3 kinase-AKT1 pathway. Br J Cancer, 2003 Mar 24, 88(6), 879 - 86 Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours; Zurita AJ et al.; This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT) . Paraffin-embedded sections from 56 previously untreated patients with metastatic TGCT were immunostained for Pgp, MRP1, BCRP, and LRP . All patients received platinum-based chemotherapy after orchidectomy . Immunostaining was related to clinicopathological parameters, response to chemotherapy, and outcome . Strong and intermediate expressions of the different MDR-related proteins were: 27 and 41% (Pgp), 54 and 37% (MRP1), 86 and 7% (BCRP), and 14 and 29% (LRP) . P-glycoprotein and MRP1 associated, respectively, to low AFP (P=0.026) and high LDH levels (P=0.014), whereas LRP expression associated with high beta-hCG levels (P=0.003) and stage IV tumours (P=0.029) . No correlation was found between Pgp, MRP1, and BCRP expression and response to chemotherapy and survival . In contrast, patients with LRP-positive tumours (strong or intermediate expression) had shorter progression-free (P=0.0006) and overall survival (P=0.0116) than LRP-negative patients, even after individual log-rank adjustments by statistically associated variables . Our data suggest that a positive LRP immunostaining at the time of diagnosis in metastatic TGCT is associated with an adverse clinical outcome. Curr Opin Neurol, 2003 Apr, 16(2), 197 - 201 Mechanisms of antiepileptic drug resistance; Sisodiya SM; PURPOSE OF REVIEW: The present review considers new developments in the study and our understanding of resistance to treatment with antiepileptic drugs in epilepsy . RECENT FINDINGS: Studies suggest that mechanisms of resistance to drug treatment that operate in other diseases may also be relevant in human and animal drug-resistant epilepsy . Immunohistochemical and molecular genetic data show that there is overexpression of a number of genes and proteins that mediate nonspecific resistance to drug treatment . In particular, there is upregulation of P-glycoprotein and multidrug resistance-associated proteins 1 and 2 . These proteins appear to be expressed in cerebral parenchymal cell populations that do not normally do so . Work in animal models suggests that these proteins are able to reduce the local concentration of antiepileptic drugs . Although these proteins are therefore good candidates for mediators of drug resistance, there is still limited proof that they are functionally important in human drug-resistant epilepsy . SUMMARY: Careful attention needs to be given to the identification and confirmation of the practical importance of mechanisms postulated to underlie drug resistance in human epilepsy . If certain mechanisms are shown to be involved, then new therapeutic options for drug-resistant human epilepsy may be forthcoming. Hepatol Res, 2003 Feb, 25(2), 99 - 104 Effect of obstructive jaundice on the regulation of hepatic cholesterol metabolism in the rat . Disappearance of abcg5 and abcg8 mRNA after bile duct ligation; Kamisako T et al.; The purpose of this study is to evaluate the effect of bile duct ligation (BDL) on changes of lipoprotein metabolism and hepatic gene expressions that are important for cholesterol metabolism . In male rats serum, very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol and apolipoproteins B and E increased after 5 days of BDL compared with those of sham operation (control) group . Serum apolipoprotein A-IV concentration in the BDL group was lower than that in the control group . In both groups, there was no difference in hepatic lipid concentrations . Hepatic mRNA expressions of scavenger receptor B1, microsomal triglyceride transfer protein (mtp), HMG-coA reductase, cholesterol 7alpha-hydroxylase and multidrug resistance gene product 2 in the BDL group were significantly higher than that in the control group . The adipocyte determination and differentiation 1 mRNA expression in the BDL group was significantly lower than that in the control group . abcg5 and abcg8 mRNA expressions were remarkably decreased in the BDL group . In conclusion, in obstructive jaundice, metabolism of lipoprotein and proteins that are important for lipid metabolism are drastically changed probably for maintaining the hepatic lipid concentration . The remarkable down-regulation of the abcg5 and abcg8 may be an adaptive change reflecting the inability of biliary cholesterol excretion. Hepatol Res, 2003 Jan, 25(1), 48 - 54 Effect of dexamethasone on biliary excretion of bile acids and organic anions in rats; Suzuki C et al.; Multidrug resistance protein 2 (Mrp2) is an important transporter for biliary excretion of organic anions, and is reported to be up-regulated by dexamethasone . In the present study, effect of dexamethazone (1 mg/kg) on biliary excretion of bile acids and organic anions was studied in rats . After bile duct cannulation, bile acids and organic anions were intravenously administered, and their biliary excretion was studied . Biliary excretion of tracer doses of taurocholate and taurolithocholate-sulfate was unchanged with dexamethasone . Dexamethasone increased the excretory maximum of taurolithocholate-sulfate and sulfobromophthalein to 1.8 and 1.5 times, respectively, whereas it did not change the excretory maximum of phenolphthalein-glucuronide and taurocholate . Dexamethasone also increased biliary glutathione excretion . These results obtained by in vivo studies can be explained by the up-regulation of Mrp2, which had been reported in in vitro studies. Arch Inst Pasteur Madagascar, 2002, 68(1-2), 44 - 7 {Mycobacterium tuberculosis resistance to antitubercular agents in Antananarivo in 2000}; Ratsirahonana O et al.; In 1991, the National Tuberculosis control Program (NTP) of Madagascar adopted the short treatment course and the Directly Observed Treatment Strategy (DOTS), according to the recommendations of the OMS/UICTMR . Development of M . tuberculosis primary resistance to the four antituberculosis drugs (streptomycin {S}, rifampicine {R}, isoniazid {H}, ethambutol {E}) is an indicator of the NTP efficiency . We report results from a five-year survey among patients with new smear positive pulmonary tuberculosis . Acquired resistance is assessed among recurrent cases . During the first survey, carried out in 1994-1995 in four large cities, multidrug resistance (MDR) rate to the major antituberculosis drug H and R was low, 0.25% for primary MDR and 5% for acquired MDR . No primary MDR was found in Antananarivo; on the other hand, acquired resistance rate was the highest there (22%) . Because of logistical reasons, the second survey (1999-2000) was only carried out in the capital, Antananarivo . Results obtained among 789 new patients with smear positive pulmonary tuberculosis and 79 recurrents cases in 9 diagnostic centres showed low primary and acquired resistance of 11.1% to any drug . Primary resistance to one drug was 10.6%, mainly due to streptomycin 8.5% . MDR rates are comparable with those observed in 1994-1995: 0.1% for primary MDR and 4% for acquired MDR . These results show that ten years after the new NTP implementation, only a few MDR strains are circulating in Antananarivo, which suggests that NTP has been effective. Cancer Biol Ther, 2002 Nov-Dec, 1(6), 652 - 60 Role of MRIT/cFLIP in protection against chemotherapy-induced apoptosis; Matta H et al.; MRIT (Mach-related inducer of toxicity)/cFLIP (cellular FADD like interlukin1-beta converting enzyme inhibitory protein) is a proteolytically inactive structural homologue of caspase-8, which is known to protect against death receptors-induced apoptosis by blocking the activation of caspase-8 . We have observed that exogenous expression of MRITalpha1/cFLIP(L) isoform also protects against cell death induced by a diverse group of chemotherapeutic drugs with different mechanisms of action, including doxorubicin, etoposide, cytosine arabinoside, daunorubicin, chlorambucil and cisplatin . However, MRITalpha1/cFLIP(L) failed to protect against apoptosis induced by paclitaxel and vincristine, two microtubule-damaging agents . Although MRITalpha1/cFLIP(L) protects against chemotherapy-induced apoptosis in both solid tumor and hematopoietic cell lines, this effect was more pronounced in the former . MRITalpha1/cFLIP(L) expression is decreased during drug-induced apoptosis and exogenous expression of MRITalpha1/cFLIP(L) delays the activation of caspase-8 and -3 during drug- induced apoptosis . These results suggest that MRITalpha1/cFLIPL may be an important determinant of both death receptor- and chemotherapy-induced apoptosis and strategies aimed at downregulating its expression deserve further study as a way to overcome multidrug resistance to cancer therapy. Drug Metab Dispos, 2003 Apr, 31(4), 482 - 90 Multiple alterations of canalicular membrane transport activities in rats with CCl(4)-induced hepatic injury; Song IS et al.; The influence of CCl(4)-induced experimental hepatic injury (CCl(4)-EHI) on the expression and transport activities of primary active transporters on the canalicular membrane, including P-glycoprotein (P-gp), a bile salt export pump (Bsep) and a multidrug resistance associated protein2 (Mrp2), was assessed . CCl(4)-EHI was induced by an intraperitoneal injection of CCl(4) to rats at a dose of 1 ml/kg 24 h prior to the preparation of canalicular liver plasma membrane (cLPM) vesicles and pharmacokinetic studies . The expression of each transporter was measured for the vesicles via Western blot analysis at 6, 12, 24, 36, and 48 h after the injection of CCl(4) . The in vivo canalicular excretion clearance (CL(exc)) of {(3)H}daunomycin, {(3)H}taurocholate and {(3)H}17beta-estradiol-17beta-D-glucuronide (E(2)17betaG), representative substrates of P-gp, Bsep, and Mrp2, respectively, was determined following an i.v . infusion to rats . The uptake of each substrate into cLPM vesicles in the presence of ATP was also measured by a rapid filtration technique . As the result of the CCl(4)-EHI, the protein level of transporters was altered as a function of time in multiple manners; it was increased by 3.6-fold for P-gp, unchanged for Bsep, and decreased by 73% for Mrp2 at 24 h . The in vivo CL(exc) and the intrinsic uptake clearance into cLPM vesicles (CL(int)) at 24 h after the CCl(4) injection (CCl(4)-EHI(24 h)) were also influenced by the EHI in a similar manner; they were increased by 1.8- and 1.9-fold for daunomycin, unchanged for taurocholate, and decreased by 41 and 39% for E(2)17betaG, respectively, consistent with multiple alterations in the expression of the relevant transporters. Am J Trop Med Hyg, 2003 Feb, 68(2), 140 - 2 Drug-resistant malaria in Bangladesh: an in vitro assessment; Noedl H et al.; Forty-four Plasmodium falciparum isolates from Bangladesh and 22 from western Thailand were successfully tested for their drug susceptibility . High degrees of resistance were observed against chloroquine with geometric mean IC50s of 114.25 and 120.5 nM, respectively, for Bangladesh and western Thailand . Most isolates from both sites were sensitive to quinine, and all were sensitive to artesunate . Many isolates were considered in vitro resistant to mefloquine, but the geometric mean IC50 for the Thai isolates (98.79 nM) was 1.6 times (P = 0.002) higher than that of isolates from Bangladesh (60.3 nM) . The high prevalence of in vitro mefloquine resistance in Bangladesh suggests that close surveillance is necessary to delay widespread multidrug resistant problems in the area. Nat Biotechnol, 2003 Apr, 21(4), 428 - 33 Epub 2003 Mar 17. Multiherbicide tolerance conferred by AtPgp1 and apyrase overexpression in Arabidopsis thaliana; Windsor B et al.; Herbicide resistance is an important trait often introduced into crop plants . Mechanisms of resistance can involve a mutant target protein that is unaffected by the herbicide, or metabolic detoxification or degradation of the herbicide . Recently, we showed that overexpression in Arabidopsis thaliana of either psNTP9, the garden pea apyrase gene, or AtPgp1, the A . thaliana homolog of the plant multidrug resistance (MDR) gene, enabled A . thaliana to germinate on the toxin cycloheximide and to grow better on toxic levels of the plant hormone N6-{2-isopentyl}adenine (2iP) . Here we report that overexpression of either MDR or apyrase proteins resulted in increased resistance to herbicides from different chemical classes . Apyrase inhibition by small molecule inhibitors reversed this resistance . Treatment of untransformed plants with an apyrase inhibitor increased their sensitivity to the same herbicides . These results indicate that the genes may be involved in a resistance mechanism relating to decreased retention or increased active efflux of herbicide from the plant cell. Pest Manag Sci, 2003 Mar, 59(3), 294 - 302 Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea; Hayashi K et al.; Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers . Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound . Tacrolimus, a macrolide compound with immunosuppressive activity, was also active . The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B . cinerea . The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole . The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants . These results indicate that chlorpromazine is a modulator of BcatrD activity in B . cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies. N Engl J Med, 2003 May 29, 348(22), 2175 - 85 Epub 2003 Mar 13. Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America; Lalezari JP et al.; BACKGROUND: The T-20 vs . Optimized Regimen Only Study 1 (TORO 1) was a randomized, open-label, phase 3 study of enfuvirtide (T-20), a human immunodeficiency virus type 1 (HIV-1) fusion inhibitor . METHODS: Patients from 48 sites in the United States, Canada, Mexico, and Brazil with at least six months of previous treatment with agents in three classes of antiretroviral drugs, resistance to drugs in these classes, or both, and with at least 5000 copies of HIV-1 RNA per milliliter of plasma were randomly assigned in a 2:1 ratio to receive enfuvirtide plus an optimized background regimen of three to five antiretroviral drugs or such a regimen alone (control group) . The primary efficacy end point was the change in the plasma HIV-1 RNA level from base line to week 24 . RESULTS: A total of 501 patients underwent randomization, and 491 received at least one dose of study drug and had at least one measurement of plasma HIV-1 RNA after treatment began . The two groups were balanced in terms of the median base-line HIV-1 RNA level (5.2 log10 copies per milliliter in both groups), median CD4+ cell count (75.5 cells per cubic millimeter in the enfuvirtide group, and 87.0 cells per cubic millimeter in the control group), demographic characteristics, and previous antiretroviral therapy . At 24 weeks, the least-squares mean change from base line in the viral load (intention-to-treat, last observation carried forward) was a decrease of 1.696 log10 copies per milliliter in the enfuvirtide group, and a decrease of 0.764 log10 copies per milliliter in the control group (P<0.001) . The mean increases in CD4+ cell count were 76 cells per cubic millimeter and 32 cells per cubic millimeter, respectively (P<0.001) . Reactions at the site of the injections were reported by 98 percent of patients receiving enfuvirtide . There were more cases of pneumonia in the enfuvirtide group than in the control group . CONCLUSIONS: The addition of enfuvirtide to an optimized antiretroviral regimen provided significant antiretroviral and immunologic benefit through 24 weeks in patients who had previously received multiple antiretroviral drugs and had multidrug-resistant HIV-1 infection . J Biol Chem, 2003 May 16, 278(20), 17664 - 71 Epub 2003 Mar 13. Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells; Wielinga PR et al.; Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues . Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles . We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5 . The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively . MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner . In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport . Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport . In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux . The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour . This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal . These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting. BMJ . 2003 Mar 15;326(7389):574. Impact of DOTS compared with DOTS-plus on multidrug resistant tuberculosis and tuberculosis deaths: decision analysis; Sterling TR et al.; OBJECTIVE: This study sought to determine the impact of the World Health Organization's directly observed treatment strategy (DOTS) compared with that of DOTS-plus on tuberculosis deaths, mainly in the developing world . DESIGN: Decision analysis with Monte Carlo simulation of a Markov decision tree . DATA SOURCES: People with smear positive pulmonary tuberculosis . DATA ANALYSIS: Analyses modelled different levels of programme effectiveness of DOTS and DOTS-plus, and high (10%) and intermediate (3%) proportions of primary multidrug resistant tuberculosis, while accounting for exogenous reinfection . MAIN OUTCOME MEASURE: The cumulative number of tuberculosis deaths per 100 000 population over 10 years . RESULTS: The model predicted that under DOTS, 276 people would die from tuberculosis (24 multidrug resistant and 252 not multidrug resistant) over 10 years under optimal implementation in an area with 3% primary multidrug resistant tuberculosis . Optimal implementation of DOTS-plus would result in four (1.5%) fewer deaths . If implementation of DOTS-plus were to result in a decrease of just 5% in the effectiveness of DOTS, 16% more people would die with tuberculosis than under DOTS alone . In an area with 10% primary multidrug resistant tuberculosis, 10% fewer deaths would occur under optimal DOTS-plus than under optimal DOTS, but 16% more deaths would occur if implementation of DOTS-plus were to result in a 5% decrease in the effectiveness of DOTS CONCLUSIONS: Under optimal implementation, fewer tuberculosis deaths would occur under DOTS-plus than under DOTS . If, however, implementation of DOTS-plus were associated with even minimal decreases in the effectiveness of treatment, substantially more patients would die than under DOTS. Pharm Res, 2003 Feb, 20(2), 324 - 7 Gene expression profiles of ABC transporters and cytochrome P450 3A in Caco-2 and human colorectal cancer cell lines; Nakumura T et al.; PURPOSE: The mRNA levels of MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), cytochrome P450 3A (CYP3A) and villin in human colorectal cell lines (HCT-15, LoVo DLD-1, HCT-116 and SW620) were quantitatively compared with those in Caco-2 cells . METHODS: The mRNA levels were determined by real time quantitative polymerase chain reaction and expressed as the relative concen trations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydro genase (GAPDH) mRNA . RESULTSl MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2 . The expression of MRP mRNA in the cell lines tested was comparable with Caco-2 . MRP . mRNA was detected only in HCT-116 and SW620 at significantly lower level than Caco-2 . CYP3A mRNA was detected in HCT-15 LoVo, DLD-1 and SW620 at similar level to Caco-2 . CONCLUSIONS: HCT-15 LoVo and DLD-1 cells express proteins important for regulating the intestinal absorption of drugs, i.e., MDR1 MRP1 and CYP3A, whereas HCT-116 and SW620 cells were not acceptable for evaluation of absorption properties of drug candidates Pharm Res, 2003 Feb, 20(2), 161 - 8 Isolation and characterization of Caco-2 subclones expressing high levels of multidrug resistance protein efflux transporter; Horie K et al.; PURPOSE: The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors . METHODS: The subclones were selected by a dilution cloning technique . The polarized efflux of {3H}-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (Papp) of {3H}-vinblastine in the basolateral (BL)-to-apical (AP) direction and in the AP-to-BL direction (Papp BL-to-AP/Papp AP-to-BL) across the cell monolayers . The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis . The kinetics parameters (Km, Vmax) of {3H}-vinblastine and the inhibitory constants (KI) of several known MDR1 substrates/inhibitors on the transport of {3H}-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared . RESULTS: Three subclones (#1, #20, #21) were selected based on their polarized efflux of {3H}-vinblastine . The Papp BL-to-AP/Papp AP-to-BL ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells . In the presence of GF-120918 (2 microM), a known MDR1-specific inhibitor, the Papp BL-to-AP/Papp AP-to-BL ratios were significantly decreased, suggesting that these cells were overexpressing MDRI . The Km values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells . In contrast, the Vmax values observed in the subclones were approximate 26-69% higher . The KI values observed for various known MDR1 substrates/inhibitors on {3H}-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones . The high functional efflux activities of these subclones were stable up to 6 months . CONCLUSIONS: Subclones #1, #20, #21 express high levels of MDR1 . These Caco-2 subclones may be useful models for profiling drugs for their MDR1 substrate activity and for establishing structure-transport relationships for this efflux transporter. Przegl Lek, 2002, 59(10), 854 - 8 {Mechanism of multidrug resistance of ovarian cancer}; Miedzinska-Maciejewska M et al.; Ovarian cancer is one of the most frequent cancers in women, and five-year survival rate from ovarian cancer is only 20% to 30% . The main problem and reason for lack of success in treatment is usually late recognition . Unfortunately, advances in surgical techniques and chemotherapy do not translate into improved survival . Despite the high response rates with initial treatment, most patients with ovarian cancer relapse . Usually then they are chemoresistant to standard chemotherapy . This article presents a review of the basic mechanisms of drug resistance in ovarian cancer and their clinical significance. Int J Oncol, 2003 Apr, 22(4), 823 - 7 Reversal of drug resistance mediated by hammerhead ribozyme against multidrug resistance-associated protein 1 in a human glioma cell line; Osada H et al.; We examined the effects of suppressing multidrug resistance-associated protein 1 (MRP1) gene expression in a human glioma cell line U87MG . Hammerhead ribozymes, designed to cleave MRP1 mRNA (alphaMRP1-Rz), were transfected into the U87MG cells . The U87MG/alphaMRP1-Rz cells were significantly sensitive to nitrosourea (ACNU) and doxorubicin (DOX) compared with the U87MG cells (p<0.01 and p<0.05, respectively, unpaired t-test) . There was no significant difference in the expression of other human genes between the U87MG/alphaMRP1-Rz and controls by cDNA array . The hammerhead ribozyme-mediated specific suppression of MRP1 was sufficient to reverse the resistance of ACNU and DOX in the human glioma cell line. Int J Oncol, 2003 Apr, 22(4), 733 - 9 The multidrug resistance modulator SDZ-PSC 833 potentiates the photodynamic activity of chlorin e6 independently of P-glycoprotein in multidrug resistant human breast adenocarcinoma cells; Merlin JL et al.; Photodynamic therapy has clinical indications in treatment of localized cancers and could be interesting for eradication of local recurrence of chemoresistant tumors . In the present study, the intracellular accumulation and distribution of chlorin e6 was investigated in MCF-7 and in P-glycoprotein overexpressing, doxorubicin resistant MCF-7/DXR cell lines . After 3-h incubation with chlorin e6 (1.7 micro mol.l(-1)), no significant difference in accumulation was observed between MCF-7 and MCF-7/DXR cells . Chlorin e6 cellular efflux did not differ in the two cell lines . The lack of influence of P-glycoprotein was confirmed since SDZ-PSC833 (PSC) had no influence in chlorin e6 accumulation and efflux in MCF-7/DXR cells . The subcellular distribution of chlorin e6 appeared different in MCF-7/DXR than in MCF-7 cells . Double staining colocalization fluorescence microscopy studies were performed to identify the subcellular localization sites for chlorin e6 using organelle probes for endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes . In MCF-7, chlorin e6 was distributed in all cytoplasmic organelles including endoplasmic reticulum and Golgi . In MCF-7/DXR, a diffuse cytoplasmic distribution was observed excepted for the endoplasmic reticulum and Golgi area in which less chlorin e6 was distributed . In MCF-7/DXR, PSC was found to restore the distribution of chlorin e6 in the endoplasmic reticulum and Golgi area while in MCF-7, no effect on the subcellular distribution of chlorin e6 was observed . Although the photodynamic activity of chlorin e6 (1.7 micro mol.l(-1), 650 nm, 8 mW.cm(-2)) was found to be lower in MCF-7/DXR than in MCF-7 cells, PSC was found to potentiate the photodynamic activity of chlorin e6 to similar extent in both cell lines . These results clearly demonstrate that PSC potentiates the photodynamic activity of Chlorin e6 independently of the expression of P-glycoprotein and further suggest that the photodynamic activity of chlorin e6 could be related to its intracellular distribution in the endoplasmic reticulum and the Golgi. Euro Surveill, 2000 Apr, 5(4), 43 - 45 The Spanish multidrug resistant tuberculosis network; Samper S et al.; The network to monitor the spread of multidrug resistance tuberculosis (MDR-TB) in Spain based on genomic typing and set up in January 1998 benefits from the participation of about 90% of the laboratories of the national health system . Of the 94 MDR-TB pa Clin Cancer Res, 2003 Mar, 9(3), 1083 - 6 Response to chemotherapy and expression of the genes encoding the multidrug resistance-associated proteins MRP2, MRP3, MRP4, MRP5, and SMRP in childhood acute myeloid leukemia; Steinbach D et al.; PURPOSE: The family of multidrug resistance-associated proteins (MRPs) belongs to the ATP-binding cassette superfamily of transporters, which have the ability to function as outward pumps for chemotherapeutic drugs . Their structure, function, and substrate specificity have been studied intensively, but little is known about their clinical relevance in malignant diseases . EXPERIMENTAL DESIGN: In this study, the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time PCR in 53 children with de novo acute myeloid leukemia . Nine patients were also analyzed in relapse . RESULTS: MRP3 gene expression was higher in patients who did not achieve remission (P = 0.023) . Expression of MRP2 (P = 0.09) or MRP3 (P = 0.041) was associated with a lower rate of survival, and patients who expressed high levels of both genes had a particularly poor prognosis (P < 0.01) . No significant association was found for overall survival or remission rate and the expression of MRP4, MRP5, and SMRP . CONCLUSIONS: This study provides first data on the clinical relevance of five MRPs in acute myeloid leukemia patients . The results strongly suggest that MRP3 and possibly also MRP2 are involved in drug resistance in this disease . Those two proteins therefore represent interesting markers for risk-adapted therapy and possible targets for the development of specific drugs to overcome multidrug resistance. J Pharm Pharmacol, 2003 Feb, 55(2), 153 - 62 The effect of food components on the absorption of P-gp substrates: a review; Deferme S et al.; P-glycoprotein (P-gp), a well characterized efflux mechanism which is functionally expressed in the intestinal epithelium, constitutes, along with intestinal metabolism, an important part of the biochemical barrier function of the intestinal mucosa . This efflux carrier may be responsible for limiting the bioavailability of several drugs after oral intake . Recently, increasing attention is being paid to the interaction of dietary components with the intestinal absorption of drugs . This review focuses on the modulating capacity of food components on the intestinal absorption of P-gp substrates . The possible P-gp inhibitory effects of several dietary constituents are discussed . In addition, this review will also focus on the effect of several bioflavonoids on the P-gp-mediated efflux of drugs . As the role of P-gp (and other efflux carriers, including multidrug resistance-associated proteins and breast cancer resistance protein) in limiting the bioavailability of drugs becomes more clear, more research is required firstly to identify the effect of dietary compounds on these efflux carriers and secondly to reveal the clinical relevance of this interaction. Kidney Int, 2003 Mar, 63(3), 976 - 86 Sulfate conjugating and transport functions of MDCK distal tubular cells; Ng KH et al.; BACKGROUND: Transfected Madin-Darby canine kidney (MDCK) cells (of distal tubular origin) have been used to study transport of organic anions . These cells have not been shown to possess sulfate-conjugating activity . Neither has transport activity been demonstrated in nontransfected MDCK cells . METHODS: Polarized and monolayers of nontransfected MDCK type II cells were incubated with prototype substrates of phenolsulfotransferase (PST) and sodium sulfate in the absence or presence of known inhibitors of multidrug resistance protein (MRP): (3-3-(2-(7-chloro-2-quinionlinyl) ethenyl)phenyl)(3-dimethylamino-3-oxopropyl)thio)methyl)thio) propanoic acid (MK571), cyclosporin A (CsA), and probenecid . Effects of glutathione (GSH) and buthionine sulfoximine (BSO), potential modulators of the organic anion transporting protein/polypeptide (OATP) isoform, OATP1 were also examined . Sulfated conjugates were identified by high-performance liquid chromatography (HPLC)-radiometry or HPLC-fluorimetry . RESULTS: Uptake, sulfate conjugation, and efflux of the sulfated conjugates of harmol, p-nitrophenol, N-acetyldopamine and acetaminophen were demonstrated . Activities in MDCK type II cells were higher than those in HepG2, human fetal liver, and Chang liver cells . A significant decrease in extracellular with a reciprocal increase in intracellular harmol sulfate was observed with MK571, CsA, and probenecid and with preloading of glutathione . Depletion of intracellular glutathione by BSO had the opposite effects . CONCLUSIONS: Normal (nontransfected) MDCK type II cells provide a suitable system for the study of the physiologic processes of uptake, sulfate conjugation, and transport of sulfated conjugates in kidney cells . Based on the action of specific inhibitors and modulators of MRP2 and OATP1, it was concluded that MRP2-like and OATP1-like transporters are possibly responsible for the transport of sulfated conjugates. Chest, 2003 Mar, 123(3), 953 - 6 Multidrug-resistant tuberculosis in pregnancy: case report and review of the literature; Lessnau KD et al.; A woman at 23 weeks' gestation was treated with rifampin, isoniazid, and ethambutol for cavitary tuberculosis (TB) . She did not respond within 3 weeks, and multidrug-resistant (MDR) TB was suspected . Direct plating on susceptibility media was performed immediately . Treatment was initiated with IV capreomycin, levofloxacin, para-aminosalicylic acid, pyrazinamide, cycloserine, and high-dose vitamin B(6) at 26 weeks' gestation . The patient delivered vaginally at week 35 . The newborn was not infected . Following delivery, ethionamide was added as a sixth drug, and levofloxacin was replaced with moxifloxacin . The patient's sputum became smear-negative and culture-negative for TB . All reported cases of MDR-TB during pregnancy are reviewed. Biochem Pharmacol, 2003 Mar 1, 65(5), 765 - 71 The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis; Hooijberg JH et al.; Previously, we reported that the multidrug resistance proteins MRP1, MRP2 and MRP3 confer resistance to therapeutic antifolates by mediating their cellular extrusion . We now determined whether MRPs also play a role in controlling cellular homeostasis of natural folates . In MRP1, MRP2 and MRP3-transfected 2008 human ovarian carcinoma cells total cellular folate content was 32-38% lower than in 2008 cells (105+/-14pmolfolate/mgprotein) when grown in medium containing 2.3 microM folic acid (FA) . Under these conditions cellular growth rates were not compromised . However, when cells were challenged under folate-depleted conditions with a short exposure (4 hr) to FA or leucovorin, MRP1 and MRP3 overexpressing cells were impaired in their growth . In contrast to wild-type cells, MRP1 transfected cells retained only 60% of the maximum growth when exposed to 500 nM leucovorin or 500 microM FA . For 2008/MRP1 and 2008/MRP3 cells FA growth stimulation capacity was dramatically decreased when, during a 4 hr exposure, metabolism into rapidly polyglutamatable and retainable dihydrofolate was blocked by the dihydrofolate reductase inhibitor trimetrexate . To retain growth under such conditions MRP1 overexpressing cells required much higher concentrations of FA (EC(50) > 500 microM) compared to 2008 cells (EC(50): 12 microM) . These results suggest that down- and up-regulation of MRP1 (and MRP3) expression can influence cellular folate homeostasis, in particular when cellular retention by polyglutamylation of folates is attenuated. Biochem Pharmacol, 2003 Mar 1, 65(5), 747 - 54 Minimally modified phosphodiester antisense oligodeoxyribonucleotide directed against the multidrug resistance gene mdr1; Brigui I et al.; In the perspective of reversing multidrug resistance through antisense strategy while avoiding non-antisense effects of all-phosphorothioate oligonucleotides which non-specifically bind to proteins, a minimally modified antisense phosphodiester oligodeoxyribonucleotide has been designed against mdr1, one of the multidrug resistance genes . Its stability in lysates prepared from NIH/3T3 cells transfected with the human mdr1 gene has already been demonstrated . Confocal microspectrofluorometry using a fluorescence resonance energy transfer technique allowed its stability inside living cells to be proven . Its internalization into the cells was achieved with different delivery agents (addition of a cholesteryl group, Superfect or an amphotericin B cationic derivative) and has been followed by fluorescence imaging . For each of the delivery systems, Western blotting allowed its antisense efficiency to be compared to that of an all-phosphorothioate antisense oligonucleotide . No antisense efficiency was demonstrated for the minimally modified ODN when internalized with Superfect . In both other cases, the best extinction of the P-glycoprotein expression has always been achieved with the all-phosphorothioate antisense . While the difference was significant in the case the amphotericin B derivative was used as delivery agent (20% remaining protein expression with the all-phosphorothioate vs . 40% with the minimally modified antisense), it was negligible for the cholesterol conjugates (2% vs . 6%) . It is of great interest to prove that an almost all-phosphodiester oligonucleotide can be an efficient antisense against an overexpressed gene . The reduction of non-antisense effects as non-specific binding to proteins are of importance in the case relatively high ODN concentrations are used, which can prove to be necessary in the case of overexpressed genes. Cancer, 2003 Mar 15, 97(6), 1481 - 7 Gemtuzumab, fludarabine, cytarabine, and cyclosporine in patients with newly diagnosed acute myelogenous leukemia or high-risk myelodysplastic syndromes; Tsimberidou A et al.; BACKGROUND: Gemtuzumab is used to treat patients with previously untreated or recurrent acute myelogenous leukemia (AML) . The fludarabine and cytarabine (ara-C) regimen is active in these patients . Resistance to gemtuzumab is associated with blast multidrug resistance (MDR) . The objectives of this study were to evaluate the efficacy and toxicity of a combination regimen of gemtuzumab, fludarabine, ara-C, and the MDR modifier (cyclosporine {CyA}) in patients with previously untreated AML, refractory anemia with excess blasts (RAEB), or RAEB in transformation (RAEBT) . METHODS: The MFAC regimen was comprised of gemtuzumab (Mylotarg trade mark ) (6 mg/m(2) intravenously {i.v.} on Day 1); fludarabine and ara-C (15 mg/m(2) and 0.5 g/m(2), respectively, twice daily on Days 2-6); and CSA (6 mg/kg loading dose before gemtuzumab, followed by 16 mg/kg continuous i.v . infusion on Days 1 and 2) . RESULTS: Fifty-nine evaluable patients were treated: 39 patients (66%) had AML and 20 patients (34%) had RAEB/RAEBT . Their median age was 57 years (range, 27-76 years) . The MFAC regimen induced complete remission (CR) in 27 patients (46%) and CR with incomplete platelet recovery (CRp) in 1 patient (2%) . The median survival period is 8 months . At 12 months, the survival rate is 38% and the event-free survival rate in patients with CR/CRp is 27% . Infections complicated 38% of the courses of chemotherapy . Grade 3/4 toxicity included hyperbilirubinemia in 31% and transaminitis in 7% of the patients . Four patients (7%) developed hepatic venoocclusive disease (VOD) . CONCLUSIONS: The MFAC regimen may merit further study in patients with AML if measures to avoid and/or treat VOD can be incorporated into the regimen . J Pharm Pharmacol, 2003 Jan, 55(1), 59 - 66 Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines; Pfrunder A et al.; Colon carcinoma cell lines are used widely as screening models for intestinal absorption of drugs . However, the expression of important transport systems and of metabolic enzymes is not completely characterized yet . The expression and inducibility of multidrug resistance gene 1 (MDR1) and cytochrome P450 isoform 3A4 (CYP3A4) was investigated in Caco-2 parental, Caco-2 TC-7 (TC-7) and LS180 cell lines . In the same three cell lines, we investigated the expression of isoforms of the multidrug resistance associated protein family (MRP1-MRP5) and the human pregnane X receptor (hPXR), which may be important for MDR1 and CYP3A4 induction . Cells were treated with rifampicin or 1alpha,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) for 72 h and the total RNA was extracted . Afterwards reverse transcription real-time polymerase chain reaction (TaqMan) assay was performed to determine the mRNA expression level . We have shown that in LS180 cells, MDR1 and CYP3A4 were inducible with both inducers . In Caco-2 parental and TC-7 cells, CYP3A4 was only inducible with 1,25(OH)(2)D(3) . Furthermore, differences were shown in gene expression of several transport proteins (MDR1 and MRP1-MRP5) and CYP3A4 in different human colon carcinoma derived cell lines . hPXR mRNA was expressed in all three cell lines but the amount of mRNA detected was significantly higher in LS180 cells than in Caco-2 and TC-7 cells . We concluded that LS180 cells were a suitable model to study MDR1 and CYP3A4 induction, but for drug transport studies Caco-2 parental and TC-7 cells would be preferred as the more physiological model. J Clin Microbiol, 2003 Mar, 41(3), 1235 - 9 Susceptibility testing with the manual mycobacteria growth indicator tube (MGIT) and the MGIT 960 system provides rapid and reliable verification of multidrug-resistant tuberculosis; Adjers-Koskela K et al.; The objective of the study was to compare the manual Mycobacteria Growth Indicator Tube (MGIT) method and the BACTEC MGIT 960 system to the BACTEC 460 method for susceptibility testing of Mycobacterium tuberculosis . The evaluation was based on testing of 36 M . tuberculosis strains with various susceptibilities to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM) . In addition, five of the strains generating discrepant results in testing for EMB were analyzed for heteroresistance . For INH, the susceptibility test results obtained by the MGIT 960 and the manual MGIT systems agreed with the BACTEC 460 results in 94 and 97% of the cases, respectively . The results of susceptibility to RMP were all in agreement . For SM, 78 and 72% of the results obtained by the MGIT 960 and the manual MGIT systems, respectively, agreed with the BACTEC 460 results . In contrast, less than 80% of the results for susceptibility to EMB obtained by the two MGIT methods agreed with the BACTEC 460 results . All five strains analyzed for EMB heteroresistance were found to consist of resistant and susceptible subpopulations . The average turnaround times were 6.4 days for the MGIT 960 system, 6.5 for the manual MGIT system, and 8.7 days for the BACTEC 460 method . Both MGIT methods can be regarded as accurate and rapid alternatives to the BACTEC 460 method for detection of strains resistant to INH and RMP . However, more studies are needed for solving the problems associated with susceptibility testing to EMB and SM. J Clin Microbiol, 2003 Mar, 41(3), 1101 - 8 Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis; Van Der Zanden AG et al.; Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB . Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced . In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears . We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the rpoB gene using reverse line blot hybridization and DNA sequencing . Analysis of the rpoB gene revealed that samples containing rifampin-resistant Mycobacterium tuberculosis carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase . Although the sensitivities of both methods were equal (84%), sequencing of the rpoB gene was more accurate in identifying mutations in the core region of the rpoB gene . Sequence analysis of the rpoB gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale . The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials. Blood, 2003 Jul 1, 102(1), 246 - 53 Epub 2003 Mar 06. Hydrolytically activated etoposide prodrugs inhibit MDR-1 function and eradicate established MDR-1 multidrug-resistant T-cell leukemia; Schroeder U et al.; Effective therapy of high-risk leukemia with established cytotoxic drugs may be limited by poor antitumor efficacy, systemic toxicity, and the induction of drug resistance . Here, we provide the first evidence that hydrolytically activated prodrugs may overcome these problems . For this purpose, VP16 was functionally blocked by hydrolytically cleavable carbonate linkers with unique characteristics to generate 2 novel prodrugs of VP16 . First, we established a more than 3-log higher efficacy of the 2 prodrugs compared with VP16 on a panel of naturally drug-resistant tumor cell lines . Second, the prodrugs did overcome VP16-induced multidrug resistance-1 gene (MDR-1)-mediated multidrug resistance in vitro in a newly established VP16-resistant T-cell leukemia cell line MOVP-3 by functionally blocking MDR-1-mediated efflux . Third, in vivo studies showed a maximum tolerated dose of ProVP16-II (> 45mg/kg), which was at least 3-fold higher than that of VP16 (15 mg/kg) . Finally, tests of ProVP16-II in a multidrug-resistant xenograft model of T-cell leukemia expressing MDR-1 indicated that only the mice treated with this prodrug revealed a complete and long-lasting regression of established, drug-resistant leukemia . In summary, the hydrolytically activated etoposide prodrugs proved effective against multidrug-resistant T-cell leukemia in vitro and in vivo and provide proof of concept for a highly promising new strategy for the treatment of MDR-1 drug-resistant malignancies. Biochem Pharmacol, 2003 Mar 15, 65(6), 969 - 77 Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein, MRP1; Saengkhae C et al.; Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane . A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport . The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU) . We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1 . The measurements were made in real time using intact cells . The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues . They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein . The active efflux of both FLU derivatives was inhibited by GSH depletion. Nucl Med Biol, 2003 Feb, 30(2), 111 - 7 Effect of P-glycoprotein and multidrug resistance associated protein gene expression on Tc-99m MIBI imaging in hepatocellular carcinoma; Chang CS et al.; P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) expressions as well as Tc-99m methoxisobutylisonitrile (MIBI) images were assessed in 25 patients hepatocellular carcinoma (HCC) . Tc-99m MIBI imaging was performed 10 minutes after intravenous injection of 20 mCi Tc-99m MIBI . Using immunohistochemical staining, 60% of the HCC lesions showed positive for Pgp and 64% showed positive for MRP . In 3 patients with MIBI uptake, immunohistochemical study of tumor tissue showed no Pgp stained cells . Nevertheless, they were all positive for MRP . The result of Tc-99m MIBI imaging is more related to the expression of Pgp than MRP gene . It is possible that other membrane transporters as well as Pgp and MRP are involved in the efflux of Tc-99m MIBI. Arch Biochem Biophys, 2003 Mar 15, 411(2), 243 - 50 Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal; Reichard JF et al.; 4-hydroxynonenal (4HNE) is a major product of peroxidative membrane lipid destruction and exerts a variety of deleterious actions through formation of covalent adducts with cellular nucleophiles . Consequently, a number of cellular enzyme systems exist that are capable of detoxifying this reactive aldehyde by oxidation, reduction, or conjugation with glutathione . In this investigation we characterize the multidrug resistance-associated protein 2 (MRP2) as the primary transmembrane transport protein in hepatocytes responsible for extracellular export of 4HNE-glutathione conjugate (HNE-SG) from the intracellular site of its formation . Suspensions of freshly isolated hepatocytes (10(6) cells/ml) prepared from either wild-type (WT) Wistar rats or TR(-) rats possessing a mutated Mrp2 gene were incubated with 4HNE (50 nmol/10(6) cells) . The formation of 4HNE metabolites, 4-hydroxynonenoic acid (HNA) and HNE-SG, was quantified in the intracellular and extracellular fractions . These studies demonstrated that freshly isolated hepatocytes from both WT and TR(-) rats formed and exported the oxidized metabolite (HNA) to similar extents . Likewise, both populations of hepatocytes displayed nearly identical rates of glutathione conjugation with 4HNE . However, the rate of HNE-SG export from TR(-) hepatocytes was approximately fourfold less than that of WT hepatocytes . In TR(-) hepatocytes, HNE-SG accumulated and remained predominantly intracellular throughout the time course, suggesting an absence of compensatory export by other hepatocellular transporters . In conclusion, these data demonstrate that although WT and TR(-) hepatocytes are similar in their conjugative and oxidative metabolism of 4HNE, export of 4HNE-SG is mediated by the MRP2 transporter, a transport system distinct from that involved in HNA efflux. Zhonghua Bing Li Xue Za Zhi, 2002 Dec, 31(6), 506 - 9 {Expression of glutathione S-transferase, P-glycoprotein, and multidrug resistance-associated protein in neuroblastoma and its clinical significance}; Lu Q et al.; OBJECTIVE: To detect expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and glutathione S-transferase (GST-pi), and to evaluate its clinical significance in neuroblastoma (NB) . METHODS: SP immunohistochemical technique was used to investigate expression of P-gp, MRP, and GST-pi in 70 cases of NB . RESULTS: The frequency of expression of P-gp, MRP, and GST-pi was 61.4%, 38.6%, and 51.4%, respectively . The coexpression rate of P-gp and MRP, P-gp and GST-pi, MRP and GST-pi, P-gp, MRP and GST-pi was 32.9%, 35.7%, 27.1%, and 24.3%, respectively . Significant positive correlation was observed between P-gp and MRP expression (P = 0.001), and between MRP and GST-pi expression (P = 0.012), but no correlation was found between P-gp and GST-pi expression . The expression of P-gp and MRP was higher in tumors from patients over 1 year old compared with those less than 1 year old at diagnosis (P = 0.01, 0.018, respectively) . MRP expression was higher in tumors from the metastatic than the non metastatic groups (P = 0.015) . All tested proteins showed significant relationship to the differentiation of the tumor (P = 0.006, 0.000, 0.019, respectively), but no correlation was found to the stage of NB or sex of the patients . MRP expression was significantly related to the reduction of both median survival time and the two-year cumulative survival (P = 0.02) . In contrast, P-gp and GST-pi expression had no correlation with survival . CONCLUSIONS: The intrinsic multidrug resistance of NB involves the combined effects of P-gp, MRP, and GST-pi . MRP expression may be an important parameter in predicting the prognosis of patients with NB. Brain Tumor Pathol, 2002, 19(2), 59 - 62 Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervous system tumors; Sasaki T et al.; Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles . It functions as a transport-associated protein that can be associated with multidrug resistance . In previous studies, expression of MVP/LRP has been documented in tumors of various types . In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis . MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized . Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors . Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1) . Various degrees and distributions of immunoreactivity to MVP/ LRP were observed . Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion. Leuk Res, 2003 May, 27(5), 413 - 23 Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype; Lopes EC et al.; Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes . We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160) . The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines . By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines . After treatment with 0.1 microg ml(-1) of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes . Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis . Our results demonstrated that CsA (1 microg ml(-1)) was able to induce apoptosis in all cell lines: 18.31% (+/-4.46) for LBR-, 25.96% (+/-5.24) for LBR-V160 and 27.36% (+/-4.12) for LBR-D160, while PSC 833 (1 microg ml(-1)) only induced apoptosis 21.51% (+/-5.73) in LBR-V160 cell line . The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x(L)) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines . Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5-8 of the tumor suppressor gene . We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 microg ml(-1)) or by direct impact on the cells through induction of apoptosis (1 microg ml(-1)), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins. J Bacteriol, 2003 Mar, 185(6), 1851 - 6 Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli; Hirakawa H et al.; In Escherichia coli, there are 32 open reading frames (ORFs) that are assumed to be response regulator genes of two-component signal transduction systems on the basis of sequence similarities . We cloned all of these 32 ORFs into a multicopy expression vector and investigated whether or not they confer drug resistance via control of drug resistance determinants . Fifteen of these ORFs, i.e., baeR, citB, cpxR, evgA, fimZ, kdpE, narL, narP, ompR, rcsB, rstA, torR, yedW, yehT, and dcuR, conferred increased single- or multidrug resistance . Two-thirds of them conferred deoxycholate resistance . Five of them, i.e., evgA, baeR, ompR, cpxR, and rcsB, modulated the expression of several drug exporter genes . The drug resistance mediated by evgA, baeR, and cpxR could be assigned to drug exporters by using drug exporter gene knockout strains. Neurosci Lett, 2003 Mar 13, 339(1), 33 - 6 Differential expression of multidrug resistance genes in naïve rat brain; Kwan P et al.; The multidrug resistance (mdr1) gene family encodes the efflux transporter P-glycoprotein (P-gp) which contributes to the functionality of the blood-brain barrier . We have characterised the regional expression of mdr1 genes in nai;ve rat brain . Adult male Sprague-Dawley rats (n=6) were sacrificed and their brains rapidly removed . Seven distinct anatomical regions were isolated by microdissection and the expression of mdr1a and mdr1b determined by quantitative reverse-transcriptase polymerase chain reaction . The mdr1a isoform was expressed in all brain regions investigated, while mdr1b was expressed to a quantifiable degree in hippocampus alone . These findings reveal a differential expression of mdr1 genes in normal rodent brain tissue and suggest that P-gp may afford a broader protection of the hippocampus than other brain structures. Indian J Gastroenterol, 2003 Jan-Feb, 22(1), 19 - 21 Multidrug resistance 1 gene expression in Indian patients with gastric carcinoma; Ramesh S et al.; BACKGROUND: Gastric carcinoma is frequently refractory to chemotherapy . The multidrug resistance 1 gene (MDR1) encodes for a protein (p-glycoprotein) that functions as a drug efflux pump and thus contributes to resistance to chemotherapeutic agents . METHODS: We studied gastric tissues from 28 patients with gastric cancer for MDR1 expression, using immunohistochemistry . RESULTS: Sixteen (57%) of 28 cases showed MDR1 expression . Sections of normal mucosa away from the tumor showed perinuclear staining for MDR1 in surface epithelial cells, whereas tumor cells showed diffuse cytoplasmic positivity . CONCLUSIONS: Over one half of gastric carcinoma specimens at our center show MDR1 gene expression. Naunyn Schmiedebergs Arch Pharmacol, 2003 Jan, 367(1), 56 - 67 Epub 2002 Nov 05. Molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree Cephalotaxus hainanensis in human tumor cell lines; Efferth T et al.; Homoharringtonine (HHT) is an ester of cephalotaxine (CET), both of which derive from the Chinese coniferous tree Cephalotaxus hainanensis . HHT inhibited tumor cell growth at molar ranges comparable to established cytostatic drugs, whereas CET was 3-4 orders of magnitude less active . Inhibition concentration 50% (IC50) values of CET and HHT were significantly correlated to doxorubicin, vincristine, methotrexate, cisplatin, or camptothecin in 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, Bethesda, Md., USA) . We tested both drugs for resistance of cell lines which selectively overexpress the multidrug resistance (MDR)-conferring genes P-glycoprotein/ MDR1 (CEM/ADR5000), MDR-related protein 1 MRP1 (HL60/AR), and breast cancer resistance protein BCRP (MDA-MB-231-BCRP) . A threefold and ninefold resistance to HHT and CET, respectively, was found in CEM/ADR5000 cells, while the other MDR cell lines did not show cross-resistance compared to their drug-sensitive counterparts . As the tumor suppressor p53 is another important factor of chemoresistance, we also analyzed the possibility that p53 affects the response of tumor cells to CET and HHT . Comparing the p53 mutational status of the 55 NCI cell lines with the IC50 values showed a significant correlation . Thus, CET and HHT were more active in cell lines without p53 mutation . We correlated the IC50 values of CET and HHT with the cell doubling times of the 55 NCI cell lines as proliferation parameter and observed that rapidly growing cells were more susceptible than slowly growing cell lines . We conducted a search mining the NCI's database for the mRNA expression of 465 genes in 55 cell lines and correlated the data with the IC50 values for CET and HHT . Of these genes 61 (=13%) correlated with the IC50 values for CET and 122 (=26%) with the IC50 values for HHT indicating the multifactorial mode of action of these drugs in cancer cells . We have chosen one example from these genes to test a causative role for drug response . U-87MG.DeltaEGFR cells transfected with an epidermal growth factor receptor ( EGFR) gene truncated in its extracellular domain through a deletion of exons 2-7 (Delta EGFR) were 14-fold more resistant to HHT than control cells transfected with mock expression vector or non-transfected cells . The present investigation presents a starting point to dissect the genes and molecular pathways involved in the tumor cells' response to CET and HHT in greater detail. Cancer Res, 2003 Mar 1, 63(5), 895 - 901 Suppression of intestinal polyposis in Mdr1-deficient ApcMin/+ mice; Yamada T et al.; Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor complex has been considered crucial for the initiation of intestinal tumorigenesis . The human multidrug resistance (MDR)1 (ABCB1) gene contains multiple beta-catenin-T-cell factor4-binding elements in its promoter and is one of the immediate targets of the complex . In the current study, we have further substantiated the biological involvement of MDR1 in intestinal tumorigenesis based on the following evidence: (a) aberrant induction of the Mdr1a (Abcb1a) gene product, P-glycoprotein, associated with nuclear accumulation of the beta-catenin protein, was observed even in nascent microscopic adenomas of Min mice; (b) Mdr1-deficient Min (Apc(Min/+)Mdr1a/b(-/-)) mice developed significantly fewer intestinal polyps than did Apc(Min/+)Mdr1a/b(+/+) mice; and (c) Inhibitors of P-glycoprotein, verapamil, and cyclosporin A had a suppressive effect on the in vitro polypoid growth of IEC6 expressing stabilized (DeltaN89) beta-catenin protein . Inhibitors of P-glycoprotein may be included in a novel class of chemopreventive agents against colorectal carcinogenesis. Arch Oral Biol, 2003 Jan, 48(1), 87 - 90 Expression of ATP-binding cassette transporter in human salivary ducts; Uematsu T et al.; P-glycoprotein expression has been observed in normal tissues as well as malignant tumours and thus does not appear to be induced by anticancer drugs . Knowledge of the distribution of ATP-binding cassette (ABC) transporters other than P-glycoprotein in normal salivary tissue is essential for understanding the physiological secretion or excretion of potentially toxic substances . Here the expression of ABC transporters was studied immunohistochemically in normal salivary gland tissue from nine patients . In striated duct cells, staining was strong for P-glycoprotein, multidrug resistance-associated protein (MRP) 1, MRP 2/canalicular multispecific organic anion transporter (cMOAT), and lung resistance-related protein (LRP) . The staining intensity of acinar and intercalated duct cells for MRP 1 expression was distinct from that for MRP2/cMOAT, but was similar to that for P-glycoprotein . LRP was observed as particles between the nuclear and luminal membranes in the cytoplasm of intercalated duct cells . The expression of ABC transporters suggests that numerous transporters other than those studied might be isolated from normal salivary tissues . These observations indicate that these ABC transporters may not arise from any previous contact with anticancer drugs but are expressed physiologically . The achieved drug resistance as well as the physiological secretory function of ABC transporters could contribute to the responsiveness to chemotherapy of malignant salivary tumours. Hum Pathol, 2003 Feb, 34(2), 150 - 5 Expression of multidrug resistance-associated proteins in rhabdomyosarcomas before and after chemotherapy: the relationship between lung resistance-related protein (LRP) and differentiation; Klunder JW et al.; Rhabdomyosarcomas generally respond well to chemotherapy, and the residual lesions often are better differentiated than their primaries . This phenomenon may be explained by selective multidrug resistance (MDR) of differentiated tumor cell populations . We assess the role of MDR proteins in chemotherapy-induced differentiation in rhabdomyosarcomas in a clinical setting . Paraffin-embedded samples of 13 pairs of primary untreated rhabdomyosarcomas and their residual, recurrent, or metastatic lesions after chemotherapy were assessed for expression of MDR proteins, including P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP-1), and lung resistance-related protein (LRP) . Expression was semiquantitatively scored based on the percentage of isolated immunoreactive tumor cells as follows: 0, negative; 0.5, <5%; 1, 5% to 25%; 2, 26% to 50%; 3, 51% to 75%, and 4, >75% . All specimens after chemotherapy, except the late recurrences, were better differentiated than their primary, untreated specimens . Pgp or MRP-1 expression did not change significantly, but LRP expression increased significantly after chemotherapy . In both untreated and treated samples, LRP was expressed primarily in differentiated cells . The findings indicate that the in vivo expression of LRP, but not of Pgp and MRP-1, is induced by chemotherapeutic treatment in rhabdomyosarcomas . The preferential expression of LRP in differentiated cells and the subsequent more extensive expression after chemotherapy suggests that LRP plays a role in therapy-induced differentiation . Biol Pharm Bull, 2003 Mar, 26(3), 303 - 7 Cyclosporine a augments P-glycoprotein expression in the regenerating rat liver; Daoudaki M et al.; In the liver, the multidrug resistance (MDR) protein P-glycoprotein (P-gp) is physiologically expressed at the bile canalicular membrane, where it participates in the biliary excretion of various lipophilic drugs and xenobiotics . Previous studies showed that the immunosuppressive agent cyclosporine A (CsA) modulates P-gp and exerts a hepatotrophic influence in the regenerating liver . Hepatocytes isolated from regenerating rat liver, after 2/3 partial hepatectomy (PH 2/3), were used as an in vivo experimental model of cells with high proliferating activity in order to investigate whether CsA influences cellular levels of P-gp in those cells . Male Wistar rats were treated with CsA (20 mg/kg body weight) for 4 d preoperatively and 1 d postoperatively, and regenerating hepatocytes were isolated by collagenase perfusion 12, 24 and 48 h after PH 2/3 . Flow cytometry and Western blotting studies with the monoclonal antibodies C494 and C219 showed that after PH 2/3, cellular levels of P-gp were initially suppressed, 12 h after PH 2/3, by 23%, but were significantly elevated thereafter, 24 and 48 h after PH 2/3 by 28% and 73%, respectively . In CsA pretreated animals, P-gp levels were increased even in normal hepatocytes by 34%, and an additional augmentation was seen in hepatocytes from 24 and 48 h regenerating livers (60% and 56%, respectively) . In summary, we demonstrate for the first time that CsA has an additive effect on the expression of P-glycoprotein during liver regeneration in the rat . Therefore, induction of P-gp might also be considered in patients receiving CsA after liver transplantation for hepatocellular carcinoma and chemotherapy as an adjuvant treatment for the prevention of tumor recurrence. Probl Tuberk, 2002, (12), 42 - 6 {Acute toxic encephalopathy in patients with tuberculosis}; Korniliva ZKh et al.; A hundred and thirty five patients admitted to Moscow Tuberculosis Hospital No . 7 for disseminated and progressive forms of tuberculosis were examined . Among neurological disorders in tuberculosis, acute toxic encephalopathy (ATE) should be placed in the first place in terms of their severity, problems of diagnosis and treatment . In patients with acutely progressive forms of tuberculosis, the development of ATE is brought about by two factors: 1) significant tuberculous toxemia concurrent, in 37% of cases, with severe alcoholic intoxication that leads to generalized toxic and allergic vasculitis and as a result DIC syndrome; 2) cerebral hypoxia with dyscirculatory disorders due to progressive cardiopulmonary failure . The status of patients with tuberculosis and ATE is generally critical or extremely critical . These are actually resuscitative patients . Most patients have disseminated bilateral lung lesions with multiple decay cavities, with massive bacterial isolation found at sputum bacterioscopy . With this, mycobacterial resistance to at least one antituberculous drug was found in 83% of cases . Primary multidrug resistance was detected in 29.6% of patients . The diagnosis of ATE in patients with tuberculosis is difficult and requires that tuberculous meningitis shall be excluded . Acute progression, no spinal fluid changes, significant signs of cooagulopathy and thrombcytopathy with multiorgan failure and progressive DIC syndrome may diagnose ATE in patients with acutely progressive tuberculosis . The specific features of treatment in patients with tuberculosis and ATE are intensive antituberculous therapy with predominantly parenteral administration of drugs and intensive therapy for the DIC syndrome . Despite the treatment, 48 (35.6%) patients died from progressive tuberculosis and ATE, in 40 (29.6%), therapeutic efficiency was low due to multidrug myobacterial resistance. Probl Tuberk, 2002, (12), 18 - 23 {Efficacy of treatment for pulmonary tuberculosis with multidrug mycobacterial resistance}; Mishin VIu et al.; The efficiency of treatment was studied in 149 patients with pulmonary tuberculosis who isolated multidrug resistance of Mycobacteria tuberculosis (MBT) . The multidrug resistance of MTB, to at least isoniazid and rifampicin can be associated with both the resistance to other essential (streptomycin, ethambutol) and that to reserve drugs . With this, patients with MBT resistance to a combination of essential and reserve drugs more frequently showed a chronic course of the disease with severe clinical manifestations and more disseminated infiltrative-and-destructive lesions in the lung . Drug treatment regimens using a combination of reserve drug were effective only in patients with MBT resistance to essential drugs while they were little effective in those with resistance to essential and reserve agents . The use of artificial pneumothorax in patients with MBT resistance to essential and reserve agents could cease bacterial isolation in 77.8% of the patients even by ingesting a small number of the drugs . Clinically, the occurrence of MBT resistance to reserve drugs is justified to determine a radically new status in patients in the context of chemotherapy and the whole further treatment in this group of patients . A clinical classification of MBT multidrug resistance is proposed, which identifies two categories of patients with pulmonary tuberculosis: those resistant to essential drugs and those resistant to a combination of essential and reserve drugs. J Biol Chem, 2003 Apr 18, 278(16), 13603 - 6 Epub 2003 Feb 27. Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein . Direct evidence for the substrate-induced fit mechanism for drug binding; Loo TW et al.; The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell . We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism . Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments . Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates . We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12) . Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12) . Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites . These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate . The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds. Blood, 2003 Mar 15, 101(6), 2368 - 73 MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models; Mahon FX et al.; Inappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs . We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp . In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp . Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 microM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture . Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833 . Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC(50)) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 microM for K562/DOX, K562/DOX plus verapamil, and K562, respectively . Retroviral-mediated transfection of the BCR-ABL(+) AR230 cell line with the MDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil . The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined . We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon. J Immunol Methods, 2003 Mar 1, 274(1-2), 129 - 37 A simplified approach to determining P-glycoprotein expression in peripheral blood mononuclear cell subsets; Ford J et al.; P-glycoprotein (P-gp), encoded by the MDR-1 (multidrug resistance) gene mediates the cellular efflux of several therapeutic agents with the potential of treatment failure . The differential expression of P-gp in many localised tissues and cells of the hematopoietic system implies diverse physiological and pharmacological roles . The exact function of P-gp involved in multidrug resistance remains unclear owing to the numerous discrepancies between different laboratories . The ability to characterise accurately P-gp expression has important clinical implications . However, a complete consensus recommendation regarding methods of P-gp detection has been difficult to reach . With the advancement in immune technology and new commercially available antibodies, we describe a simplified direct immunofluorescent assay capable of detecting surface P-gp expression in peripheral blood mononuclear cells (PBMCs) and subpopulations of lymphocytes in vivo by dual colour flow cytometry . Results were expressed as mean increase in fluorescence (MI) compared to isotypically matched controls . Using this assay, differential basal P-gp expression was found to exist in the following significant hierarchy CD56+ (MI=0.684+/-0.273; n=15)>CD8+ (MI=0.312+/-0.117; n=15)>CD4+ (MI=0.194+/-0.086; n=15) . This method is rapid and reproducible and has potential use for in vitro and in vivo application. Pharm Res, 2003 Jan, 20(1), 31 - 7 Impact of Mrp2 on the biliary excretion and intestinal absorption of furosemide, probenecid, and methotrexate using Eisai hyperbilirubinemic rats; Chen C et al.; PURPOSE: This study assesses the impact of rat multidrug resistance-associated protein 2 (Mrp2) on the biliary excretion and oral absorption of furosemide, probenecid, and methotrexate using Eisai hyperbilirubinemic rats (EHBR) . METHODS: To assess Mrp2-mediated biliary excretion, rats received a 2-h intravenous infusion of furosemide, probenecid, or methotrexate . Blood and bile samples were collected at specified intervals . To assess Mrp2's impact on oral absorption, rats received furosemide, probenecid, or methotrexate orally at 5 mg/kg . Jugular and portal blood samples were obtained at timed intervals . All samples were analyzed by LC-MS/MS . Pharmacokinetic parameters were estimated using WinNonlin and standard pharmacokinetic equations . RESULTS: Thirty seven- and 39-fold reductions in biliary clearance were observed in EHBR as compared to control rats for probenecid and methotrexate, respectively . Biliary clearance was comparable between EHBR and control rats for furosemide . In all cases, no significant difference in absorption was observed between EHBR and control rats . CONCLUSIONS: This study provides the first evidence that Mrp2 mediates the biliary excretion of probenecid but not furosemide . Additionally, Mrp2 apparently has a less profound impact on intestinal absorption than biliary excretion of its substrates . Furthermore, alteration in systemic clearance in EHBR indicates that a potential compensatory mechanism may occur in EHBR. Kekkaku, 2002 Dec, 77(12), 771 - 5 {Clinical evaluation of the cause of death in patients with active pulmonary tuberculosis}; Kobashi Y et al.; We made a clinical analysis of the cause of death of forty deceased patients with active pulmonary tuberculosis who were admitted to Kawasaki Medical School Hospital, Kawasaki Medical School Kawasaki Hospital, and Asahigaoka Hospital during the period from January 1996 to December 2001 . The age of 40 deceased patients (29 males/11 females) ranged from 55 to 93 years old, and were mostly bedridden . Underlying diseases existed in all except one case, and they were respiratory diseases in 9 patients and non-respiratory diseases in 34 patients . Laboratory findings revealed poor nutritional conditions . The diagnosis of pulmonary tuberculosis was established within one month from the appearance of symptoms in over half of these patients because most of them were smear positive for Mycobacterium tuberculosis . None of the strains of Mycobacterium tuberculosis isolated from these patients were multidrug resistant for antituberculous drugs and only one strain was completely resistant for Rifampicin . Radiological findings of the tuberculosis were bilateral in 30 patients . Consolidation shadows without cavity were noted in 22 patients, and extension within the unilateral lung field was observed in 24 patients . Regarding the cause of death, advanced pulmonary tuberculosis was the cause in 17 patients and non-tuberculous diseases were the cause in 23 patients . There were 15 patients with bacterial superinfections such as bacterial pneumonia, 4 with malignancy, and 4 with other disease . The number of pulmonary tuberculosis patients in poor general and nutritional condition has been increasing with the aging of the Japanese population . Treatment for pulmonary tuberculosis has been successful in most cases, however, the number of the deaths unrelated to tuberculosis including those due to bacterial superinfection has been increasing . Therefore, treatment should be considered against resistant microoganisms such as MRSA. J Pharmacol Exp Ther, 2003 May, 305(2), 515 - 24 Epub 2003 Jan 24. Effect of ursodeoxycholic acid on the impairment induced by maternal cholestasis in the rat placenta-maternal liver tandem excretory pathway; Serrano MA et al.; We investigated the effects of ursodeoxycholic acid (UDCA; 60 microg/day/100 g b.wt.) on the impairment induced by maternal obstructive cholestasis during pregnancy (OCP) in the rat placenta-maternal liver tandem excretory pathway . A blunted catheter was implanted in the common bile duct on day 14 of pregnancy, and the tip was cut on day 21 . {(14)C}Glycocholate (GC) was then administered through the umbilical artery of "in situ" perfused placenta (placental transfer test) or through the maternal jugular vein (biliary secretion test), and GC bile output was measured . OCP impaired both GC placental transfer and maternal biliary secretion . UDCA moderately improved the latter but had a more marked beneficial effect on GC placental transfer . Histological examination revealed trophoblast atrophy and structural alterations, e.g., loss of apical membrane microvilli in OCP placentas . Gene expression level was investigated by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis . OCP reduced both placental lactogen II (a trophoblast-specific gene) mRNA and the functional amount of epithelial tissue, determined by transplacental diffusion of antipyrin . Using a rapid filtration technique, impairment in the ATP-dependent GC transport across trophoblast apical plasma membranes obtained from OCP placentas was found . UDCA partially prevented all these changes . The expression level of organic anion transporters Oatp1, Oatp2, and Oatp4, and multidrug resistance-associated proteins Mrp1, Mrp2, and Mrp3 in whole placenta were not affected or were moderately affected by OCP but greatly enhanced by UDCA . In summary, UDCA partially prevents deleterious effects of OCP on the rat placenta-maternal liver tandem excretory pathway, mainly by preserving trophoblast structure and function. Cancer Genet Cytogenet, 2003 Mar, 141(2), 120 - 7 Combined cytogenetic and array-based comparative genomic hybridization analyses of Wilms tumors: amplification and overexpression of the multidrug resistance associated protein 1 gene (MRP1) in a metachronous tumor; Goldstein M et al.; Tumor samples from a variety of Wilms tumors (WT) obtained from three patients were analyzed by cytogenetic and array-based comparative genomic hybridization (CGH) methods . The tumors represented different stages of tumorigenesis and included a unilateral primary WT and contralateral nephrogenic rest (case 1), a primary WT and a contralateral metachronous WT (case 2), and a recurrent WT with lung metastases (case 3) . All six specimens exhibited abnormal karyotypes characteristic of different WT levels of progression . Array-based CGH examinations of 57 genes that are commonly amplified in various cancers revealed a 2.6-fold genomic amplification of the multidrug resistance-associated protein 1 (MRP1) gene in the metachronous WT, but no amplification in the primary tumor . This sole amplification event in our series was also confirmed by Southern blot analysis . Furthermore, quantitative reverse transcriptase polymerase chain reaction showed a sixfold overexpression of the MRP1 gene in this metachronous WT relative to the primary tumor . Our findings suggest that for most of the genes examined in this series genomic amplification does not play a role in WT pathogenesis . Isolated amplification and overexpression of the MRP1 gene in the metachronous WT, however, suggest that this gene may be an important factor in the development and progression of metachronous tumors. Int J Radiat Oncol Biol Phys, 2003 Mar 15, 55(4), 1051 - 65 Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes; Schoenlein PV et al.; PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines . MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy . To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes . In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels . Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed . RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation . Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells . CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype . Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei. J Cardiovasc Pharmacol, 2003 Mar, 41(3), 406 - 13 Desbutylhalofantrine: evaluation of QT prolongation and other cardiovascular effects after intravenous administration in vivo; McIntosh MP et al.; Desbutylhalofantrine (Hfm) is an active and equipotent metabolite of halofantrine (Hf) . Both compounds are effective in the treatment of sensitive and multidrug-resistant and In vitro data and interpretation of some clinical studies of Hf have suggested that, unlike Hf, Hfm may be devoid of adverse cardiac effects . The aim of these investigations was to provide the first in vivo examination of the intrinsic capacity of Hfm to affect repolarization in the heart, using an anesthetized rabbit model . Using a dose-rising regimen, Hfm was administered IV at doses of 1, 1, 2, 4, and 8 mg/kg and the baseline rate-corrected QT interval (QTc) value of 377 +/- 13 ms rose to 394 +/- 16, 396 +/- 12, 429 +/- 18, 433 +/- 16, and 489 +/- 15 ms, respectively . There were no significant changes in blood pressure, heart rate, or PR or QRS intervals . The Hfm plasma concentrations were quantitated after high-performance liquid chromatographic analysis, the results indicating a significant correlation between Hfm plasma concentration and QT(c) prolongation . The study also identified a concentration-dependent hemolysis of erythrocytes after administration of Hfm . The conclusions from this study are that IV administration of Hfm does cause a significant prolongation of the QT(c) interval in a rabbit model. J Pharmacol Exp Ther, 2003 Mar, 304(3), 1258 - 67 Effects of the flavonoids biochanin A, morin, phloretin, and silymarin on P-glycoprotein-mediated transport; Zhang S et al.; Flavonoids are constituents of fruits, vegetables, and plant-derived beverages, as well as components in herbal-containing dietary supplements . The objective of this investigation was to characterize the effect of flavonoids on P-glycoprotein (P-gp)-mediated cellular efflux and to determine the molecular mechanism(s) of the flavonoid-drug interaction . Studies were conducted in the sensitive and multidrug resistant human breast cancer cell lines MCF-7 and MDA435/LCC6 and examined the effects of the flavonoids biochanin A, morin, phloretin, and silymarin on daunomycin (DNM) accumulation and doxorubicin cytotoxicity . The potential mechanism(s) involved in the interaction was evaluated by determining flavonoid effects on 1) P-gp ATPase activity, 2) {(3)H}azidopine photoaffinity labeling of P-gp, and 3) cellular P-gp levels . The flavonoids increased {(3)H}DNM accumulation in P-gp positive cells, but not P-gp negative cells, and these effects were both flavonoid concentration- and P-gp expression level-dependent . Biochanin A and silymarin potentiated doxorubicin cytotoxicity in P-gp positive cells . Biochanin A and phloretin stimulated, whereas morin and silymarin inhibited P-gp ATPase activity, confirming that these flavonoids interact with P-gp . Morin and silymarin significantly inhibited {(3)H}azidopine photoaffinity labeling of P-gp, suggesting a direct interaction with P-gp substrate binding . A 24-h preincubation with all flavonoids, followed by flavonoid removal, did not alter cellular P-gp level in P-gp positive cells . In conclusion, biochanin A, morin, phloretin, and silymarin all inhibited P-gp-mediated cellular efflux and the mechanism of the interaction involved, at least in part, a direct interaction . The findings of this study indicate a potential for significant flavonoid-drug interactions with P-gp substrates. J Pharmacol Exp Ther, 2003 Mar, 304(3), 1161 - 71 Pharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein; Kumar S et al.; The mechanisms of pharmacokinetic interactions of a novel anti-human immunodeficiency virus (anti-HIV-1) antagonist of chemokine receptor 5 (CCR5) {2-(R)-{N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-fluorophenyl)cyclopent-1-yl)amino}-3-methylbutanoic acid (MRK-1)} with ritonavir were evaluated in rats and monkeys . MRK-1 was a good substrate for the human (MDR1) and mouse (Mdr1a) multidrug resistance protein transporters and was metabolized by CYP3A isozymes in rat, monkey, and human liver microsomes . Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir . Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with an increase in dose from 2 to 10 mg/kg . The area under the plasma concentration-time curve (AUC) of MRK-1 was increased 4- to 6-fold when a 2 or 10 mg/kg dose was orally coadministered with 10 mg/kg ritonavir . Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2 mg/kg MRK-1 maximally by approximately 30 to 40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes . Oral coadministration of quinidine (10 and 30 mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2 mg/kg) by approximately 40 to 50% and approximately 100 to 300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species . At the 10 mg/kg MRK-1 dose, however, the entire increase in its AUC upon coadministration with ritonavir or quinidine could be attributed to a reduced systemic clearance, and no effects on intestinal absorption were apparent . In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose. Antimicrob Agents Chemother, 2003 Mar, 47(3), 917 - 22 Clinical concentrations of thioridazine kill intracellular multidrug-resistant Mycobacterium tuberculosis; Ordway D et al.; The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis . These compounds have not been used as anti-M . tuberculosis agents because their in vitro activities take place at concentrations which are bey