Hugues Chanteux

Comparative Stability Studies of Antipseudomonal β-Lactams for Potential Administration through Portable Elastomeric Pumps (Home Therapy for Cystic Fibrosis Patients) and Motor-Operated Syringes (Intensive Care Units)

ABSTRACT The stability of antipseudomonal β-lactams in concentrated solutions was examined in view of their potential administration by continuous infusion with external pumps (for intensive care patients) or with portable pumps carried under clothing (for cystic fibrosis patients). Aztreonam (100 g/liter), piperacillin (128 g/liter, with tazobactam), and azlocillin (128 g/liter) remained 90% stable for up to more than 24 h at 37°C (mezlocillin [128 g/liter] was stable at 25°C but not at 37°C). Ceftazidime (120 g/liter), cefpirome (32 g/liter), and cefepime (50 g/liter) remained 90% stable for up to 24, 23.7, and 20.5 h at 25°C but only for 8, 7.25, and 13 h at 37°C, respectively. The control of temperature therefore appears to be critical for all three cephalosporins that cannot be recommended for use in portable pumps carried under clothes for prolonged periods for reasons of stability. Cefpirome and cefepime solutions developed an important color change (from light yellow to dark red) upon exposure when stored at 30°C or higher. Degradation of ceftazidime was accompanied by the liberation of pyridine which, at 37°C, was in excess of what is allowed by the U.S. Pharmacopeia, i.e., 1.1 mg/liter, after 8 and 12 h for drug concentrations of 12 and 8.3%, respectively. Imipenem and meropenem are too unstable (10% degradation at 25°C after 3.5 and 5.15 h, respectively) to be recommended for use by continuous infusion. Faropenem, examined in comparison with imipenem and meropenem, proved as stable as aztreonam or piperacillin.

Intracellular pharmacodynamics of antibiotics.

This article establishes the pharmacokinetic-pharmacodynamic parameters that are important when considering the intracellular activity of antibiotics. Generally speaking, the main classes of antibiotics seem to share globally the same properties against extracellular and intracellular organisms. The specific cellular pharmacokinetic properties may modulate those parameters so as to let other ones to become critical. Simple rules, such as equating accumulation and activity, are certainly incorrect, and other determinants need to be added to the equation. Finally, this article emphasizes the fact that much remains to be done in this area before rational therapeutic choices can be made.

LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms.

BackgroundIL-10 is a cytokine mainly produced by macrophages that plays key roles in tolerance to inhaled antigens and in lung homeostasis. Its regulation in alveolar macrophages (HAM), the resident lung phagocytes, remains however unknown.MethodsThe present study investigated the role of intracellular signalling and transcription factors controlling the production of IL-10 in LPS-activated HAM from normal nonsmoking volunteers.ResultsLPS (1–1000 pg/ml) induced in vitro IL-10 production by HAM, both at mRNA and protein levels. LPS also activated the phosphorylation of ERK, p38 and JNK MAPkinases (immunoblots) and Sp-1 nuclear activity (EMSA). Selective inhibitors of MAPKinases (respectively PD98059, SB203580 and SP600125) and of Sp-1 signaling (mithramycin) decreased IL-10 expression in HAM. In addition, whilst not affecting IL-10 mRNA degradation, the three MAPKinase inhibitors completely abolished Sp-1 activation by LPS in HAM.ConclusionThese results demonstrate for the first time that expression of IL-10 in lung macrophages stimulated by LPS depends on the concomitant activation of ERK, p38 and JNK MAPKinases, which control downstream signalling to Sp-1 transcription factor. This study further points to Sp-1 as a key signalling pathway for IL-10 expression in the lung.

Cellular Pharmacokinetics and Pharmacodynamics of the Glycopeptide Antibiotic Oritavancin (LY333328) in a Model of J774 Mouse Macrophages

ABSTRACT The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells. Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells. In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels. Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h). In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation). The rates of clearance of oritavancin and latex beads were comparable (150 and 120 μl × mg of protein−1 × h−1, respectively) and were approximately 200 times higher than that of horseradish peroxidase. Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation). Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation. Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter). We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity.

Influence of P-Glycoprotein Inhibitors on Accumulation of Macrolides in J774 Murine Macrophages

ABSTRACT The influence of inhibitors of P-glycoprotein (verapamil [VE], cyclosporine [CY], and GF120918 [GF]) on the cell handling of macrolides (erythromycin [ERY], clarithromycin [CLR], roxithromycin [ROX], azithromycin [AZM], and telithromycin [TEL]) was examined in J774 murine macrophages. The net influx rates of AZM and TEL were increased from 2- to 3.5-fold in the presence of these inhibitors, but their efflux was slowed only marginally. At 3 h, the inhibitors increased the levels of AZM, ERY, and TEL accumulation approximately three- to fourfold (the effect of VE, however, was lower) but did not influence CLR accumulation (the inhibitors had an intermediate behavior on ROX accumulation). The effect was concentration dependent (half-maximal increases in the level of accumulation of AZM were obtained with GF, CY, and VE at 0.5, 5, and 10μ M, respectively). ATP depletion also caused an approximately threefold increase in the level of accumulation of AZM. Two inhibitors of MRP (probenecid [2.5 mM] and gemfibrozil [0.25 mM]) had no effect. Monensin (a proton ionophore) completely suppressed the accumulation of AZM in control cells as well as in cells incubated in the presence of VE, demonstrating that transmembrane proton gradients are the driving force causing the accumulation of AZM in both cases. Yet, VE did not alter the pH of the lysosomes (approximately 5) or of the cytosol (approximately 7.1). P-glycoprotein was detected by immunostaining at the cell surface as well as in intracellular vacuoles (endosomes and lysosomes). The data suggest that the influx of AZM, ERY, TEL, and ROX is adversely influenced by the activity of P-glycoprotein in J774 macrophages, resulting in suboptimal drug accumulation.

Accumulation and Oriented Transport of Ampicillin in Caco-2 Cells from Its Pivaloyloxymethylester Prodrug, Pivampicillin

ABSTRACT Pivampicillin (PIVA), an acyloxymethylester of ampicillin, is thought to enhance the oral bioavailability of ampicillin because of its greater lipophilicity compared to that of ampicillin. The fate of PIVA in intestinal cells and the exact location of its conversion into ampicillin have, however, never been unambiguously established. Polarized Caco-2 cells have been used to examine the handling of PIVA and the release of ampicillin from PIVA by the intestinal epithelium. Experiments were limited to 3 h. Cells incubated with PIVA (apical pole) showed a fast accumulation of ampicillin and transport toward the basolateral medium, whereas PIVA itself was only poorly accumulated and transported. Cells incubated with free ampicillin accumulated and transported only minimal amounts of this drug. Release of ampicillin from cells incubated with PIVA was unaffected by PEPT1 and OCTN2 inhibitors but was sharply decreased after ATP depletion or addition of bis(4-nitrophenyl)-phosphate (BNPP; an esterase inhibitor). PIVA incubated with Caco-2 lysates released free ampicillin, and this release was inhibited by BNPP. Efflux studies showed that the ampicillin that accumulated in cells after incubation with PIVA was preferentially transported out of the cells through the basolateral pole. This efflux was decreased by multidrug resistance-associated protein (MRP) inhibitors (probenecid, MK-571) and by ATP depletion. A phthalimidomethylester of ampicillin that resists cellular esterases failed to cause any significant release (cell lysate) or transport (polarized Caco-2 cells) of ampicillin. These results show that when PIVA is given to Caco-2 cells from their apical pole, ampicillin is released intracellularly and that ampicillin is thereafter preferentially effluxed into the basolateral medium through an MRP-like transporter.

Cell Handling, Membrane-Binding Properties and Membrane-Penetration Modeling Approaches of Pivampicillin and Phthalimidomethylampicillin, Two Basic Esters of Ampicillin, in Comparison with Chloroquine and Azithromycin

AbstractPurpose. The purpose of this work was to examine and understand the cellular pharmacokinetics of two basic esters of ampicillin, pivaloyloxymethyl (PIVA) and phthalimidomethyl (PIMA), in comparison with lysosomotropic drugs (chloroquine, azithromycin). Methods. Cell culture studies (J774 macrophages) were undertaken to study uptake and release kinetics and to assess the influence of concentration, pH, proton ionophore (monensin), and MRP and P-gp inhibitors (probenecid, gemfibrozil, cyclosporin A, GF 120918). Equilibrium dialysis with liposomes were performed to directly asses the extent of drug binding to bilayers. Conformational analysis modeling of the drug penetration in bilayers was conducted to rationalize the experimental observations. Results. PIVA and PIMA showed properties in almost complete contrast with those of chloroquine and azithromycin, i.e., fast apparent accumulation and fast release at 4°C as well as at 37°C, saturation of uptake (apparent Kd 40 μM), no influence of monensin, MRP, or P-gp inhibitors; tight binding to liposomes (Kd approx. 40 μM); and sharp increase in calculated free energy when forced in the hydrophobic domain. Conclusions. Although they are weak organic bases, PIVA and PIMA show none of the properties of lysosomotropic agents. We hypothesize that they remain locked onto the pericellular membrane and may never penetrate cells as such in significant amounts.