Showing papers by "Paul M. Tulkens published in 2006"
TL;DR: The intracellular activities of antibiotics against S. aureus are critically dependent upon their extracellular concentrations and the duration of cell exposure (within the 0- to 24-h time frame) to antibiotics and are always lower than those that can be observedextracellularly.
Abstract: The pharmacodynamic properties governing the activities of antibiotics against intracellular Staphylococcus aureus are still largely undetermined. Sixteen antibiotics of seven different pharmacological classes (azithromycin and telithromycin [macrolides]; gentamicin [an aminoglycoside]; linezolid [an oxazolidinone]; penicillin V, nafcillin, ampicillin, and oxacillin [beta-lactams]; teicoplanin, vancomycin, and oritavancin [glycopeptides]; rifampin [an ansamycin]; and ciprofloxacin, levofloxacin, garenoxacin, and moxifloxacin [quinolones]) have been examined for their activities against S. aureus (ATCC 25923) in human THP-1 macrophages (intracellular) versus that in culture medium (extracellular) by using a 0- to 24-h exposure time and a wide range of extracellular concentrations (including the range of the MIC to the maximum concentration in serum [C(max); total drug] of humans). All molecules except the macrolides caused a net reduction in bacterial counts that was time and concentration/MIC ratio dependent (four molecules tested in detail [gentamicin, oxacillin, moxifloxacin, and oritavancin] showed typical sigmoidal dose-response curves at 24 h). Maximal intracellular activities remained consistently lower than extracellular activities, irrespective of the level of drug accumulation and of the pharmacological class. Relative potencies (50% effective concentration or at a fixed extracellular concentration/MIC ratio) were also decreased, but to different extents. At an extracellular concentration corresponding to their C(max)s (total drug) in humans, only oxacillin, levofloxacin, garenoxacin, moxifloxacin, and oritavancin had truly intracellular bactericidal effects (2-log decrease or more, as defined by the Clinical and Laboratory Standards Institute guidelines). The intracellular activities of antibiotics against S. aureus (i) are critically dependent upon their extracellular concentrations and the duration of cell exposure (within the 0- to 24-h time frame) to antibiotics and (ii) are always lower than those that can be observed extracellularly. This model may help in rationalizing the choice of antibiotic for the treatment of S. aureus intracellular infections.
247 citations
Journal Article•
TL;DR: It appears important to evaluate the intracellular activity of antibiotics in appropriate models, despite the fact that all antibiotics reach intrACEllular concentrations that are at least equal to, and more often higher than, the extracellular concentrations.
Abstract: The treatment of intracellular infections requires the use of antibiotics presenting appropriate cellular pharmacokinetic and pharmacodynamic properties. These properties, however, cannot be predicted on the simple basis of cellular drug accumulation and minimum inhibitory concentration in broth. In most cases, intracellular activity is actually lower than extracellular activity, despite the fact that all antibiotics reach intracellular concentrations that are at least equal to, and more often higher than the extracellular concentrations. This discrepancy may result from impairment of the expression of antibiotic activity or a change in bacterial responsiveness inside the cells. It therefore appears important to evaluate the intracellular activity of antibiotics in appropriate models.
134 citations
TL;DR: Involving a trained clinical pharmacist in a geriatric team led to clinically relevant and well-accepted optimization of medicine use and this initiative may be a springboard for further development of clinical pharmacy services.
Abstract: BACKGROUND: Patient-centered clinical pharmacy services are still poorly developed in Europe, despite their demonstrated advantages in North America and the UK. Reporting European pilot experiences is, therefore, important to assess the usefulness of clinical pharmacy services in this specific context. OBJECTIVE: To report the results of the first implementation of Belgian clinical pharmacy services targeting patients at high risk of drug-related problems. METHODS: An intervention study was conducted by a trained clinical pharmacist providing pharmaceutical care to 101 patients (mean age 82.2 y; mean +/- SD number of prescribed drugs 7.8 +/- 3.5) admitted to an acute geriatric unit, over a 7 month period. All interventions to optimize prescribing, and their acceptance, were recorded. An external panel (2 geriatricians, 1 clinical pharmacist) assessed the interventions' clinical significance. Persistence of interventions after discharge was assessed through telephone calls. RESULTS: A total of 1066 interventions were made over the 7 month period. The most frequent drug-related problems underlying interventions were: underuse (15.9%), wrong dose (11.9%), inappropriate duration of therapy (9.7%), and inappropriate choice of medicine (9.6%). The most prevalent consequences were to discontinue a drug (24.5%), add a drug (18.6%), and change dosage (13.7%). Acceptance rate by physicians was 87.8%. Among interventions with clinical impact, 68.3% and 28.6% had moderate and major clinical significance, respectively. Persistence of chronic treatment changes 3 months after discharge was 84%. CONCLUSIONS: Involving a trained clinical pharmacist in a geriatric team led to clinically relevant and well-accepted optimization of medicine use. This initiative may be a springboard for further development of clinical pharmacy services.
108 citations
TL;DR: The data support the use of telavancin in infections where intracellular and extracellular S. aureus are present and could indicate multiple mechanisms of action for telvancin.
Abstract: OBJECTIVES: To compare extracellular and intracellular activities of telavancin (versus vancomycin) against Staphylococcus aureus (MSSA, MRSA, VISA and VRSA). METHODS: Determination of cfu changes (3-24 h) in culture medium and in macrophages at concentrations ranging from 0.01 to 1000x MIC. RESULTS: Extracellularly, telavancin displayed a fast, concentration-dependent bactericidal activity against all strains. The concentration-effect relationship was bimodal for MSSA and MRSA [two successive sharp drops in bacterial counts (0.3-1x MIC and 100-1000x MIC) separated by a zone of low concentration dependency]. When compared at human total drug Cmax (vancomycin, 50 mg/L; telavancin, 90 mg/L) towards MSSA, MRSA and VISA, telavancin caused both a faster and more marked decrease of cfu, with the limit of detection (>5 log decrease) reached already at 6 versus 24 h for vancomycin. Intracellularly, the bactericidal activity of telavancin was less intense [-3 log (MSSA) to -1.5 log (VRSA) at Cmax and at 24 h]. A bimodal relationship with respect to concentration (at 24 h) was observed for both MSSA and MRSA. In contrast, vancomycin exhibited only marginal intracellular activity towards intraphagocytic MSSA, MRSA and VISA (max. -0.5 log decrease at 24 h and at Cmax). CONCLUSIONS: Telavancin showed time- and concentration-dependent bactericidal activity against both extracellular and intracellular S. aureus with various resistance phenotypes. The data support the use of telavancin in infections where intracellular and extracellular S. aureus are present. Bimodality of dose responses (MSSA and MRSA) could indicate multiple mechanisms of action for telavancin.
100 citations
TL;DR: The weak activity of gentamicin and oxacillin towards intraphagocytic S. aureus compared with extracellular forms is not related to an overall decrease of their relative potencies but to impaired efficacy, suggesting the need for new approaches to improve the eradication of intracellular S.Aureus.
Abstract: Background: Staphylococcus aureus survives in acid media, including phagolysosomes. Conflicting in vitro/in vivo data exist on its susceptibility to antibiotics in such environments. Methods: Oxacillin and gentamicin activities against methicillin-susceptible S. aureus ATCC 25923 were compared extracellularly (broth; different pH) and assessed intracellularly (THP-1 macrophages), using a pharmacological approach (antibiotic concentrations: 0.01‐1000 3 MIC). Antibiotic cellular contents were determined by microbiological assay. Results: MICs and MBCs increased 72-fold for gentamicin, and decreased 8-fold for oxacillin between pH 7.4 and 5.0. Plots of log10 colony-forming unit changes at 24 h versus log10 of antibiotic concentration followed sigmoidal shapes, allowing calculation of EC50 (relative potency) and apparent Emax (relative efficacy) in all conditions. In broth, the EC50 of gentamicin rose 316-fold and that of oxacillin decreased 15-fold with unchanged apparent Emax [25 log (limit of detection)] between pH 7.4 and 5. Intracellularly, EC50s were similar to those observed extracellularly at pH 7.4, but Emax values were much lower (21 log) for both antibiotics. Calculations based on the assumed pH in phagolysosomes (5.4) and on local accumulation of antibiotics (gentamicin, 23-fold; oxacillin, 0.05-fold) suggest that the contrasting effects of acid pH on relative potencies of gentamicin and oxacillin could be almost exactly compensated for by differences in accumulation. Conclusions: The weak activity of gentamicin and oxacillin towards intraphagocytic S. aureus compared with extracellular forms is not related to an overall decrease of their relative potencies but to impaired efficacy, suggesting the need for new approaches to improve the eradication of intracellular S. aureus.
90 citations
TL;DR: Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubation cells, indicating that cytosolic gentamic in triggers apoptosis.
Abstract: Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can be reproduced with cultured renal cells, but only at high extracellular concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK1 cells involves a rapid (2-h) permeabilization of lysosomes and activation of the mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery of gentamicin to the cytosol by electroporation would sensitize LLC-PK1 cells to apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square waves) in the presence of gentamicin (3 microM to 3 mM; 1.2 mg/liter to 1.2 g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax (a marker of mitochondrial pathway activation) contents by Western blotting and competitive reverse transcriptase PCR and at 24 h for apoptosis by 4',6'-diamidino-2'-phenylindole staining (confirmed by electron microscopy) and for necrosis (by determination of lactate dehydrogenase release). Nonelectroporated cells were incubated with gentamicin for 8 and 24 h. Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the Bax level was not associated with an increase in the level of its mRNA but was associated with the accumulation of ubiquitinated forms (probably as a result of impairment of its degradation by the proteasome). Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubated cells. The data indicate that cytosolic gentamicin triggers apoptosis. Sequestration of gentamicin in lysosomes would, to some extent, protect against apoptosis.
77 citations
TL;DR: Water-soluble complexes of AmB and polyvinylpyrrolidone (AmB-PVP) compared favourably with AmB for antifungal activity, were less haemolytic and cytotoxic than AmB, and show a similar cytotoxicity profile to AmBisome.
Abstract: OBJECTIVES: Poor solubility and toxicity severely hinder the clinical use of amphotericin B (AmB), in spite of its attractive chemotherapeutic properties. Water-soluble complexes of AmB and polyvinylpyrrolidone (AmB-PVP) could display lower cytotoxicity while maintaining antifungal activity. METHODS: AmB-PVP [with PVP of 10, 24 and 40 kDa (AC1, AC2 and AC4)] were compared with free AmB for (i) activity against Candida spp. (five albicans; nine non-albicans) and Aspergillus spp. (four strains), (ii) haemolysis of sheep red blood cells, and (iii) release of lactate dehydrogenase from J774 macrophages [with further comparison with free PVP and a liposomal formulation of amphotericin (AmBisome)]. RESULTS: MICs and MFCs of AC1, AC2 and AC4 against Candida spp. and of AC2 and AC4 against Aspergillus spp. were similar to those of AmB (and even lower for some Candida strains). Killing kinetics (24 h) were also similar. Haemolytic activity of AC2 and AC4 was 2-fold lower than that of free AmB. Cytotoxicity of AC2 towards J774 macrophages was 8-fold lower, and that of AC4 5-fold lower than that of AmB and not significantly different from that of AmBisome. The lower cytotoxicity of AC2, AC4 was correlated with a lower cellular accumulation of amphotericin. Spectroscopic analysis shows that the lower toxicity of AmB-PVP was not owing to significant change in the monomeric/polymeric forms ratio of the drug. CONCLUSIONS: AmB-PVP complexes compared favourably with AmB for antifungal activity, were less haemolytic and cytotoxic than AmB, and show a similar cytotoxicity profile to AmBisome.
54 citations
TL;DR: It is proposed that this 3D model may be an important tool to understand the structure–function relationship of P‐glycoprotein and the finding of different positions both for a single ligand and for different ligands corroborates the experimental evidence indicating the existence of multiple drug binding sites.
Abstract: P-glycoprotein is a membrane protein involved in the phenomenon of multidrug resistance. Its activity and transport function have been largely characterized by various biochemical studies and a low-resolution image has been obtained by electron microscopy. Obtaining a high-resolution structure is, however, still remote due to the inherent difficulties in the experimental determination of membrane protein structures. We present here a three-dimensional (3D) atomic model of P-glycoprotein in absence of ATP. This model was obtained using a combination of computational techniques including comparative modeling and rigid body dynamics simulations that embody all available cysteine disulfide crosslinking data characterizing the whole protein in absence of ATP. The model features rather well most of the experimental interresidue distances derived both in the transmembrane domains and in the nucleotide binding domains. The model is also in good agreement with electron microscopy data, particularly in terms of size and topology. It features a large cavity detected in the protein core into which seven ligands were successfully docked. Their predicted affinity correlates well with experimental values. Locations of docked ligands compare favorably with those suggested by cysteine-scanning data. The finding of different positions both for a single ligand and for different ligands corroborates the experimental evidence indicating the existence of multiple drug binding sites. The interactions identified between P-glycoprotein and the docked ligands reveal that different types of interactions such as H-bonds, pi-pi and cation-pi interactions occur in agreement with a recently proposed pharmacophore model of P-glycoprotein ligands. Furthermore, the model also displays a lateral opening located in the transmembrane domains connecting the lipid bilayer to the central cavity. This feature supports rather well the commonly admitted mechanism of substrate uptake from the lipid bilayer. We propose that this 3D model may be an important tool to understand the structure-function relationship of P-glycoprotein.
31 citations
TL;DR: The data suggest that the resistance mechanism is mediated by an overexpression and/or increased activity of the MRP-like ciprofloxacin transporter expressed at a basal level in wild-type J774 macrophages, which modulates both the intracellular pharmacokinetics and activity of cipprofloxACin.
Abstract: Ciprofloxacin is the substrate for a multidrug resistance-related protein (MRP)-like multidrug transporter in J774 mouse macrophages, which also modestly affects levofloxacin but only marginally affects garenoxacin and moxifloxacin (J.-M. Michot et al., Antimicrob. Agents Chemother. 49:2429-2437, 2005). Two clones of ciprofloxacin-resistant cells were obtained by a stepwise increase in drug concentration (from 34 to 51 to 68 mg/liter) in the culture fluid. Compared to wild-type cells, ciprofloxacin-resistant cells showed (i) a markedly reduced ciprofloxacin accumulation (12% of control) and (ii) a two- to threefold lower sensitivity to the enhancing effect exerted by MRP-inhibitors (probenecid and MK571) on ciprofloxacin accumulation or by ciprofloxacin itself. ATP-depletion brought ciprofloxacin accumulation to similarly high levels in both wild-type and ciprofloxacin-resistant cells. Garenoxacin and moxifloxacin accumulation remained unaffected, and levofloxacin showed an intermediate behavior. DNA and protein synthesis were not impaired in ciprofloxacin-resistant cells for ciprofloxacin concentrations up to 100 mg/liter (approximately 85 and 55% inhibition, respectively, in wild-type cells). In Listeria monocytogenes-infected ciprofloxacin-resistant cells, 12-fold higher extracellular concentrations of ciprofloxacin were needed to show a bacteriostatic effect in comparison with wild-type cells. The data suggest that the resistance mechanism is mediated by an overexpression and/or increased activity of the MRP-like ciprofloxacin transporter expressed at a basal level in wild-type J774 macrophages, which modulates both the intracellular pharmacokinetics and activity of ciprofloxacin.
28 citations
01 Jan 2006
TL;DR: The combination of the local experience with the demonstrated advantages of Clinical Pharmacy (as seen from foreign studies) makes this project more and more realistic in Belgium.
Abstract: Summary Clinical Pharmacy, also called Pharmaceutical care, aims to maximise therapeutic effect, to minimise risk, to minimise cost, and to respect patient choice. North American countries and the United Kingdom have a large experience of pharmaceutical care, and this service is complementary to the activities performed by physicians, nurses, and other healthcare professionals. Experimental studies have shown that clinical pharmacy brings a definite added value in terms of the quality of use of medicines in various settings (hospitals, retail pharmacies, and outpatient clinics. These studies also demonstrate the economical benefits of pharmaceutical care. Clinical pharmacy is now under development in Belgium, and the pilot projects launched since 2000 have been well accepted. This has triggered the University Hospitals and the Faculties to launch actions oriented towards both education and research. Changes introduced in the educational curriculum of pharmacy students make them more alert to their future pharmaceutical care activities. A specific post-graduate program is now developed and implemented in two universities. The professional organizations also organize postgraduate training in pharmaceutical care. In the near future, an official recognition at the National level will be important to create the necessary framework in which Clinical Pharmacy can develop in Belgium. The combination of our local experience with the demonstrated advantages of Clinical Pharmacy (as seen from foreign studies) makes this project more and more realistic in our country. La pharmacie clinique, encore appelee “soins pharmaceutiques”, vise a assurer une therapie medicamenteuse sure, efficace, et d’un bon rapport cout/benefice, tout en respectant les choix du patient. L’Angleterre, les Etats-Unis et le Canada ont une grande experience des soins pharmaceutiques. La pharmacie clinique y est un element complementaire et souvent juge essentiel aux soins prodigues par les medecins et le personnel infirmier. Les etudes montrent que la mise en place de services de pharma cie clinique apporte une valeur ajoutee reelle tant en milieu hospitalier que dans le cadre des officines ouvertes au public et des consultations pharmaceutiques pour patients ambu
5 citations