The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by highresolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.
INTRODUCTIONFast and reliable in vitro growth assessment of malaria parasites is the key to the assessment of in vitro drug susceptibility of malaria parasites, and to high throughput screening of newly developed drugs and drug combinations. Although there are several highly sensitive assays available today, the time and effort necessary to test a single microtiter plate remains substantial. Our goal was to assess the value of the SYBR Green I dye for drug susceptibility testing in Plasmodium falciparum cultures, both, under controlled, simulated field conditions and under optimal laboratory conditions.Recently, several new in vitro flourescene assays have been reported. 1 -4 The assays detect the presence of malaria DNA in infected erythrocytes as a measure of parasite growth and propagation and parasite growth inhibition by antimalarial drugs .Although fast and relatively inexpensive, growth assessment using nucleic acid stains has inherent limitations because these stains are not specific for malaria DNA. The SYBR Green I binds to any double-stranded DNA, including the DNA inherently present in whole blood samples, thereby resulting in high background readings.SYBR Green I has been used in molecular biology as a substitute for ethidium bromide for several years. It is an asymmetrical cyanine dye, binding to double stranded DNA, preferring G and C base pairs.2 When intercalated into DNA, it is highly fluorescent, absorbing light at a wavelength between 390 and 505 nm, with a peak at 497 nm and a secondary peak near 254 nm. It emits light at 505 to 615 nm, with a peak at 520 nm. MATERIALS AND METHODSA series of experiments were conducted to evaluate the usefulness of SYBR Green I for in vitro drug susceptibility testing of malaria parasites, not only at the previously tested higher, 4 but also at lower parasite densities. For all tests 20 µL of a 10× concentration of SYBR Green I plus 80 µL either undiluted or, if postincubation parasitemia exceeded ~3.5% infected red blood cells (IRBC), diluted (1:2) sample was used. The dye was obtained as 10,000X stock in DMSO (Sigma-Aldrich GesmbH, Vienna, Austria) and diluted in sterilized distilled water. The enzymelinked immunosorbent assay (ELISA) assays were done after dilution of the samples in distilled water as previously described. 6 Background fluorescence of the medium was analyzed by comparing regular RPMI 1640 medium containing HEPES, gentamycin, and phenol red to RPMI medium with HEPES and gentamycine, but without phenol red. The latter medium was then measured with and without addition of gentamycine.Following initial testing the phenol red-free medium containing HEPES and gentamycine was used as standard medium. To assess the influence of uninfected erythrocytes on the measurements, these were tested against the standard medium at different hematocrit levels. In addition, whole blood collected in heparinized tubes and fresh erythrocyte concentrate, both washed three times in RPMI medium, were analyzed. Two-fold serial dilutions of infected blood, with and w...
Critically ill patients often experience acute kidney injury and the need for renal replacement therapy in the course of their treatment in an intensive care unit (ICU). These patients are at an increased risk for candidiasis. Although there have been several reports of micafungin disposition during renal replacement therapy, to this date there are no data describing the elimination of micafungin during high-dose continuous venovenous hemodiafiltration with modified AN69 membranes. The aim of this prospective open-label pharmacokinetic study was to assess whether micafungin plasma levels are affected by continuous hemodiafiltration in critical ill patients using the commonly employed AN69 membrane. A total of 10 critically ill patients with micafungin treatment due to suspected or proven candidemia were included in this trial. Prefilter/postfilter micafungin clearance was measured to be 46.0 ml/min (Ϯ21.7 ml/min; n ϭ 75 individual time points), while hemofilter clearance calculated by the sieving coefficient was 0.0038 ml/min (Ϯ0.002 ml/min; n ϭ 75 individual time points). Total body clearance was measured to be 14.0 ml/min (Ϯ7.0 ml/min; n ϭ 12). The population area under the curve from 0 to 24 h (AUC 0 -24 ) was calculated as 158.5 mg · h/liter (Ϯ79.5 mg · h/liter; n ϭ 13). In spite of high protein binding, no dose modification is necessary in patients receiving continuous venovenous hemodiafiltration with AN69 membranes. A dose elevation may, however, be justified in certain cases. (This study has been registered at ClinicalTrials.gov under identifier NCT02651038.)
Doripenem is a broad-spectrum parenteral carbapenem with enhanced activity against and Current dosing regimens recommend the administration of 0.25 to 0.5 g once daily in patients undergoing intermittent renal replacement therapy. As patients are usually dialyzed thrice weekly, we aimed to investigate a 1-g posthemodialysis regimen, thus reducing treatment costs and enhancing patient compliance. A second objective of this trial was to describe the pharmacokinetics of intradialytic doripenem. Ten oliguric or anuric patients in need of intermittent renal replacement therapy were included in this trial. All patients suffered from a septic episode. The mean hemofilter clearance was 123.46 ± 42.03 ml/min, and the total body clearance between hemodialysis sessions was 16.79 ± 6.02 ml/min. The average prehemodialysis trough concentration was 2.4 ± 1.3 mg/liter, while the EUCAST resistance breakpoint for is set at 2 mg/liter. The interpatient variability was considerably higher than the intrapatient variability. Apart from one patient who suffered an allergic reaction, doripenem was tolerated well by all patients. Our data indicate that posthemodialysis administration of 1 g of doripenem results in sufficient plasma levels in anuric but not oliguric patients during the entire dosing interval. (This trial was registered with EudraCT under registration no. 2009-018010-18 and at ClinicalTrials.gov under registration no. NCT02018939.).
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