Drug Efflux Mechanism Research Articles

Reversal of resistance in microorganisms by help of non-antibiotics

Intracellular efflux pumps have been largely the research focus in multidrug-resistant (MDR) Gram-positive and Gram-negative microorganisms and parasites including cancers. However, drug efflux mechanisms other than pumps per se have been observed, supported by the effects of isomeric, non-antibiotic depressant (DPR), phenothiazines and thixenes, and antidepressant (ADPR) phenylpiperidine neurotropic drugs, alone or in combination with classical antimicrobials on MDR Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes and Streptococcus pneumoniae. Of the non-antibiotics we investigated, the DPR l-thioridazine, trans-clopenthixol and isomers of phenylpiperidines NNC 20-4962 (isomer of femoxetine) and NNC 20-7052 (isomer of paroxetine) were potent antimicrobials with the least neurotropic activity, pointing to a possible general isomeric structure-activity relationship. These compounds may be regarded as new efflux inhibitors. Moreover, these isomers have considerably reduced, in some cases absent, neurotropism and reduced mammalian toxicity. This may alleviate concerns about adverse effects and therapeutic safety for infected patients in life-threatening situations where the non-antibiotic dosage would be in the lower, non-chronic dosage ranges generally prescribed for individuals with mild mental health problems. The results point to the prokaryotic and eukaryotic microorganisms' phospholipid/protein domain involvement of the cationic, amphiphilic, non-antibiotic DPR and ADPR, with the phospholipids being the initial sites attracting and concentrating the neurotropes to induce a form of suspended animation, followed by gross changes of cell wall and membrane structure, and thereby potentiating their destructive or immobilizing effects on various as yet only hinted at resistance and efflux mechanisms. Combination of appropriate isomeric non-antibiotic DPR and ADPR of low neurotropism and toxicity with conventional and classical antimicrobials promises early, new therapeutic strategies salutary against microbial resistance, resistance development, pathogenicity and virulence.

Molecular graphics approach to bacterial AcrB protein–β-lactam antibiotic molecular recognition in drug efflux mechanism

AcrAB–TolC is the most important multidrug efflux pump system of Gram-negative bacteria, responsible for their resistance to lipophilic and amphiphilic drugs. In this work, a molecular graphics study of the pump components AcrB and TolC, 16 β-lactam antibiotics and 7 other substrates, as well as of AcrB–substrate complexes, was performed in order to give a mechanistic proposal for the efflux process at molecular level. AcrAB–TolC is a proton-dependent electromechanical device which opens to extrude drugs from the bacterial periplasm and perhaps cytoplasm, by means of a series of structural changes within the complex and its components AcrA, AcrB and TolC. These changes are initiated by protonation and disruption of salt bridges and certain hydrogen bonds, and are followed by conformational changes in which a number of intra- and interchain interactions are rearranged. Molecular properties of β-lactams accounting for their lipophilicity, shape/conformation and other sterical features, polar/charge group distribution and other electronic properties, and hydrogen bonding potency determine their interaction with polar headpieces of the inner membrane, recognition and binding to receptors of AcrB and TolC. The orientation of the β-lactam molecular dipoles with respect the efflux system is maintained during the drug efflux. Elongated cylinder-like β-lactam antibiotics with lipophylic side chains, a significantly negative component of the dipole moment and low hydrogen bonding capacity seem to be good substrates of AcrAB–TolC.

X-ray Structure of the EmrE Multidrug Transporter in Complex with a Substrate

EmrE is a prototype of the Small Multidrug Resistance family of efflux transporters and actively expels positively charged hydrophobic drugs across the inner membrane of Escherichia coli. Here, we report the x-ray crystal structure, at 3.7 angstrom resolution, of one conformational state of the EmrE transporter in complex with a translocation substrate, tetraphenylphosphonium. Two EmrE polypeptides form a homodimeric transporter that binds substrate at the dimerization interface. The two subunits have opposite orientations in the membrane and adopt slightly different folds, forming an asymmetric antiparallel dimer. This unusual architecture likely confers unidirectionality to transport by creating an asymmetric substrate translocation pathway. On the basis of available structural data, we propose a model for the proton-dependent drug efflux mechanism of EmrE.

ATP-binding cassette (ABC) proteins in untreated acute myeloid leukemia (AML) in patients 60 years and older (CALGB 9760)

6551 Background: Drug efflux mediated by the ABC protein P-glycoprotein (Pgp) is an adverse prognostic factor and a presumed mechanism of treatment failure in older AML patients, but little is known about the other multidrug resistance (MDR)-associated ABC protein drug efflux pumps multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP). We studied these additional drug efflux mechanisms and the effects of diverse MDR modulators. Methods: Pre-treatment samples from 106 untreated AML patients 60 years and older [ages, 67 to 84 (median, 71) years; 82 (77%) de novo AML, 24 (23%) following myelodysplastic syndromes, and 4 (4%) therapy-related] were studied for expression of Pgp, MRP-1 and BCRP, for drug efflux and for modulation of drug efflux by the Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C (FTC) and by cyclosporine A (CsA) which has activity against all three proteins, by flow cytometry. Pgp, MRP-1 and BCRP expression was studied with the MRK-16, MRPm6 and BXP-21 antibodies. Flow cytometry data were analyzed by the Kolmogorov-Smirnov statistic, expressed as a D-value, with cutoffs defining expression, efflux and modulation established in cell lines. Results: Pgp, MRP-1 and BCRP were expressed in 43%, 30% and 52% of samples, and at least one protein in 86%. Pgp and BCRP, Pgp and MRP-1, and MRP-1 and BCRP were coexpressed in 38%, 16% and 31%, and all three proteins in 13% of samples. Efflux of mitoxantrone, a substrate for Pgp, MRP-1 and BCRP, was seen in 77% of samples, and was reduced by PSC-833, probenecid and FTC in 30%, 8% and 15%,and by CsA in 34%. Effects of MDR protein-specific modulators correlated poorly with MDR protein expression. Conclusions: AML cells in older patients frequently express diverse ABC proteins and frequently exhibit drug efflux, but effects of specific modulators on efflux in vitro correlate poorly with expression of specific proteins, and the modulators studied were effective in no more than half of cases in which efflux was seen. These data support ongoing efforts to develop and test broad-spectrum ABC protein modulators with efficacy in modulating all of the ABC proteins expressed in AML cells. No significant financial relationships to disclose.

Effect of bisphenol A on drug efflux in BeWo, a human trophoblast-like cell line

Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein.

International clone of Neisseria meningitidis serogroup A with tetracycline resistance due to tet(B).

Thirteen Neisseria meningitidis clinical isolates from Africa, Asia, and the United States for which the tetracycline MICs were elevated (> or =8 microg/ml) were examined for 14 recognized resistance genes. Only the drug efflux mechanism encoded by tet(B) was detected. All isolates were in serogroup A, belonged to complex ST-5, and were closely related by pulsed-field gel electrophoresis analysis.

Resistance in cancer: a target for drug discovery.

Resistance remains a major problem in the clinical utility of cancer chemotherapy. However, it also represents a tumour cell phenotype that is in many ways different, and thus distinguishable, from the majority of normal cells. Two approaches to the targeting of resistant cells are described involving intratumoral P450 expression, mechanisms of drug-efflux and defective DNA repair. It is suggested that the view of the solid tumour as a complex organ rather than a collection of individual cells will inform future drug development and both overcome and target multiple resistance mechanisms.

Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy.

Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.

Expression of P-glycoprotein (ABCB1) and Mrp1 (ABCC1) in adult rat brain: focus on astrocytes

P-glycoprotein (P-gp, ABCB1) and the multidrug resistance-associated protein 1 (Mrp1, ABCC1) are two ATP-driven pumps that mediate the export of organic anions from cells and may confer cellular resistance to many cytotoxic hydrophobic drugs. Immunohistochemistry has shown that P-gp is expressed in rat brain capillary vessels forming the blood–brain barrier (BBB). Mrp1 mRNAs have been detected by RT-PCR in rat brain isolated capillaries. Although many studies have been published in this field, very little information is available on the expression, distribution and physiological functions of the two pumps in rat brain. To characterize the cerebral expression of both P-gp and Mrp1 transporters, we studied immunoreactions of rat brain sections with the two most commonly used antibodies: the monoclonal C219 (anti-P-gp) and the polyclonal 6KQ (anti-Mrp1). Immunological analyses revealed heterogeneity of the P-gp and Mrp1 expressions in rat brain. Indeed, choroidal and ependymal cells expressed Mrp1 rather than P-gp. However, tanycytes lining the third ventricle were strongly immunoreactive with both antibodies, suggesting a particular role for these cells in drug efflux mechanisms. Because of the detection of a 70-kDa component with 6KQ antibodies, immunoreactions obtained in rats were compared with these obtained in wild type and mrp1(−/−) mice. It showed that a positive reaction at the apical surface of the ependymal layer remained obvious, showing that 6KQ antibodies recognize an ependymal molecule, differing from the Mrp1. In addition, a continuous expression of C219-labeled epitopes, similar to endothelial labeling, was detected at the blood–brain barrier, whereas a discontinuous labeling, co-localized with glial fibrillary acidic protein (GFAP) immunostaining, was obtained with 6KQ antibodies. We showed that P-gp was preferentially expressed in the endothelial component and Mrp1 in the astroglial component of the blood–brain barrier. Moreover, Mrp1 was rather expressed than P-gp in parenchyma astrocytes and in glia limitans lining the meninges. These findings provide new insights into the cerebral distribution of two ABC transporters linked to multidrug resistance (MDR).

KINETIC CHARACTERIZATION OF P-GLYCOPROTEIN-MEDIATED EFFLUX OF RHODAMINE 6G IN THE INTACT RABBIT LUNG

P-Glycoprotein (P-gp) is an ATP-dependent drug efflux transporter involved in multidrug resistance and drug disposition in many organ systems. A majority of P-gp substrates are lipophilic amine drugs which also exhibit rapid extensive accumulation in lung tissue. P-gp is expressed in lung tissue, and the very nature of this drug efflux mechanism suggests a moderating role in pulmonary drug disposition. Little is known about P-gp-mediated efflux out of lung tissue or its kinetic characteristics as they may relate to the impact of P-gp on pulmonary drug accumulation. The present study develops an experimental and kinetic model to characterize the kinetics of P-gp-mediated efflux of rhodamine 6G dye (R6G) out of the intact rabbit lung. The perfusate concentration of R6G with time during recirculation through an isolated perfused rabbit lung was measured, and 66.6 +/- 2.6% (S.E.) of the perfusate R6G was taken up by the lung. In the presence of P-gp inhibitors, R6G uptake increased significantly to 87.5 +/- 1.1% (P < 0.002), indicating a functional pulmonary P-gp efflux transporter. Fractional lung accumulation of R6G increased with increasing R6G perfusate concentration, a result consistent with saturation of an efflux transporter. A parsimonious three-compartment kinetic model of R6G pulmonary disposition was used to interpret data sets from experiments with different perfusion variables and to estimate parameters descriptive of the dominant kinetic processes involved in R6G pulmonary accumulation. The estimated value of the kinetic parameter, k(pgp), rate constant for P-gp-mediated R6G efflux, indicates that this transporter plays a significant role in moderating R6G pulmonary disposition.

The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability

Many orally administered drugs must overcome several barriers before reaching their target site. The first major obstacle to cross is the intestinal epithelium. Although lipophilic compounds may readily diffuse across the apical plasma membrane, their subsequent passage across the basolateral membrane and into blood is by no means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein (Pgp; MDR1) and MRP2, may drive compounds from inside the cell back into the intestinal lumen, preventing their absorption into blood. Drugs may also be modified by intracellular phase I and phase II metabolising enzymes. This process may not only render the drug ineffective, but it may also produce metabolites that are themselves substrates for Pgp and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are subject to further metabolism and biliary excretion, often by a similar system of ATP-binding cassette (ABC) transporters and enzymes to that present in the intestine. Thus a synergistic relationship exists between intestinal drug metabolising enzymes and apical efflux transporters, a partnership that proves to be a critical determinant of oral bioavailability. The effectiveness of this system is optimised through dynamic regulation of transporter and enzyme expression; tissues have a remarkable capacity to regulate the amounts of protein both at transcriptional and post-transcriptional levels in order to maintain homeostasis. This review addresses the progress to date on what is known about the role and regulation of drug efflux mechanisms in the intestine and liver.

Liposome-based approaches to overcome anticancer drug resistance.

Drug resistance remains an important obstacle towards better outcomes in the treatment of cancer. One general approach to overcome this problem has been to inhibit specific resistance mechanisms, such as P-glycoprotein (PGP)-mediated drug efflux, using small molecule agents or other therapeutic strategies. Alternatively, drug delivery approaches using liposomes or other carriers can in principle target drugs to tumor tissue, tumor cells, or even compartments within tumor cells. By increasing bioavailability of drugs at sites of action, these approaches may provide therapeutic advantages, including enhanced efficacy against resistant tumors. Current liposomal anthracyclines have achieved clinical use in cancer treatment by providing efficient encapsulation of drug in stable and non-reactive carriers, and there is evidence indicating potential benefit in some clinical settings involving resistant tumors. Other liposome-based strategies include constructs designed to be taken up by tumor cells, as well as further modifications to allow triggered drug release. These approaches seek to overcome drug resistance by more efficient delivery to tumor cells, and in some cases by concomitant avoidance or inhibition of drug efflux mechanisms. Newer agents employ molecular targeting, such as immunoliposomes using antibody-directed binding and internalization. These agents selectively deliver drug to tumor cells, can efficiently internalize for intracellular drug release, and can potentially enhance both efficacy and safety.

P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement

Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to posses functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route. Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux. Broad substrate recognization by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors. In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest. Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination. Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors. In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.

Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole.

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Clonal relationships among isolates of erythromycin-resistant Streptococcus pyogenes of different geographical origin.

The clonal relationships among 261 erythromycin-resistant Streptococcus pyogenes isolates collected in 1986-1997 from nine countries in Europe and North and South America were studied by using two molecular typing methods: Vir typing and random amplified polymorphic DNA (RAPD) analysis. A total of 49 different Vir genotypes (VTs) and 33 different RAPD patterns were noted among the 261 isolates. Isolates that shared the same VT and RAPD pattern were considered to belong to the same clone. Although as many as 60 different clones were found among the isolates studied, only seven clones, comprising 157 of the 261 (60%) isolates, were found in more than one country. Five of these seven clones expressed the M phenotype known to be associated with the drug efflux mechanism, and only two clones expressed the macrolide-lincosamide-streptogramin B-resistance phenotype. The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes. Furthermore, predominance of the seven clones indicates that erythromycin-resistant Streptococcus pyogenes of the same clonal origin may be widely distributed and found in several different countries.

CmeABC functions as a multidrug efflux system in Campylobacter jejuni.

Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.

Rapid induction of long-lasting drug efflux activity in brain vascular endothelial cells but not malignant glioma following irradiation.

The influence of radiotherapy on malignant glioma multidrug resistance to chemotherapy was evaluated because patients with glioma often are treated with a combination of radiotherapy and chemotherapy. Multidrug resistance gene (MDR1, mdr1a, and mdr1b) transcripts were found in human and rat glioma cell lines. P-Glycoprotein (Pgp) was immunohistochemically detected in glioma cell lines and in the rat brain vascular endothelial cell line (RBE4). A multidrug resistance pump efflux activity assay demonstrated increased calcein efflux of RBE4 endothelial cells, but not glioma cells, 2 h after irradiation and still increased 14 d after irradiation. The increased efflux was equally inhibited by verapamil with or without irradiation. In the rat intracranial glioma model (BT4C), Pgp was demonstrated in capillary endothelial cells of the tumor tissue and surrounding normal brain, but not in tumor cells. The expression of gene transcripts or Pgp was not affected by irradiation. The results indicate that long-lasting verapamil-resistant drug efflux mechanisms are activated in brain endothelial cells after irradiation. The results might explain the poor efficacy of chemotherapy following radiotherapy and contribute to consideration of new treatment strategies in the management of malignant glioma.

CYP3A4, CYP3A5, and MDR1 in Human Small and Large Intestinal Cell Llines Suitable for Drug Transport Studies

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.

Managing therapy in older adult patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is predominantly a disease of older adults, with more than 50% of cases occurring in adults over 60 years of age. Treatment of AML in older adults is complicated not only by comorbidities that are common in this patient population, but also by the prevalence, in this age group, of forms of AML with a poor prognosis. The problems encountered and the strategies that have been used to improve the outlook in older adults with AML are presented. The two main strategies for improving outcomes in older adults with AML are to develop effective chemotherapeutic regimens with improved tolerability, and to reduce drug resistance. In studies to identify optimal chemotherapeutic regimens in older adults, a satisfactory balance between efficacy and toxicity has not yet been achieved. Also, the use of growth factors to promote hematopoietic recovery has yet to yield consistent reductions in treatment-related morbidity or mortality. Drug resistance can be modified by inhibiting drug efflux mechanisms or by increasing sensitivity to cytotoxic agents, but these strategies have not yet been shown to significantly affect outcomes. Novel approaches including antibody-targeted and molecular-targeted chemotherapy may have the potential to improve the prognosis for older adults with AML.

Physicochemical Evaluation of a Stability-Driven Approach to Drug Entrapment in Regular and in Surface-Modified Liposomes

The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10–90% were, excepting sucrose, either similar in the N- and T-liposomes or better in the N- than in the T-liposomes, for both regular and bioadhesive liposomes. (2) For all liposome types and methods of preparation, fluorescein, histidine, and MMC did not adsorb to the liposomal surface. Sucrose and MMC did adsorb to the liposomal surface irrespective of the liposome preparation mode, sucrose favoring bioadhesive over regular liposomes and MMC having the opposite trend. (3) For both regular and bioadhesive liposomes, the mechanism of drug efflux from the N-liposomes was found to be governed by a single rate constant, as previously found for the T-liposomes. The magnitudes obtained, ranging from 3.5(±0.2) × 10−3 to 400(±17) × 10−3 h−1, were always drug specific and occasionally also liposome type (i.e., regular or bioadhesive) specific. For MMC and CAM, the novel approach rendered liposomes with improved sustained release. The results reported here attest, overall, to the potential of this novel approach, meriting further investigations. Studies currently underway with MMC indicate N-liposomes also have functional advantages.