Abstract: The small-colony-variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureus strain and its counterpart with an SCV phenotype both in vitro and in vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log(10) in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.
Abstract: Co-trimoxazole, clindamycin and linezolid are used to treat community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, but little is known about intracellular activity. Moxifloxacin is active against intracellular methicillin-susceptible S. aureus (MSSA), but CA-MRSA has not been studied.
Abstract: In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log(2) dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log(2) dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.
Abstract: The aim of this study was to investigate the stability of a mixture of temocillin 20mg/ml in 5% dextrose and in 0.9% sodium chloride polyolefin bags after freezing, microwave thawing and long-term storage at 5±3°C.
Abstract: Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.
Abstract: Fluoroquinolones enter eukaryotic cells but the correlation between cellular accumulation and activity remains poorly established. Gemifloxacin is known to accumulate to a larger extent than most other fluoroquinolones in tissues. Using murine J774 macrophages and human THP-1 monocytes, we show that gemifloxacin accumulates more than ciprofloxacin and even moxifloxacin. Whilst showing indistinguishable kinetics of accumulation in J774 macrophages, gemifloxacin was released at an approximately two-fold slower rate than ciprofloxacin and its release was only partial. Gemifloxacin was also a weaker substrate than ciprofloxacin for the efflux transporter Mrp4 active in J774 macrophages. In cells infected with Listeria monocytogenes or Staphylococcus aureus (typical cytoplasmic and phagolysosomal organisms, respectively), gemifloxacin was equipotent to moxifloxacin and ciprofloxacin in concentration-dependent experiments if data are normalised based on the minimum inhibitory concentration (MIC) in broth. Thus, larger cellular concentrations of gemifloxacin than of moxifloxacin or ciprofloxacin were needed to obtain a similar target effect. Fractionation studies showed a similar subcellular distribution for all three fluoroquinolones, with approximately two-thirds of the cell-associated drug recovered in the soluble fraction (cytosol). These data suggest that cellular accumulation of fluoroquinolones is largely a self-defeating process as far as activity is concerned, with the intracellular drug made inactive in proportion to its accumulation level. Whilst these observations do not decrease the intrinsic value of fluoroquinolones for the treatment of intracellular infections, they indicate that ranking fluoroquinolones based on cell accumulation data without measuring the corresponding intracellular activity may lead to incorrect conclusions regarding their real potential.
Abstract: Antibiotics used by general practitioners frequently appear in adverse-event reports of drug-induced hepatotoxicity. Most cases are idiosyncratic (the adverse reaction cannot be predicted from the drug's pharmacological profile or from pre-clinical toxicology tests) and occur via an immunological reaction or in response to the presence of hepatotoxic metabolites. With the exception of trovafloxacin and telithromycin (now severely restricted), hepatotoxicity crude incidence remains globally low but variable. Thus, amoxicillin/clavulanate and co-trimoxazole, as well as flucloxacillin, cause hepatotoxic reactions at rates that make them visible in general practice (cases are often isolated, may have a delayed onset, sometimes appear only after cessation of therapy and can produce an array of hepatic lesions that mirror hepatobiliary disease, making causality often difficult to establish). Conversely, hepatotoxic reactions related to macrolides, tetracyclines and fluoroquinolones (in that order, from high to low) are much rarer, and are identifiable only through large-scale studies or worldwide pharmacovigilance reporting. For antibiotics specifically used for tuberculosis, adverse effects range from asymptomatic increases in liver enzymes to acute hepatitis and fulminant hepatic failure. Yet, it is difficult to single out individual drugs, as treatment always entails associations. Patients at risk are mainly those with previous experience of hepatotoxic reaction to antibiotics, the aged or those with impaired hepatic function in the absence of close monitoring, making it important to carefully balance potential risks with expected benefits in primary care. Pharmacogenetic testing using the new genome-wide association studies approach holds promise for better understanding the mechanism(s) underlying hepatotoxicity.
Abstract: Finafloxacin, an 8-cyano-substituted fluoroquinolone, expresses enhanced activity at acidic pH and is less susceptible to several fluoroquinolone resistance determinants. In this study, we compared finafloxacin and ciprofloxacin for (i) activity against ciprofloxacin-susceptible and -resistant Staphylococcus aureus as well as wild-type and Lde efflux-positive (Lde+) Listeria monocytogenes, (ii) accumulation in THP-1 macrophages and (iii) intracellular activity towards phagocytised S. aureus, L. monocytogenes and Legionella pneumophila (developing in acidic, neutral and mildly acidic environments, respectively), using a pharmacological approach assessing drug potencies and maximal relative efficacies (E(max)). Finafloxacin minimum inhibitory concentrations (MICs) were two-fold lower than those of ciprofloxacin against meticillin-susceptible S. aureus ATCC 25923, were only modestly increased in an isogenic strain overexpressing NorA and were ≤0.25 mg/L for community-acquired meticillin-resistant S. aureus. No loss of activity was seen in Lde+ L. monocytogenes. An acidic pH decreased the MIC of finafloxacin and increased that of ciprofloxacin both for S. aureus and L. monocytogenes, in parallel with corresponding changes in drug accumulation (tested with S. aureus ATCC 25923 only). Finafloxacin accumulated less than ciprofloxacin in THP-1 cells, but the situation was reversed by exposure of cells to acid pH. In S. aureus-infected cells, acid pH increased the potency of finafloxacin without change of E(max), whilst decreasing the potency and the maximal relative efficacy of ciprofloxacin (less negative E(max)). Finafloxacin was more potent and showed larger E(max) than ciprofloxacin against phagocytised L. pneumophila, but was less potent against phagocytised L. monocytogenes. Finafloxacin appears to be an acid-pH-favoured antibiotic that may find useful applications in infections where the local pH is low.
Abstract: The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable but can nevertheless be controlled, and it must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of 30 scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, USA, from 16 to 18 May 2011. From these discussions there emerged a priority list of steps that need to be taken to resolve this global crisis.
Abstract: Long-term exposure to pharmacological agents can select for cells that overexpress efflux transporters. We previously showed that mouse J774 macrophages cultivated for a prolonged period of time with toxic concentrations of the fluoroquinolone ciprofloxacin overexpress the efflux transporter Mrp4 and display a reduced accumulation of this antibiotic, but no change in the accumulation of moxifloxacin, a closely related molecule (Antimicrob. Agents Chemother. [2006] 50, 1689-1695 and [2009] 53, 2410-2416). Because of this striking difference between the two fluoroquinolones, we have now examined the modifications in the expression of ABC efflux transporters induced by the prolonged exposure of J774 macrophages to high concentrations of moxifloxacin. The resulting cell line showed (i) no difference in the accumulation of moxifloxacin but an increased accumulation and decreased efflux of ciprofloxacin; (ii) an overexpression of the multidrug transporters Abcb1a (P-gp), Abcc2 (Mrp2) and Abcg2 (Bcrp1), and a decreased expression of Abcc4 (Mrp4). While P-gp and Bcrp1 were functional, they did not modify the cellular accumulation of fluoroquinolones. The data show that exposing cells to high concentrations of a drug that is not affected by active efflux can trigger a pleiotropic response leading to a modulation in the expression of several transporters. These changes, however, are not sufficient to protect cells against the toxicity that fluoroquinolones may exert at large concentrations. They could also cause unanticipated drug interactions in vivo, should the drug exposure grossly exceed what is anticipated from its current registered use.
Abstract: Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT(MIC)]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT(MIC) is the index that is the most predictive of the outcome of infection both intra- and extracellularly.
Abstract: Pseudomonas aeruginosa causes severe nosocomial pneumonia in Intensive Care Unit (ICU) patients, with an increased prevalence of multiresistant strains. We examined the impact of the use of antipseudomonal antibiotic(s) on the susceptibility of P. aeruginosa isolated from ICU patients with clinically suspected hospital-acquired pneumonia collected in five teaching hospitals (110 non-duplicate initial isolates; 62 clonal pairs of initial and last isolates during treatment). Minimum inhibitory concentrations (MICs) were determined for amikacin, ciprofloxacin, meropenem, piperacillin/tazobactam (TZP), cefepime and ceftazidime (used in therapy) as well as five reporter antibiotics (aztreonam, colistin, gentamicin, piperacillin and ticarcillin) using Clinical and Laboratory Standards Institute (CLSI) methodology. Susceptibility was assessed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints. Resistance rates prior to treatment exceeded 25% for cefepime, ceftazidime, piperacillin, ticarcillin and aztreonam (EUCAST and CLSI) and for gentamicin, TZP and colistin (EUCAST only). The highest rates of cross-resistance were noted for ceftazidime and cefepime and the lowest rate for amikacin. Mean MIC values were systematically higher in isolates from patients previously exposed (1 month) to the corresponding antibiotic. For clonal pairs, a systematic increase in MIC between initial and last isolates (significant for amikacin, cefepime, meropenem and TZP) was noted. There was a significant correlation between the use of antibiotics (adjusted for respective proportional use of each drug) and loss of susceptibility at the population level when using EUCAST breakpoints. The high level of resistance of P. aeruginosa in ICU patients with nosocomial pneumonia as well as its further increase during treatment severely narrows the already limited therapeutic options. Further observational studies and the development of early diagnosis for resistant isolates are warranted.
Abstract: Radezolid is a novel biaryloxazolidinone in clinical development which shows improved activity, including against linezolid-resistant strains. In a companion paper (29), we showed that radezolid accumulates about 11-fold in phagocytic cells, with approximately 60% of the drug localized in the cytosol and approximately 40% in the lysosomes of the cells. The present study examines its activity against (i) bacteria infecting human THP-1 macrophages and located in different subcellular compartments (Listeria monocytogenes, cytosol; Legionella pneumophila, vacuoles; Staphylococcus aureus and Staphylococcus epidermidis, mainly phagolysosomal), (ii) strains of S. aureus with clinically relevant mechanisms of resistance, and (iii) isogenic linezolid-susceptible and -resistant S. aureus strains infecting a series of phagocytic and nonphagocytic cells. Radezolid accumulated to similar levels ( approximately 10-fold) in all cell types (human keratinocytes, endothelial cells, bronchial epithelial cells, osteoblasts, macrophages, and rat embryo fibroblasts). At equivalent weight concentrations, radezolid proved consistently 10-fold more potent than linezolid in all these models, irrespective of the bacterial species and resistance phenotype or of the cell type infected. This results from its higher intrinsic activity and higher cellular accumulation. Time kill curves showed that radezolid's activity was more rapid than that of linezolid both in broth and in infected macrophages. These data suggest the potential interest of radezolid for recurrent or persistent infections where intracellular foci play a determinant role.
Abstract: Treatment of Staphylococcus aureus infections remains problematic (slow responses and frequent recurrences). Intracellular persistence of the S. aureus could explain those difficulties because of impaired intracellular efficacy of antibiotics. Our aim was to study linezolid for its intracellular activity.
Abstract: Treatment of chronic or recurrent Staphylococcus aureus infections may require using antibiotics with activity against intracellular multiresistant organisms. Quinupristin/dalfopristin (3:7) has been examined in this context.
Abstract: Radezolid (RX-1741) is the first biaryloxazolidinone in clinical development. It shows improved activity, including against linezolid-resistant strains. Radezolid differs from linezolid by the presence of a biaryl spacer and of a heteroaryl side chain, which increases the ionization and hydrophilicity of the molecule at physiological pH and confers to it a dibasic character. The aim of this study was to determine the accumulation and subcellular distribution of radezolid in phagocytic cells and to decipher the underlying mechanisms. In THP-1 human macrophages, J774 mouse macrophages, and human polymorphonuclear neutrophils, radezolid accumulated rapidly and reversibly (half-lives of approximately 6 min and 9 min for uptake and efflux, respectively) to reach, at equilibrium, a cellular concentration 11-fold higher than the extracellular one. This process was concentration and energy independent but pH dependent (accumulation was reduced to 20 to 30% of control values for cells in medium at a pH of <6 or in the presence of monensin, which collapses pH gradients between the extracellular and intracellular compartments). The accumulation at equilibrium was not affected by efflux pump inhibitors (verapamil and gemfibrozil) and was markedly reduced at 4 degrees C but was further increased in medium with low serum content. Subcellular fractionation studies demonstrated a dual subcellular distribution for radezolid, with approximately 60% of the drug colocalizing to the cytosol and approximately 40% to the lysosomes, with no specific association with mitochondria. These observations are compatible with a mechanism of transmembrane diffusion of the free fraction and partial segregation of radezolid in lysosomes by proton trapping, as previously described for macrolides.
Abstract: Multidrug transporters of the ATP-binding cassette family export a wide variety of compounds across membranes in both prokaryotes and eukaryotes, using ATP hydrolysis as energy source. Several of these membrane proteins are of clinical importance. Although biochemical and structural studies have provided insights into the mechanism underlying substrate transport, many key questions subsist regarding the molecular and structural nature of this mechanism. In particular, the detailed conformational changes occurring during the catalytic cycle are still elusive. We explored the conformational changes occurring upon ATP/Mg(2+) binding using molecular dynamics simulations starting from the nucleotide-bound structure of SAV1866 embedded in an explicit lipid bilayer. The removal of nucleotide revealed a major rearrangement in the outer membrane leaflet portion of the transmembrane domain (TMD) resulting in the closure of the central cavity at the extracellular side. This closure is similar to that observed in the crystal nucleotide-free structures. The interface of the nucleotide-binding domain dimer (NDB) is significantly more hydrated in the nucleotide-free trajectory though it is not disrupted. This finding suggests that the TMD closure could occur as a first step preceding the dissociation of the dimer. The transmission pathway of the signal triggered by the removal of ATP/Mg(2+) mainly involves the conserved Q-loop and X-loop as well as TM6.
Abstract: Active efflux is a common mechanism of resistance to fluoroquinolones in Streptococcus pneumoniae. Two efflux systems have been described so far in this species: PmrA, a member of the major facilitator superfamily; and the two ABC transporters PatA and PatB. We studied the inducibility of expression of pmrA, patA and patB by using subinhibitory concentrations of fluoroquinolones.
Abstract: Staphylococcus aureus survives inside eukaryotic cells. Our objective was to assess the activity of NZ2114, a novel peptidic antibiotic, against intracellular S. aureus in comparison with established antistaphylococcal agents acting on the bacterial envelope with a distinct mechanism.
Abstract: Small-colony variant (SCV) strains of Staphylococcus aureus show reduced antibiotic susceptibility and intracellular persistence, potentially explaining therapeutic failures. The activities of oxacillin, fusidic acid, clindamycin, gentamicin, rifampin, vancomycin, linezolid, quinupristin-dalfopristin, daptomycin, tigecycline, moxifloxacin, telavancin, and oritavancin have been examined in THP-1 macrophages infected by a stable thymidine-dependent SCV strain in comparison with normal-phenotype and revertant isogenic strains isolated from the same cystic fibrosis patient. The SCV strain grew slowly extracellularly and intracellularly (1- and 0.2-log CFU increase in 24 h, respectively). In confocal and electron microscopy, SCV and the normal-phenotype bacteria remain confined in acid vacuoles. All antibiotics tested, except tigecycline, caused a net reduction in bacterial counts that was both time and concentration dependent. At an extracellular concentration corresponding to the maximum concentration in human serum (total drug), oritavancin caused a 2-log CFU reduction at 24 h; rifampin, moxifloxacin, and quinupristin-dalfopristin caused a similar reduction at 72 h; and all other antibiotics had only a static effect at 24 h and a 1-log CFU reduction at 72 h. In concentration dependence experiments, response to oritavancin was bimodal (two successive plateaus of -0.4 and -3.1 log CFU); tigecycline, moxifloxacin, and rifampin showed maximal effects of -1.1 to -1.7 log CFU; and the other antibiotics produced results of -0.6 log CFU or less. Addition of thymidine restored intracellular growth of the SCV strain but did not modify the activity of antibiotics (except quinupristin-dalfopristin). All drugs (except tigecycline and oritavancin) showed higher intracellular activity against normal or revertant phenotypes than against SCV strains. The data may help rationalizing the design of further studies with intracellular SCV strains.
Abstract: BACKGROUND AND AIMS: Optimal treatment of infections caused by Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila requires antibiotics with intracellular activity. Linezolid accumulates poorly within cells. Torezolid (TR-700) is a novel methyltetrazolyl oxazolidinone with potentially different cellular pharmacokinetic properties. Our aim was to examine the accumulation and intracellular activities of torezolid in this context. METHODS: Measurement of torezolid cell content and antibacterial activity in comparison with linezolid using human macrophages (THP-1) and human endothelial cells [human umbilical vein endothelial cells (HUVECs)], applying models allowing for the quantitative evaluation of the pharmacodynamics of antibiotics towards intracellular bacteria. RESULTS: Torezolid accumulated rapidly in THP-1 macrophages, reaching a stable intracellular to extracellular ratio of approximately 10 (compared with approximately 1-2 for linezolid) within 15 min. On a weight concentration basis (mg/L), torezolid was approximately 5- to 10-fold more potent intracellularly (lower concentration needed to achieve a bacteriostatic effect) than linezolid against phagocytosed S. aureus, L. monocytogenes and L. pneumophila, with no change in maximal efficacy (approximately 1 log(10) reduction of the original, post-phagocytosis inoculum). When drugs were compared at equipotent concentrations (multiples of the MIC), no difference was seen between linezolid and torezolid, but the higher potency of torezolid allowed control of intracellular infections caused by linezolid-resistant S. aureus. CONCLUSIONS: Torezolid exerts intracellular activity at lower extracellular concentrations than linezolid because of its greater potency independent of its greater intracellular accumulation. This may confer an advantage to torezolid in vivo if the drug can be used at dosages creating serum concentrations similar to those achieved with linezolid.
Abstract: Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [E(max)], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E(max), >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an E(max) of a 1.1-log CFU reduction. The parameter most important for activity was fC(peak)/MIC, where fC(peak) is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.
Abstract: DD-ligases catalyze the synthesis of the D-Ala-D-Ala and D-Ala-D-Ser dipeptides or the D Ala-D-Lac depsipeptide in an early step of peptidoglycan synthesis. Their function is essential for bacterial growth and specific to bacteria, making them attractive targets for the development of novel antibiotics. This review examines the biochemical and structural features of these enzymes and presents the main families of inhibitors described so far. Over the last 20 years, 7 structures of DD-ligases have been solved by X-ray crystallography, giving a detailed view of the general topology of the active site and of the residues in the catalytic pocket that play a central role in substrate recognition. This has paved the way to the rational design of inhibitors, which can be classified as (i) analogues of substrates, (ii) analogues of the product of the reaction, (iii) analogues of the transition state, and (iv) original scaffolds discovered by screening or by rational computer-aided design. The three first strategies have led to molecules that are polar by nature and have therefore poor access to their cytosolic target. The fourth one is potentially most promising as it yields more diverse structures. The most active molecules show affinity constants in the microM range, but microbiological evaluation remains scarce (typical MIC 1-8 mg/L for the tested compounds). These data strongly suggest targeting DD-ligases is a promising approach for discovery of new antibiotics. Future research should, however, aim at finding more potent inhibitors endowed with the appropriate pharmacokinetic properties that ensure access to their intracellular target.
Abstract: Routine therapeutic drug monitoring (TDM) reports only total vancomycin (VAN) concentrations, although protein binding varies and it is generally accepted that only free VAN is active. The aims of this study were to examine the correlation between free and total VAN concentrations in order to estimate whether free VAN levels can be predicted based on its total concentration. A high-performance liquid chromatography (HPLC) method was set up and validated (against routine laboratory immunoassays) for measurement of free [ultrafiltration (Centrifree); cut-off 30 kDa] and total [solid-phase extraction (Oasis MCX cartridge)] VAN in serum. Samples (n=65) from patients (n=15) treated by continuous infusion were analysed. There was a wide variation in free to total VAN ratios [range 12-100%; mean 63.6+/-25.8%, with 59 values falling outside the 95% confidence interval (57.3-69.9%); median 70.2%]. The correlation between free and total VAN was poor (R(2)=0.55). Artefacts such as pH variation of sera could be excluded. Both intrapatient and interpatient variabilities were large and no correlation could be made with patients' clinical conditions. Total VAN concentration is not predictive of free VAN concentration, suggesting that actual determination of free VAN might be recommended as an improved method of TDM.
Abstract: In Staphylococcus aureus, rsbU down-regulates agr and stimulates production of staphyloxanthin (STX), an antioxidant that may contribute to intracellular survival after phagocytosis. Using isogenic rsbU(-) and rsbU(+) strains, we show that rsbU causes increased internalization and intracellular growth in both THP-1 macrophages and human umbilical vein endothelial cells (more so for the latter) without change in subcellular localization and that inhibition of STX biosynthesis markedly reduces intracellular growth of the rsbU(+) strain (and of clinical isolates, including USA300; tested with macrophages only) without affecting internalization. Thus, rsbU is important for uptake and for STX biosynthesis and is critical for intracellular multiplication of S. aureus.
Abstract: While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity against Listeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity in L. monocytogenes while PBP4 additionally has a protective affect against both penicillin and carbapenem.
Abstract: The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP-1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2-D-DIGE approach followed by MALDI-TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D-Ala-D-Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde-3-phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti-Listeria therapy.
Abstract: CEM-101 is a novel fluoroketolide with lower MICs than those of telithromycin and macrolides. Our aim was to assess the cellular accumulation and intracellular activity of CEM-101 using models developed for analyzing the pharmacokinetics and pharmacological properties of antibiotics against phagocytized bacteria. We used THP-1 macrophages and Staphylococcus aureus (ATCC 25923 [methicillin (meticillin) sensitive]), Listeria monocytogenes (strain EGD), and Legionella pneumophila (ATCC 33153). CEM-101 reached cellular-to-extracellular-concentration ratios of about 350 within 24 h (versus approximately 20, 30, and 160 for telithromycin, clarithromycin, and azithromycin, respectively). This intracellular accumulation was suppressed by incubation at a pH of < or = 6 and by monensin (proton ionophore) and was unaffected by verapamil (P-glycoprotein inhibitor; twofold accumulation increase for azithromycin) or gemfibrozil. While keeping with the general properties of the macrolide antibiotics in terms of maximal efficacy (Emax; approximately 1-log10-CFU decrease compared to the postphagocytosis inoculum after a 24-h incubation), CEM-101 showed significantly greater potency against phagocytized S. aureus than telithromycin, clarithromycin, and azithromycin (for which the 50% effective concentration [EC50] and static concentrations were about 3-, 6-, and 15-fold lower, respectively). CEM-101 was also about 50-fold and 100-fold more potent than azithromycin against phagocytized L. monocytogenes and L. pneumophila, respectively. These differences in EC50s and static concentrations between drugs were minimized when data were expressed as multiples of the MIC, demonstrating the critical role of intrinsic drug activity (MIC) in eliciting the antibacterial intracellular effects, whereas accumulation per se was unimportant. CEM-101 should show enhanced in vivo potency if used at doses similar to those of the comparators tested here.
Abstract: The rational selection of antibiotics for the treatment of meningitis must take into account several criteria, among which their intrinsic activity against the causative bacteria, and their pharmacokinetic and pharmacodynamic properties. The intrinsic activity is evaluated by the Minimal Inhibitory Concentration (MIC), which, however, does not give any information on the bactericidal potency of the drug (important property for infections localized in compartments with low immune defense such as the CSF). The capacity of the antibiotic to reach the infected compartment depends on its physicochemical properties (molecular weight, lipophilicity) and its protein binding capacity, but also on the properties of the blood-CSF barrier (permeability modulated by inflammation and activity of active transporters). Pharmacodynamics correlate intrinsic activity to pharmacokinetics by determining the optimal value of the ratio between MIC and time of exposure, area under the curve, or peak concentration. On these bases, beta-lactams appear as first-line antibiotics, if used with large and repeated doses (or even as a continuous infusion), because of their time-dependent activity. The choice of the molecule is based on the susceptibility of the bacterium. Potential alternatives include chloramphenicol (limited however by its toxicity), moxifloxacin (showing high bactericidal effect, a low MIC, and appropriate penetration) but little clinically documented, linezolid and vancomycin for Methicillin-Resistant Staphylococcus aureus (MRSA), and vancomycin for penicillin non-susceptible pneumococci. Other molecules in clinical development are being evaluated for this indication.
Abstract: In a companion paper (H. A. Nguyen et al., Antimicrob. Agents Chemother. 53:1434-1442, 2009), we showed that vancomycin, oxacillin, fusidic acid, clindamycin, linezolid, and daptomycin are poorly active against the intracellular form of a thymidine-dependent small-colony variant (SCV) strain isolated from a cystic fibrosis patient and that the activity of quinupristin-dalfopristin, moxifloxacin, rifampin, and oritavancin remains limited (2- to 3-log CFU reduction) compared to their extracellular activity. Antibiotic combination is a well-known strategy to improve antibacterial activity, which was examined here against an intracellular SCV strain using combinations with either rifampin or oritavancin. Time-kill curve analysis using either concentrations that caused a static effect for each antibiotic individually or concentrations corresponding to the maximum concentration in human serum showed largely divergent effects that were favorable when antibiotics were combined with rifampin at low concentrations only and with oritavancin at both low and high concentrations. The nature of the interaction between rifampin, oritavancin, and moxifloxacin was further examined using the fractional maximal effect method, which allows categorization of the effects of combinations when dose-effect relationships are not linear. Rifampin and oritavancin were synergistic at all concentration ratios investigated. Oritavancin and moxifloxacin were also synergistic but at high oritavancin concentrations only. Rifampin and moxifloxacin were additive. This approach may help in better assessing and improving the activity of antibiotics against intracellular SCV strains.
Abstract: BACKGROUND: P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies. RESULTS: We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants. CONCLUSION: Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs), within the transmembrane domains (TMDs) or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission. Our models may characterize different steps in the catalytic cycle and may be important tools to understand the structure-function relationship of P-glycoprotein.
Abstract: Resistance in Gram-negative pathogens is an increasing concern, with carbapenems often appearing as the only acceptable treatment option in serious infections. Reviving older compounds that have fallen into disuse may help to alleviate this burden. Temocillin (6-alpha-methoxy-ticarcillin) is resistant to most if not all classical and extended-spectrum beta-lactamases and to AmpC enzymes. It is also chemically stable, allowing administration by continuous infusion. Pharmacokinetic/pharmacodynamic analysis, aided by Monte-Carlo simulations, suggests a breakpoint of 8 mg/L for the registered maximum dosage of 4 g daily. Temocillin's weaknesses, explaining its limited previous use, are a lack of activity against Gram-positive organisms, anaerobes and Pseudomonas. In settings where these are unlikely or are covered by other agents, temocillin may be useful, potentially 'sparing' carbapenems and having little apparent potential to select for Clostridium difficile.
Abstract: Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, beta-lactams show activity against intracellular methicillin (methicillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these beta-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (E(max)), relative potency (the concentration causing a reduction of the inoculum halfway between E(0) and E(max) [EC(50)]), and static concentration (C(s))} were measured. All strains showed sigmoidal dose-responses, with E(max) being about a 1 log(10) CFU decrease from the postphagocytosis inoculum, and EC(50) and C(s) being 0.2 to 0.3x and 0.6 to 0.9x the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.
Abstract: Ciprofloxacin, the most widely used totally synthetic antibiotic, is subject to active efflux mediated by a MRP-like transporter in wild-type murine J774 macrophages. To identify the transporter among the seven potential Mrps, we used cells made resistant to ciprofloxacin obtained by long-term exposure to increasing drug concentrations (these cells show less ciprofloxacin accumulation and provide a protected niche for ciprofloxacin-sensitive intracellular Listeria monocytogenes). In the present paper, we first show that ciprofloxacin-resistant cells display a faster efflux of ciprofloxacin which is inhibited by gemfibrozil (an unspecific MRP inhibitor). Elacridar, at a concentration known to inhibit P-glycoprotein and breast cancer resistance protein (BCRP), only slightly increased ciprofloxacin accumulation, with no difference between resistant and wild-type cells. Analysis at the mRNA (real-time PCR) and protein (Western blotting) levels revealed an overexpression of Mrp2 and Mrp4. Mrp4 transcripts, however, were overwhelmingly predominant (45% [wild-type cells] to 95% [ciprofloxacin-resistant cells] of all Mrp transcripts tested [Mrp1 to Mrp7]). Silencing of Mrp2 and Mrp4 with specific small interfering RNAs showed that only Mrp4 is involved in ciprofloxacin transport in both ciprofloxacin-resistant and wild-type cells. The study therefore identifies Mrp4 as the most likely transporter of ciprofloxacin in murine macrophages but leaves open a possible common upregulation mechanism for both Mrp4 and Mrp2 upon chronic exposure of eukaryotic cells to this widely used antibiotic.
Abstract: Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (<5 min) and complete (>95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0-600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 microM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin.
Abstract: Moxifloxacin, a fluoroquinolone with potent activity against respiratory pathogens, is approved and considered as an alternative to beta-lactams and macrolides for the treatment of acute bacterial sinusitis and lower respiratory tract infections. In this review, we critically examine its safety profile in comparison with other fluoroquinolones and other antibacterial classes sharing similar indications. Data were extracted from published clinical trials, meta-analyses, postmarketing studies, spontaneous report systems and case reports for rare effects. Global analysis did not reveal significantly higher incidences of drug-related adverse effects than for comparators. Tendon rupture was infrequent with moxifloxacin, including when used in elderly patients with chronic obstructive pulmonary disease. Severe toxic cutaneous reactions and allergies were very rare. Phototoxicity and CNS adverse effects were less common than with other fluoroquinolones. Although causing a 4-7 msec corrected QT interval prolongation, severe cardiac toxicity was neither seen in large cohorts or clinical trials nor reported to pharmacovigilance systems. Hepatotoxicity was not different from what was observed for other fluoroquinolones (excluding trovafloxacin) and less frequent than reported for amoxicillin-clavulanic acid or telithromycin. The data show that using moxifloxacin, in its accepted indications and following the corresponding guidelines, should not be associated with an excessive incidence of drug-related adverse reactions, provided the clinician takes care in identifying patients with known risk factors and pays due attention to the contraindications and warnings mentioned in the labelling.
Abstract: The mechanisms that might explain the antibiotic use in ambulatory practices go widely beyond microbiology and epidemiology. Sociocultural factors (help-seeking behaviour) and structural factors (liberal fee-for system), similar in France and Belgium, explain how an inappropriate health demand and an inappropriate supply are supporting each other, to keep the two countries high in European ranking. Beyond these collective factors of antibiotic use, the general practitioners' individual responsibility is also discussed.
Abstract: Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target.
Abstract: Staphylococcus aureus invades eukaryotic cells. When methicillin-resistant S. aureus (MRSA) ATCC 33591 is phagocytized by human THP-1 macrophages, complete restoration of susceptibility to cloxacillin and meropenem is shown and the strain becomes indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (S. Lemaire et al., Antimicrob. Agents Chemother. 51:1627-1632, 2007). We examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (three hospital-acquired MRSA [HA-MRSA] strains, two community-acquired MRSA [CA-MRSA] strains, two HA-MRSA strains with the vancomycin-intermediate phenotype, one HA-MRSA strain with the vancomycin-resistant phenotype, and one animal [porcine] MRSA strain), (ii) activated THP-1 cells and nonprofessional phagocytes (keratinocytes, Calu-3 bronchial epithelial cells), and (iii) other beta-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction in MICs in broth at pH 5.5 compared with the MIC at pH 7.4 and (ii) sigmoidal dose-response curves with cloxacillin (0.01x to 100x MIC, 24 h of incubation) after phagocytosis by THP-1 macrophages that were indistinguishable from each other and from the dose-response curve for methicillin-susceptible S. aureus (MSSA) ATCC 25923 (relative potency [50% effect], 6.09x MIC [95% confidence interval {CI}, 4.50 to 8.25]; relative efficacy [change in bacterial counts over the original inoculum for an infinitely large cloxacillin concentration, or maximal effect], -0.69 log CFU [95% CI, -0.79 to -0.58]). Similar dose-response curves for cloxacillin were also observed with MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lower level of restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when the data were normalized for differences in MICs. We conclude that the restoration of MRSA susceptibility to beta-lactams after phagocytosis is independent of the strain and the types of cells but varies between beta-lactams.
Abstract: Levels of apoptosis induction (4',6'-diamidino-2-phenylindole staining, activation of caspase 3) for aminoglycosides were compared by using renal LLC-PK1 cells. Amikacin caused less apoptosis than gentamicin in incubated cells. In electroporated cells, neomycin B and gentamicin caused apoptosis in the 0.03 to 0.1 mM range, isepamicin required larger concentrations (0.2 mM), and amikacin was without effect.
Abstract: BACKGROUND AND AIMS: Listeria monocytogenes and Staphylococcus aureus invade and multiply in THP-1 monocytes. Fluoroquinolones accumulate in these cells, but are less active against intracellular than extracellular forms of L. monocytogenes and S. aureus. We examined whether differentiation of THP-1 monocytes into adherent, macrophage-like cells increases fluoroquinolone uptake and activity. METHODS: THP-1 monocytes were differentiated with phorbol myristate acetate (PMA) and compared with unstimulated cells for: (i) moxifloxacin and levofloxacin accumulation; and (ii) activity against phagocytosed L. monocytogenes and S. aureus (5 h contact). RESULTS: The differentiation of THP-1 monocytes caused: (i) a 3- to 4-fold increase in moxifloxacin uptake and a significant increase in its activity against intracellular L. monocytogenes (from 1.3 log(10) to 2.1 log(10) cfu decrease compared with the post-phagocytosis inoculum), but not against S. aureus (1.0-1.2 log(10) cfu decrease throughout); and (ii) no change in levofloxacin accumulation and intracellular activity against either L. monocytogenes or S. aureus. CONCLUSIONS: Although differentiation of monocytes enhances the uptake and activity of moxifloxacin against L. monocytogenes, this cannot be extended to other intracellular bacteria and to levofloxacin. These results further demonstrate that antibiotic intracellular accumulation and activity are not necessarily linked and suggest that intracellular drug and pathogen combinations must be studied individually.
Abstract: Decreased susceptibility of Staphylococcus aureus to antistaphylococcal agents may be associated with inability to eradicate intracellular forms, which could explain therapeutic failures. This hypothesis was tested using clinical isolates obtained from a patient with persistent staphylococcal bacteraemia under therapy. Four isogenic isolates (three from tissue, one from blood) with increased MICs for vancomycin (1-4 mg/L) and for daptomycin (1-4 mg/L) were collected after an initial 16-day treatment with vancomycin-rifampicin-gentamicin, followed by 13-20 days of treatment with daptomycin-rifampicin-gentamicin. Isolates were tested for MICs and for: (i) vancomycin (BODIPY-FL-vancomycin) and daptomycin binding; (ii) cell wall turnover (loss of N-acetyl-d-[1-(14)C]glucosamine in 30 min after 1 h of labelling); and (iii) Triton X-100-induced autolysis. Extracellular (broth) and intracellular (THP-1 macrophages) activities of rifampicin, linezolid and fusidic acid at C(max), and of vancomycin, daptomycin, quinupristin-dalfopristin and oritavancin over a wide range of extracellular concentrations (with pharmacological modelling to determine E(max)), were measured at 24 h. Increases in vancomycin MICs correlated with increased drug binding, and decreased cell wall turnover and detergent-induced autolysis. Increases in daptomycin MICs correlated with decreased daptomycin binding. Intracellular activity was weak (E(max) <1 log(10) CFU decrease) for vancomycin against all isolates, and for daptomycin against isolates with MICs >1 mg/L. Among all antibiotics tested, only quinupristin-dalfopristin and oritavancin provided close to bactericidal intracellular activities (1.6-2.5 log(10) CFU decreases at C(max)). Determination of the intracellular susceptibility of S. aureus, combined with improved methods of diagnosis, could be useful when dealing with persistent staphylococcal infections and could improve therapy.
Abstract: The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.
Abstract: BACKGROUND: Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin). METHODS: J774 macrophages and rat embryo fibroblasts were exposed for up to 24 and 72 h to telavancin (5-90 mg/L). The following studies were performed: measurement of (14)C-labelled telavancin cellular uptake and subcellular distribution (cell fractionation), determination of pericellular membrane integrity (lactate dehydrogenase release), electron microscopy with morphometric analysis of changes in lysosome size and determination of total phospholipid and cholesterol content. RESULTS: The uptake of telavancin proceeded linearly as a function of time and concentration in both cell types (clearance rate of approximately 10 mL/g of protein/h). Efflux (macrophages) was approximately 5.7-fold slower. Telavancin subcellular distribution was superimposable on that of a lysosomal marker (N-acetyl-beta-hexosaminidase). It did not cause an increase in the release of lactate dehydrogenase and did not induce significant increases in total phospholipid or cholesterol content. It caused only mild morphological lysosomal alterations (similar to vancomycin and much less than oritavancin by morphometric analysis). CONCLUSIONS: Telavancin is taken up by eukaryotic cells and localizes in lysosomes, causing mild morphological alterations without evidence of lipid metabolism alterations. These data support our observations that telavancin is active against intracellular S. aureus.
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of beta-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by beta-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) beta-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.
Abstract: Over the last decades, numerous papers have appeared--and still are appearing--that describe concentrations in tissues in an effort to predict the efficacy of an antimicrobial agent based on these concentrations and MICs for microorganisms. A common method is to use measurements of concentrations in tissue homogenates, comparing these with values derived from the corresponding blood samples and on that basis draw conclusions with respect to the potential clinical use of the drug. This approach is not justifiable for a number of reasons that includes both pharmacokinetic as well as pharmacodynamic causes. This way of presenting data with the derived conclusions is often misleading and may ultimately be harmful in patient care.
Abstract: BACKGROUND AND AIMS: Temocillin, a 6alpha-methoxy-penicillin stable towards most beta-lactamases (including extended-spectrum beta-lactamase), is presented as an alternative to carbapenems for susceptible Enterobacteriaceae in microbiological surveys. We aimed at documenting its potential clinical usefulness in intensive care (IC) patients using pharmacokinetic/pharmacodynamic approaches applied to conventional (twice daily) and continuous infusion (CI) modes of administration. METHODS: (i) In vitro evaluation of temocillin stability and compatibility with other drugs under conditions pertinent of CI in IC patients; (ii) pharmacokinetic study in patients treated by CI (4 g/day; n = 6) versus [twice daily (2 g every 12 h); n = 6]; (iii) population pharmacokinetic analysis of twice daily with Monte Carlo simulations to determine 95% probability of target attainment (PTA(95)) versus MIC (based on time above MIC > or = 40% for measured free drug). RESULTS: Temocillin was stable at 37 degrees C in 8.34% solutions for 24 h and compatible with flucloxacillin and aminoglycosides, but not with several other antibiotic and non-antibiotic drugs. With CI, stable total serum concentrations were 73.5 +/- 3.0 mg/L (SEM) and free concentration 29.3 +/- 2.8 mg/L. With twice daily, Cmax (total drug) was 147 +/- 12.3 mg/L (SEM; free drug: 50.3 +/- 15.8 mg/L), lowest trough (total drug) 12.3 mg/L, and PTA(95) (free drug) obtained for MIC < or = 8 mg/L. CONCLUSIONS: Temocillin (4 g/day) by CI yields stable free serum concentrations above the current breakpoint (16 mg/L), although individual variations may suggest lowering the breakpoint to 8 mg/L (as for twice daily) unless the daily dose or the frequency of administration is increased.
Abstract: Ketolides differ from macrolides by removal of the 3-O-cladinose (replaced by a keto group), a 11,12- or 6,11-cyclic moiety and a heteroaryl-alkyl side chain attached to the macrocyclic ring through a suitable linker. These modifications allow for anchoring at two distinct binding sites in the 23S rRNA (increasing activity against erythromycin-susceptible strains and maintaining activity towards Streptococcus pneumoniae resistant to erythromycin A by ribosomal methylation), and make ketolides less prone to induce methylase expression and less susceptible to efflux in S. pneumoniae. Combined with an advantageous pharmacokinetic profile (good oral bioavailability and penetration in the respiratory tract tissues and fluids; prolonged half-life allowing for once-a-day administration), these antimicrobial properties make ketolides an attractive alternative for the treatment of severe respiratory tract infections such as pneumonia in areas with significant resistance to conventional macrolides. For telithromycin (the only registered ketolide so far), pharmacodynamic considerations suggest optimal efficacy for isolates with minimum inhibitory concentration values < or = 0.25 mg/l (pharmacodynamic/pharmacokinetic breakpoint), calling for continuous and careful surveys of bacterial susceptibility. Postmarketing surveillance studies have evidenced rare, but severe, side effects (hepatotoxicity, respiratory failure in patients with myasthenia gravis, visual disturbance and QTc prolongation in combination with other drugs). On these bases, telithromycin indications have been recently restricted by the US FDA to community-acquired pneumonia, and caution in patients at risk has been advocated by the European authorities. Should these side effects be class related, they may hinder the development of other ketolides such as cethromycin (in Phase III, but on hold in the US) or EDP-420 (Phase II).
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate S. aureus (VISA) are spreading worldwide, making the search for antibiotics directed against new targets a high priority. Drugs that anchor in the bacterial membrane (e.g. ceragenins and lipopeptides) or that target the bacterial membrane and proteic (lipoglycopeptides) or lipidic (glycodepsipeptides) cell wall precursors seem to have the most potential because they show a fast and extensive bactericidal effect and are probably less prone to select for resistance owing to the difficulty in modifying their targets in a way that is compatible with bacterial survival. The efficacy of lipopeptides and lipoglycopeptides has been demonstrated in the treatment of skin and skin structure infections, and bacteremia caused by resistant S. aureus. Ceragenins and glycodepsipeptides are restricted to topical applications because of their unsatisfactory safety profile. The mode of action, pharmacological and microbiological properties and target indications of these anti-MRSA agents, which function by disturbing membrane integrity, are reviewed in this article.
Abstract: Apoptosis plays a central role not only in the physiological processes of kidney growth and remodeling, but also in various human renal diseases and drug-induced nephrotoxicity. We present in a synthetic fashion the main molecular and cellular pathways leading to drug-induced apoptosis in kidney and the mechanisms regulating it. We illustrate them using three main nephrotoxic drugs (cisplatin, gentamicin, and cyclosporine A). We discuss the main regulators and effectors that have emerged as key targets for the design of therapeutic strategies. Novel approaches using gene therapy, antisense strategies, recombinant proteins, or compounds obtained from both classical organic and combinatorial chemistry are examined. Finally, key issues that need to be addressed for the success of apoptosis-based therapies are underlined.
Abstract: P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC(50); relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy (E(max); CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 microM) and elacridar (GF 120918; 0.5 microM), two known inhibitors of P-gp, decreased daptomycin EC(50) (about threefold) in THP-1 and MDCK cells without affecting E(max). Daptomycin EC(50) was about three- to fourfold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr1 [the gene encoding P-gp] expression) decreased the accumulation of daptomycin in parallel with that of DiOC(2) (a known substrate of P-gp); (ii) silencing mdr1 with duplex human mdr1 siRNAs reduced the cell content in immunoreactive P-gp to 15 to 30% of controls and caused an eight- to 13-fold increase in daptomycin accumulation. We conclude that daptomycin is subject to efflux from THP-1 macrophages and MDCK cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus.
Abstract: BACKGROUND: Mex efflux pumps contribute to multidrug resistance in Pseudomonas aeruginosa. Evidencing their expression in clinical isolates would help in rationalizing antibiotic selection. METHODS: We have developed a combined phenotypic and genotypic approach for the differential diagnosis of resistance mediated by four major transporters (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM). The methodology was validated using reference strains harbouring only one specific transporter and its applicability evaluated towards seven selected clinical isolates, the resistance mechanisms of which could not be assigned by conventional techniques. Phenotypic detection used MIC measurements with reporter antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin (MexXY-OprM)] with and without Phe-Arg-beta-naphthylamide. Genotypic detection was made by semi-quantitative reverse transcription PCR (RT-PCR) for mexC and mexE, and by quantitative competitive RT-PCR and real-time PCR for mexA and mexX (correlation between both methods: > 88 % ; overexpression levels ranging between 4.8 and 8.1). RESULTS: Convergence between phenotypic and genotypic methods was observed in control strains for all pumps. For clinical isolates, convergence was obtained in 6 of 7 strains for MexXY-OprM and MexEF-OprM, and in 5 of 7 for MexAB-OprM and MexCD-OprJ, mostly due to hard to interpret phenotypic data. CONCLUSIONS: The data plead for combining phenotypic and genotypic approaches in the diagnosis of efflux-mediated resistance in P. aeruginosa.
Abstract: Pseudomonas aeruginosa is a major cause of nosocomial infections. This organism shows a remarkable capacity to resist antibiotics, either intrinsically (because of constitutive expression of beta-lactamases and efflux pumps, combined with low permeability of the outer-membrane) or following acquisition of resistance genes (e.g., genes for beta-lactamases, or enzymes inactivating aminoglycosides or modifying their target), over-expression of efflux pumps, decreased expression of porins, or mutations in quinolone targets. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. Susceptibility testing is therefore crucial in clinical practice. Empirical treatment usually involves combination therapy, selected on the basis of known local epidemiology (usually a beta-lactam plus an aminoglycoside or a fluoroquinolone). However, therapy should be simplified as soon as possible, based on susceptibility data and the patient's clinical evolution. Alternative drugs (e.g., colistin) have proven useful against multiresistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful to date. Among broad-spectrum antibiotics in development, ceftobiprole, sitafloxacin and doripenem show interesting in-vitro activity, although the first two molecules have been evaluated in clinics only against Gram-positive organisms. Doripenem has received a fast track designation from the US Food and Drug Administration for the treatment of nosocomial pneumonia. Pump inhibitors are undergoing phase I trials in cystic fibrosis patients. Therefore, selecting appropriate antibiotics and optimising their use on the basis of pharmacodynamic concepts currently remains the best way of coping with pseudomonal infections.
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 x 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 log(10) colony-forming unit changes at 24 h versus log(10) of antibiotic concentration followed sigmoidal shapes, allowing calculation of EC(50) (relative potency) and apparent E(max) (relative efficacy) in all conditions. In broth, the EC(50) of gentamicin rose 316-fold and that of oxacillin decreased 15-fold with unchanged apparent E(max) [-5 log (limit of detection)] between pH 7.4 and 5. Intracellularly, EC(50)s were similar to those observed extracellularly at pH 7.4, but E(max) values were much lower (-1 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.
Abstract: Early studies showed that methicillin-resistant Staphylococcus aureus (MRSA) strains are susceptible to beta-lactams when they are exposed to pH < or = 5.5 in broth. Because S. aureus survives in the phagolysosomes of macrophages, where the pH may be acidic, we have examined the susceptibility of MRSA ATCC 33591 phagocytized by human THP-1 macrophages to meropenem (MEM) and cloxacillin (CLX). Using a pharmacodynamic model assessing key pharmacological (50% effective concentration and maximal efficacy) and microbiological (static concentration) descriptors of antibiotic activity, we show that intraphagocytic MRSA strains are as sensitive to MEM and CLX as methicillin-susceptible S. aureus (MSSA; ATCC 25923). This observation was replicated in broth if the pH was brought to 5.5 and was confirmed with clinical strains. Electron microscopy showed that both the MRSA and the MSSA strains localized and multiplied in membrane-bounded structures (phagolysosomes) in the absence of beta-lactams. Incubation of the infected macrophages with ammonium chloride (to raise the phagolysosomal pH) made MRSA insensitive to MEM and CLX. No difference was seen in mec, mecA, mecI, mecR1, femA, and femB expression (reversed transcription-PCR) or in PBP 2a content (immunodetection) in MRSA grown in broth at pH 5.5 compared with that in MRSA grown in broth at 7.4. The level of [(14)C]benzylpenicillin binding to cell walls prepared from a non-beta-lactamase-producing MRSA clinical isolate was two times lower than that to cell walls prepared from MSSA ATCC 25923 at pH 7.4, but the levels increased to similar values for both strains at pH 5.5. These data suggest that the restoration of susceptibility of intraphagocytic of MRSA to MEM and CLX is due to the acidic pH prevailing in phagolysosomes and is mediated by an enhanced binding to penicillin-binding proteins.
Abstract: Antibacterial resistance in Streptococcus pneumoniae is increasing worldwide, affecting principally beta-lactams and macrolides (prevalence ranging between approximately 1% and 90% depending on the geographical area). Fluoroquinolone resistance has also started to emerge in countries with high level of antibacterial resistance and consumption. Of more concern, 40% of pneumococci display multi-drug resistant phenotypes, again with highly variable prevalence among countries. Infections caused by resistant pneumococci can still be treated using first-line antibacterials (beta-lactams), provided the dosage is optimised to cover less susceptible strains. Macrolides can no longer be used as monotherapy, but are combined with beta-lactams to cover intracellular bacteria. Ketolides could be an alternative, but toxicity issues have recently restricted the use of telithromycin in the US. The so-called respiratory fluoroquinolones offer the advantages of easy administration and a spectrum covering extracellular and intracellular pathogens. However, their broad spectrum raises questions regarding the global risk of resistance selection and their safety profile is far from optimal for wide use in the community. For multi-drug resistant pneumococci, ketolides and fluoroquinolones could be considered. A large number of drugs with activity against these multi-drug resistant strains (cephalosporins, carbapenems, glycopeptides, lipopeptides, ketolides, lincosamides, oxazolidinones, glycylcyclines, quinolones, deformylase inhibitors) are currently in development. Most of them are only new derivatives in existing classes, with improved intrinsic activity or lower susceptibility to resistance mechanisms. Except for the new fluoroquinolones, these agents are also primarily targeted towards methicillin-resistant Staphylococcus aureus infections; therefore, demonstration of their clinical efficacy in the management of pneumococcal infections is still awaited.
Abstract: OBJECTIVES: To evaluate the effect of pharmaceutical care provided in addition to acute Geriatric Evaluation and Management (GEM) care on the appropriateness of prescribing. DESIGN: Randomized, controlled trial, with the patient as unit of randomization. SETTING: Acute GEM unit. PARTICIPANTS: Two hundred three patients aged 70 and older. INTERVENTION: Pharmaceutical care provided from admission to discharge by a specialist clinical pharmacist who had direct contacts with the GEM team and patients. MEASUREMENTS: Appropriateness of prescribing on admission, at discharge, and 3 months after discharge, using the Medication Appropriateness Index (MAI), Beers criteria, and Assessing Care of Vulnerable Elders (ACOVE) underuse criteria and mortality, readmission, and emergency visits up to 12 months after discharge. RESULTS: Intervention patients were significantly more likely than control patients to have an improvement in the MAI and in the ACOVE underuse criteria from admission to discharge (odds ratio (OR)=9.1, 95% confidence interval (CI)=4.2-21.6 and OR=6.1, 95% CI=2.2-17.0, respectively). The control and intervention groups had comparable improvements in the Beers criteria. CONCLUSION: Pharmaceutical care provided in the context of acute GEM care improved the appropriate use of medicines during the hospital stay and after discharge. This is an important finding, because only limited data exist on the effect of various strategies to improve medication use in elderly inpatients. The present approach has the potential to minimize risk and improve patient outcomes.
Abstract: Four small molecular receptors of vancomycin have been designed to make part of a novel biosensor device based on the FTIR-ATR detection: N-Boc (2a) or N-Ac (2b)-6-aminocaproyl-D-Ala-D-Ala and N-Boc (3a) or N-Ac (3b)-6-aminocaproyl-D-Ala-d-Ser. Using an original microbiological approach to assess the competition of compounds with the natural target of vancomycin in bacteria, EC(50) values of 6.3-8.0 x 10(-5)M (2a-b) and 7.1-9.3 x 10(-4)M (3a-b) were determined. Vancomycin:2b complex was characterized by MS.
Abstract: OBJECTIVES: Does exposure to subinhibitory concentrations of quinolones favour overexpression of efflux pumps or selection of target site mutations? METHODS: ATCC 49,619 (fully susceptible) and SP32 (clinical isolate with PmrA-mediated efflux and mutation in ParE) were exposed for 24 h in broth to ciprofloxacin, levofloxacin, moxifloxacin or garenoxacin at concentrations of 0.5x the MIC, with daily re-adjustments for up to 13 days. Efflux was detected phenotypically (decrease in MIC in the presence of reserpine), and expression of pmrA and patA/patB was measured by real-time PCR and comparative RT-PCR, respectively. Target site mutations were detected by sequencing of the quinolone resistance determining regions in parC, parE and gyrA. The clonal identity of isolates was checked by PFGE of genomic DNA. RESULTS: Ciprofloxacin selected for stable mutants with 2.5-5-fold MIC increases for ciprofloxacin, 2-3-fold for levofloxacin and 1.3-2-fold for garenoxacin and moxifloxacin [partial reversion with reserpine for ciprofloxacin, gemifloxacin and levofloxacin (SP32 strain only), but not for garenoxacin and moxifloxacin]. Increased MICs were associated with overexpression of patA/B but not pmrA. In contrast, exposure to levofloxacin, moxifloxacin or garenoxacin selected target site mutations (gyrA, parC, parE) in both strains. Increases in MIC caused by efflux were similar to those caused by target site mutations. CONCLUSIONS: Exposure of Streptococcus pneumoniae to subinhibitory MICs of ciprofloxacin, a substrate for efflux pumps, results in patA/B-mediated efflux whatever the initial level of expression of pmrA of the strain. Quinolones that are poorly (levofloxacin) or not affected (moxifloxacin, garenoxacin) in their activity by efflux transporters preferentially select for target site mutants.
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.
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.
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.
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.
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.
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.
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.
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.
Abstract: OBJECTIVES: Assessment of the activity of three beta-lactams [ertapenem (a carbapenem with a prolonged half-life), meropenem and ampicillin] against intraphagocytic Listeria monocytogenes and Staphylococcus aureus. METHODS: Quantitative measurements of cfu changes in broth and in THP-1 macrophages (post-phagocytosis) over time (5 and 24 h) at concentrations spanning from sub-MICs to C(max) (maximal concentration typically observed in patients' serum upon administration of conventional doses); morphological studies using an electron microscope; evaluation of drug stability (HPLC), protein binding (equilibrium dialysis) and measurement of drug cellular accumulation (microbiological assay). RESULTS: Ertapenem was unable to control L. monocytogenes growth in THP-1 macrophages at all concentrations and times tested, even under conditions where ampicillin and meropenem were bactericidal. This behaviour could not be ascribed to drug instability, protein binding or lack of cell accumulation in comparison with ampicillin or meropenem. Ertapenem, ampicillin and meropenem were equally effective at reducing the post-phagocytosis inoculum of S. aureus ( approximately 1 log cfu), and caused conspicuous changes in the morphology of intracellular bacteria consistent with their lysis. These effects were obtained, however, only at large multiples (100-fold or more) of the MIC maintained over 24 h. Because of the high intrinsic antimicrobial potency of the beta-lactams studied, these concentrations were below the C(max). CONCLUSIONS: Ertapenem will probably be ineffective against intraphagocytic forms of L. monocytogenes for reasons that remain to be discovered. Conversely, ertapenem could be an alternative to ampicillin and meropenem against intraphagocytic S. aureus since its longer half-life may allow high concentrations to be maintained for more prolonged times.
Abstract: Ciprofloxacin is subject to efflux from J774 macrophages through a multidrug resistance-related protein-like transporter (J. M. Michot, F. Van Bambeke, M. P. Mingeot-Leclercq, and P. M. Tulkens, Antimicrob. Agents Chemother. 48:2673-2682, 2004). Here, we compare ciprofloxacin to levofloxacin, garenoxacin, and moxifloxacin for transport. At 4 mg/liter, an apparent steady state in accumulation was reached after 30 to 60 min for all quinolones but to quite different levels (approximately 3, 5, 10, and 16 fold). Accumulation of ciprofloxacin was increased (to about 16 to 20 fold) by ATP depletion, increase in extracellular concentration, and the addition of probenecid, gemfibrozil, or MK571 (but not verapamil or GF120918). These treatments did not affect the accumulation of moxifloxacin. Levofloxacin and garenoxacin showed an intermediate behavior. Efflux of ciprofloxacin was slowed down by probenecid (half-life, 7.2 versus 1.6 min). Moxifloxacin efflux was faster and unaffected by probenecid (half-lifes, 0.27 versus 0.33 min). Efflux of levofloxacin and garenoxacin was modestly decreased by probenecid (1.5 and 2.1 fold). Accumulation of 14C-labeled ciprofloxacin was increased by unlabeled ciprofloxacin and moxifloxacin, but moxifloxacin was two times less potent. Accumulation of moxifloxacin at 4 degrees C was almost identical to that at 37 degrees C, whereas that of ciprofloxacin was minimal (levofloxacin and garenoxacin showed intermediate behaviors). Cells subjected to thermal shock (56 degrees C; 10 min) accumulated all quinolones at a similar level (16 to 23 fold). We conclude that moxifloxacin is apparently not subject to efflux from J774 macrophages, even though it can interact with the ciprofloxacin transporter. Levofloxacin and garenoxacin are partially effluxed. Data suggest that efflux plays an important role in the differential accumulation of quinolones by J774 macrophages.
Abstract: Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubules and renal cell lines. Using LLC-PK1 cells, we have examined the concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM; 0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway; (iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive necrosis as assessed by the measurement of lactate dehydrogenase release). Within 2 h, gentamicin induced a partial relocalization [from lysosomes to cytosol] of the weak organic base acridine orange. We thereafter observed (a) a loss of mitochondrial membrane potential (as from 10 h, based on spectrophotometric and confocal microscopy using JC1 probe) and (b) the release of cytochrome c from granules to cytosol, and the activation of caspase-9 (as from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity (assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of calcein from negatively-charged liposomes at pH 5.4, which was slowed down by raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin (to mimic the inner mitochondrial membrane). The present data provide temporal evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration of the permeability of lysosomes, triggering of the mitochondrial pathway, and activation of caspase-3.
Abstract: Oritavancin, a semisynthetic derivative of vancomycin endowed with a cationic amphiphilic character, accumulates to large extent in the lysosomes of eukaryotic cells (F. Van Bambeke, S. Carryn, C. Seral, H. Chanteux, D. Tyteca, M. P. Mingeot-Leclercq, and P. M. Tulkens, Antimicrob. Agents Chemother. 48:2853-2860, 2004). In the present study, we examined whether this accumulation could cause cell alterations in phagocytic (J774 mouse macrophages) and nonphagocytic (rat embryo fibroblasts) cells exposed to clinically meaningful (0- to 40-mg/liter) concentrations of oritavancin. Optical and electronic microscopy evidenced conspicuous alterations of the vacuolar apparatus in both cell types, characterized by the deposition of concentric lamellar structures, finely granular material, or other less-defined osmiophilic material, often deposed in giant vesicles. Biochemical studies showed an accumulation of phospholipids (1.5 x control values) and free and esterified cholesterol (3 to 4 x control values for total cholesterol). Accumulation of these lipids was in close relation to that of oritavancin (excess phospholipid/oritavancin and excess cholesterol/oritavancin molar ratios of 2 to 3 and 3 to 5, respectively). Cholesterol accumulation was rapid and reversible, and that of phospholipids was slower and poorly reversible. Vancomycin and teicoplanin, used as controls (50 and 100 mg/liter, respectively), did not cause any significant change in the lipid content of fibroblasts. The data therefore suggest that oritavancin has the potential to cause a mixed-lipid storage disorder in eukaryotic cells.
Abstract: OBJECTIVES: Quinolones accumulate in eukaryotic cells and show activity against a large array of intracellular organisms, but systematic studies aimed at examining their pharmacodynamic profile against intracellular bacteria are scarce. The present work aims at comparing intracellular-to-extracellular activities in this context. METHODS: We assessed the activities of ciprofloxacin, levofloxacin, moxifloxacin and garenoxacin against the extracellular (broth) and intracellular (infected J774 macrophages) forms of Listeria monocytogenes (cytosolic infection) and Staphylococcus aureus (phagolysosomal infection) using a range of clinically meaningful extracellular concentrations (0.06-4 mg/L). RESULTS: All four quinolones displayed concentration-dependent bactericidal activity against extracellular and intracellular L. monocytogenes and S. aureus for extracellular concentrations in the range 1-4-fold their MIC. Compared at equipotent extracellular concentrations, intracellular activities against L. monocytogenes were roughly equal to those that were extracellular, but were 50-100 times lower against S. aureus. Because quinolones accumulate in cells (ciprofloxacin, approximately 3 times; levofloxacin, approximately 5 times; garenoxacin, approximately 10 times, moxifloxacin, approximately 13 times), these data show that, intracellularly, quinolones are 5-10 times less potent against L. monocytogenes (P=0.065 [ANCOVA]), and at least 100 times less potent (P < 0.0001) against S. aureus. Because of their lower MICs and higher accumulation levels, garenoxacin and moxifloxacin were, however, more active than ciprofloxacin and levofloxacin when compared at similar extracellular concentrations. CONCLUSIONS: Quinolone activity is reduced intracellulary. This suggests that either only a fraction of cell-associated quinolones exert an antibacterial effect, or that intracellular activity is defeated by the local environment, or that intracellular bacteria only poorly respond to the action of quinolones.
Abstract: Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1<phosphatidylinositol approximately phosphatidylserine approximately phosphatidylglycerol approximately phosphatidylpropanol<phosphatidic acid). For liposomes containing phosphatidylinositol, this increase of activity was not modified by the presence of phosphatidylethanolamine and enhanced by cholesterol only when the phosphatidylinositol content was lower than 18%. Our results, therefore show that both the surface-negative charge and the nature of the acidic lipid included in bilayers modulate the activity of phospholipase A1 towards phosphatidylcholine, while the change in lipid hydration or in fluidity of membrane are less critical. These observations may have physiological implications with respect to the rate of degradation of cellular membranes after their lysosomal segregation.
Abstract: OBJECTIVES: To explore the processes leading to inappropriate use of medicines for elderly patients admitted for acute care. DESIGN: Qualitative study with semistructured interviews with doctors, nurses, and pharmacists; focus groups with inpatients; and observation on the ward by clinical pharmacists for one month. SETTING: Five acute wards for care of the elderly in Belgium. PARTICIPANTS: 5 doctors, 4 nurses, and 3 pharmacists from five acute wards for the interviews; all professionals and patients on two acute wards for the observation and 17 patients (from the same two wards) for the focus groups. RESULTS: Several factors contributed to inappropriate prescribing, counselling, and transfer of information on medicines to primary care. Firstly, review of treatment was driven by acute considerations, the transfer of information on medicines from primary to secondary care was limited, and prescribing was often not tailored to elderly patients. Secondly, some doctors had a passive attitude towards learning: they thought it would take too long to find the information they needed about medicines and lacked self directed learning. Finally, a paternalistic doctor-patient relationship and difficulties in sharing decisions about treatment between prescribers led to inappropriate use of medicines. Several factors, such as the input of geriatricians and good communication between members of the multidisciplinary geriatric team, led to better use of medicines. CONCLUSIONS: In this setting, improvements targeted at the abilities of individuals, better doctor-patient and doctor-doctor relationships, and systems for transferring information between care settings will increase the appropriate use of medicines in elderly people.
Abstract: Quinolones are one of the largest classes of antimicrobial agents used worldwide. This review considers the quinolones that are available currently and used widely in Europe (norfoxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin) within their historical perspective, while trying to position them in the context of recent and possible future advances based on an understanding of: (1) their chemical structures and how these impact on activity and toxicity; (2) resistance mechanisms (mutations in target genes, efflux pumps); (3) their pharmacodynamic properties (AUC/MIC and Cmax/MIC ratios; mutant prevention concentration and mutant selection window); and (4) epidemiological considerations (risk of emergence of resistance, clonal spread). Their main indications are examined in relation to their advantages and drawbacks. Overall, it is concluded that these important agents should be used in an educated fashion, based on a careful balance between their ease of use and efficacy vs. the risk of emerging resistance and toxicity. However, there is now substantial evidence to support use of the most potent drug at the appropriate dose whenever this is required.
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.
Abstract: The accumulation and efflux kinetics of ciprofloxacin have been examined by using murine J774 macrophages. Accumulation (at equilibrium) was increased (three- to fourfold) (i) when cells were incubated with high extracellular drug concentrations (typically 200 mg/liter) as opposed to clinically meaningful concentrations (10 mg/liter or lower), (ii) during ATP- depletion and at acid pH, and (iii) during coincubation with probenecid, gemfibrozil and the preferential multidrug resistance-related protein (MRP) inhibitor MK571. All these conditions were also associated with a marked decrease in ciprofloxacin efflux (half-lives increased from <2 min in controls to up to 10 min). Monensin (a proton ionophore), verapamil, and the preferential P-glycoprotein (P-gp) inhibitor GF120918 had no or only minimal effect, while cyclosporin A, which is not specific for P-gp but also acts on MRP, had an intermediate effect. Short-term uptake studies showed that the influence of the modulators on the apparent drug influx was almost immediate (delay of < or =1 min). Cells made resistant to probenecid and showing a marked overexpression of MRP1 (by Western blot analysis and confocal microscopy) accumulated ciprofloxacin to almost the same extent as did control cells, but efflux was inhibited less by probenecid, gemfibrozil, and MK571. We conclude that ciprofloxacin is subject to constitutive efflux in J774 macrophages through the activity of an MRP-related transporter which is probably distinct from MRP1. We also suggest that the cellular accumulation of ciprofloxacin in wild-type cells is constitutively impaired at therapeutically meaningful concentrations.
Abstract: BACKGROUND: Listeria monocytogenes tends to survive in phagocytes. Granulocyte macrophage colony-stimulating factor (GM-CSF) protects mice against L. monocytogenes infection, and mice knocked out for the GM-CSF gene are more susceptible to these infections. METHODS: THP-1 cells were used to characterize the GM-CSF receptor (binding isotherms; STAT5 phosphorylation), measure the intracellular growth of L. monocytogenes (5 h after phagocytosis), examine the influence of a 24-h incubation with GM-CSF before infection, measure the production of tumor necrosis factor (TNF)-alpha and the expression of nitric oxide synthase (iNOS), and evaluate the influence of anti-GM-CSF receptor (GM-CSFR alpha ) and anti-TNF-alpha antibodies and the addition of N(omega)-nitro-L-arginine methyl ester (L-NAME) and catalase. RESULTS: THP-1 cells display functional GM-CSFR alpha. GM-CSF impairs the intracellular growth of L. monocytogenes to approximately 65% of its value in unstimulated cells. This effect is abolished by anti-GM-CSFR alpha, anti-TNF-alpha antibodies, and catalase (and, to a lesser extent, by L-NAME). GM-CSF stimulates the release of TNF-alpha and the expression of iNOS. TNF-alpha added to unstimulated cells (even in large amounts) does not fully reproduce the impairment in the growth of L. monocytogenes caused by GM-CSF. CONCLUSIONS: GM-CSF impairs the intracellular growth of L. monocytogenes by a synergistic action of the GM-CSF-triggered release of autocrine TNF-alpha and hydrogen peroxide and the production of NO (associated with the stimulation of the expression of iNOS).
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 microl x mg of protein(-1) x 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.
Abstract: Vancomycin and teicoplanin are still the only glycopeptide antibiotics available for use in humans. Emergence of resistance in enterococci and staphylococci has led to restriction of their use to severe infections caused by Gram-positive bacteria for which no other alternative is acceptable (because of resistance or allergy). In parallel, considerable efforts have been made to produce semisynthetic glycopeptides with improved pharmacokinetic and pharmacodynamic properties, and with activity towards resistant strains. Several molecules have now been obtained, helping to better delineate structure-activity relationships. Two are being currently evaluated for skin and soft tissue infections and are in phases II/III. The first, oritavancin (LY333328), is the 4'-chlorobiphenylmethyl derivative of chloroeremomycin, an analogue to vancomycin. It is characterised by: i) a spectrum covering vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and to some extent glycopeptide-intermediate S. aureus (GISA); ii) rapid bactericidal activity including against the intracellular forms of enterococci and staphylococci; and iii) a prolonged half-life, allowing for daily administration. The second molecule is dalbavancin (BI397), a derivative of the teicoplanin analogue A40926. Dalbavancin has a spectrum of activity similar to that of oritavancin against vancomycin-sensitive strains, but is not active against VRE. It can be administered once a week, based on its prolonged retention in the organism. Despite these remarkable properties, the use of these potent agents should be restricted to severe infections, as should the older glycopeptides, with an extension towards resistant or poorly sensitive bacteria, to limit the risk of potential selection of resistance.
Abstract: This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 microM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A1. At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in Vmax with identical Km. Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis.
Abstract: Antibiotic efflux pumps expressed in eukaryotic cells can decrease the intracellular accumulation of the corresponding drugs and therefore impair their activity against intracellular bacteria. We have investigated whether verapamil (an inhibitor of P-glycoprotein) and gemfibrozil (an inhibitor of multidrug resistance proteins (MRP) and other organic anion transporters), can modulate the intracellular activity of azithromycin and ciprofloxacin against Listeria monocytogenes and Staphylococcus aureus in J774 macrophages. In parallel, we have measured the cell accumulation and subcellular distribution of both drugs. Antibiotics were used at equipotent extracellular concentrations (from 0.5 x to 10 x MIC) to allow for pharmacological comparisons. Azithromycin was bacteriostatic against L. monocytogenes and slightly bactericidal against S. aureus. Verapamil did not improve the maximal activity of azithromycin but allowed it to reach a similar effect at extracellular concentrations about seven-fold lower in both models. Azithromycin was predominantly localized in cell granules (66%), the remainder being in the cytosol and in the 'nuclei/unbroken cells' fraction. Verapamil increased the cellular accumulation of azithromycin by almost 2.4-fold without modifying its subcellular distribution. Ciprofloxacin displayed a strong concentration-dependent bactericidal activity in both models. Gemfibrozil increased ciprofloxacin activity almost 2.5-fold against L. monocytogenes, but not against S. aureus. Ciprofloxacin was predominantly (65%) distributed in the cytosol. Gemfibrozil increased ciprofloxacin total accumulation by approximately 2.4-fold, but the excess was only found in the cytosol. Inhibition of efflux pumps may be a useful strategy to improve antibiotic efficacy against intracellular bacteria when increased accumulation can be obtained in the compartment where bacteria sojourn.
Abstract: PURPOSE: 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 degrees C as well as at 37 degrees C, saturation of uptake (apparent Kd 40 microM), no influence of monensin, MRP, or P-gp inhibitors; tight binding to liposomes (Kd approx. 40 microM); 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.
Abstract: The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/ phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.
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 10 micro 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.
Abstract: 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.
Abstract: AIMS: To determine the intracellular accumulation in a macrophage cell line of ampicillin and ampicillin esters, and to measure their activity against intracellular Listeria monocytogenes. METHODS: Quantitative evaluation of the activity of ampicillin, phthalimidomethylampicillin (PIMA) or pivaloyloxymethylampicillin (PIVA) against intracellular L. monocytogenes, and direct measurement of cellular ampicillin concentration in J774 macrophages. RESULTS: Ampicillin, PIMA and PIVA caused a 0.5 log decrease in cell-associated cfu within 5 h when used at an extracellular concentration of 3.6 microM [10 x MIC of ampicillin (1.25 mg/L); 1.83 mg/L for PIMA and 1.67 mg/L for PIVA]. Addition of beta-lactamase in the extracellular milieu abolished the activity of ampicillin and of PIMA but not that of PIVA. At low extracellular concentrations [0.5 x MIC ampicillin (62.5 microg/L); equimolar concentrations for PIMA (91.5 microg/L) and PIVA (83.5 microg/L)], ampicillin and PIMA lost all activity (compared with controls), but PIVA remained as active as at the higher concentration. Incubation of cells with PIVA at the low concentration (83.5 microg/L) for 20 h caused a 2 log reduction of cfu if the medium was changed every 5 h (to compensate for the degradation of extracellular PIVA). Incubation of cells with PIVA allowed for a marked (four- to 25-fold) cell accumulation of ampicillin, whereas no ampicillin accumulation was seen for cells incubated with ampicillin or with PIMA. CONCLUSIONS: This is the first demonstration that PIVA (a prodrug of ampicillin) can be used to promote ampicillin cellular accumulation and, thereby to increase ampicillin intracellular activity. PIVA could be useful for control of the intracellular multiplication of L. monocytogenes.
Abstract: Cefepime has been examined for stability, potential liberation of degradation products and compatibility with other drugs under conditions mimicking its potential use by continuous infusion in cystic fibrosis and intensive care patients (5-12% w/v solutions; temperatures from 20 to 37 degrees C; 1 h contact at 25 degrees C with other drugs frequently co-administered by intravenous route to these types of patients). Ceftazidime was used as a comparator based on a previous normative study with this antibiotic for the same indications. Based on a limit of max. 10% degradation, cefepime can be considered stable for a maximum of 24 h at 25 degrees C, but for only approximately 14 h at 30 degrees C, and for <10 h at 37 degrees C. Cefepime released so far unidentified degradation products if maintained at >30 degrees C for >12 h as shown from a marked increase in pH and from the development of a strong red-purple colour. Incompatibilities were observed with erythromycin, propofol, midazolam, phenytoin, piritramide, theophylline, nicardipine, N-acetylcysteine and a concentrated solution of dobutamine. We conclude that: (i) cefepime cannot be used safely by continuous infusion if containers are kept for more than a few hours at 37 degrees C (as will be the case for cystic fibrosis patients if using portable pumps carried under clothes); (ii) caution must be exercised in intensive care patients if the temperature and co-administration of other drugs is not kept under tight control. The nature and safety of the cefepime degradation products need to be studied further.
Abstract: Using J774 macrophages, the intracellular activities of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) against Staphylococcus aureus (strain ATCC 25923) have been quantitatively assessed in a 24-h model. S. aureus was positively localized in phagolysosomes by confocal and electron microscopy, and extracellular growth was prevented with 0.5 mg of gentamicin/liter (1x MIC) in controls. When tested at extracellular concentrations equivalent to their maximum concentrations in human serum, all antibiotics except azithromycin caused a significant reduction of the postphagocytosis inoculum within 24 h, albeit to markedly different extents (telithromycin [2 mg/liter], 0.60 log; ciprofloxacin [4.3 mg/liter], 0.81 log; gentamicin [18 mg/liter], 1.21 log; moxifloxacin [4 mg/liter], 1.51 log; oritavancin [25 mg/liter], 3.49 log). Intracellular activities were not systematically related to drug accumulation (apparent cellular-to-extracellular concentration ratios in infected cells: ciprofloxacin, 3.2; gentamicin, 6.8; telithromycin, 8.7; moxifloxacin, 13.4; azithromycin, 50; oritavancin, 348). Intracellular activity was not directly correlated to extracellular activity as measured in broth. Conditions of pH 5 (i.e., mimicking that of phagolysosomes) markedly reduced the activity of gentamicin, azithromycin, and telithromycin (>or=32 x) and fairly extensively reduced that of ciprofloxacin and moxifloxacin (>or=4 x) but did not affect oritavancin activity. We conclude that the cellular accumulation of antibiotics is not the only parameter to take into account for intracellular activity but that local environmental conditions (such as pH) and other factors can also prove critical.
Abstract: Pretreatment of J774 mouse macrophages by the dicationic macrolide antibiotic, azithromycin (AZ), selectively inhibited fluid-phase endocytosis of horseradish peroxidase and lucifer yellow, but not phagocytosis of latex beads. AZ delayed sequestration of receptor-bound transferrin and peroxidase-anti-peroxidase immune complexes into cell-surface endocytic pits and vesicles, but did not slow down the subsequent rate of receptor-mediated endocytosis. AZ down-regulated cell surface transferrin receptors, but not Fc gamma receptors, by causing a major delay in the accessibility of internalized transferrin receptors to the recycling route, without slowing down subsequent efflux, resulting in redistribution of the surface pool to an intracellular pool. Acidotropic accumulation of AZ was associated with an extensive vacuolation of late endosomes/lysosomes, and these compartments became inaccessible to horseradish peroxidase and immune complexes, but not to latex beads. The inhibitory profile of AZ cannot be solely accounted for by vacuolation and interference with acidification. AZ may help in dissecting various steps of the endocytic apparatus such as lateral mobility of receptors at the plasma membrane, formation of clathrin-independent endocytic vesicles, orientation of transferrin receptors into the recycling route, and fusogenicity with lysosomes.
Abstract: The activities of ampicillin, meropenem, azithromycin, gentamicin, ciprofloxacin, and moxifloxacin against intracellular hemolysin-positive Listeria monocytogenes were measured in human THP-1 macrophages and were compared with the extracellular activities observed in broth. All extracellular concentrations were adjusted to explore ranges that are clinically achievable in human serum upon conventional therapy. In broth, ampicillin, meropenem, and azithromycin were only bacteriostatic, whereas gentamicin, ciprofloxacin, and moxifloxacin were strongly bactericidal in a concentration-dependent manner. In cells, ampicillin, meropenem, azithromycin, and ciprofloxacin were slightly bactericidal (0.3- to 0.8-log CFU reductions), moxifloxacin was strongly bactericidal (2.1-log CFU reduction), and gentamicin was virtually inactive. The difference in the efficacies of moxifloxacin and ciprofloxacin in cells did not result from a difference in levels of accumulation in cells (6.96 +/- 1.05 versus 7.75 +/- 1.03) and was only partially explainable by the difference in the MICs (0.58 +/- 0.04 versus 1.40 +/- 0.17 mg/liter). Further analysis showed that intracellular moxifloxacin expressed only approximately 1/7 of the activity demonstrated against extracellular bacteria and ciprofloxacin expressed only 1/15 of the activity demonstrated against extracellular bacteria. Gentamicin did not increase the intracellular activities of the other antibiotics tested. The data suggest (i) that moxifloxacin could be of potential interest for eradication of the intracellular forms of L. monocytogenes, (ii) that the cellular accumulation of an antibiotic is not the only determinant of its intracellular activity (for fluoroquinolones, it is actually a self-defeating process as far as activity is concerned), and (iii) that pharmacodynamics (activity-to-concentration relationships) need to be considered for the establishment of efficacy against intracellular bacteria, just as they are for the establishment of efficacy against extracellular infections.
Abstract: The stability of antipseudomonal beta-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 degrees C (mezlocillin [128 g/liter] was stable at 25 degrees C but not at 37 degrees 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 degrees C but only for 8, 7.25, and 13 h at 37 degrees 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 degrees C or higher. Degradation of ceftazidime was accompanied by the liberation of pyridine which, at 37 degrees 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 degrees 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.
Abstract: The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.
Abstract: The stability and compatibility of ceftazidime have been examined in the context of its potential use in concentrated solutions for continuous infusion in patients suffering from severe nosocomial pneumonia and receiving other intravenous medications by the same route. Ceftazidime stability in 4 to 12% solutions was found satisfactory (<10% degradation) for 24 h if kept at a temperature of 25 degrees C (77 degrees F) maximum. Studies mimicking the simultaneous administration of ceftazidime and other drugs as done in clinics showed physical incompatibilities with vancomycin, nicardipine, midazolam, and propofol and a chemical incompatibility with N-acetylcystein. Concentrated solutions (50 mg/ml) of erythromycin or clarithromycin caused the appearance of a precipitate, whereas gentamicin, tobramycin, amikacin, isepamicin, fluconazole, ketamine, sufentanil, valproic acid, furosemide, uradipil, and a standard amino acid solution were physically and chemically compatible.
Abstract: Three pharmacokinetic/pharmacodynamic parameters--(i) the peak concentration to the minimum inhibitory concentration ratio (C(max)/MIC); (ii) the area under the concentration-time curve to MIC ratio (AUC(24h)/MIC); and (iii) the time the concentration exceeds the MIC (T>MIC)--are important predictors of the clinical efficacy of antibiotics. For antibiotics with pronounced concentration-dependent killing, such as the fluoroquinolones or the aminoglycosides, C(max)/MIC and AUC(24)/MIC are the main factors that establish efficacy. Antibiotics with a weak, or no, concentration dependency generally have their efficacy linked to T>MIC, and these include the beta-lactams and the conventional macrolides. Antibiotics with weak concentration-dependent effects, but with prolonged persistent effects, such as tetracyclines and azithromycin, have their activity mostly related to the AUC(24)/MIC. By applying these concepts to current antibiotics, and also to the development of novel agents, it is possible to optimise their dosages and administration schedules. This will maximise therapeutic efficacy, may prevent or delay the emergence of bacterial resistance to antibiotics, and can certainly minimise side-effects.
Abstract: Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.
Abstract: Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.
Abstract: Kidney cortex apoptosis was studied with female Wistar rats treated for 10 days with gentamicin and netilmicin at daily doses of 10 or 20 mg/kg of body weight and amikacin or isepamicin at daily doses of 40 mg/kg. Apoptosis was detected and quantitated using cytological (methyl green-pyronine) and immunohistochemical (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining, in parallel with a measurement of drug-induced phospholipidosis (cortical phospholipids and phospholipiduria), cortical proliferative response ((3)H incorporation in DNA and histoautoradiography after in vivo pulse-labeling with [(3)H]thymidine), and kidney dysfunction (blood urea nitrogen and creatinine). Gentamicin induced in proximal tubules a marked apoptotic reaction which (i) was detectable after 4 days of treatment but was most conspicuous after 10 days, (ii) was dose dependent, (iii) occurred in the absence of necrosis, and (iv) was nonlinearly correlated with the proliferative response (tubular and peritubular cells). Comparative studies revealed a parallelism among the extents of phospholipidosis, apoptosis, and proliferative response for three aminoglycosides (gentamicin >> amikacin congruent with isepamicin). By contrast, netilmicin induced a marked phospholipidosis but a moderate apoptosis and proliferative response. We conclude that rats treated with gentamicin develop an apoptotic process as part of the various cortical alterations induced by this antibiotic at low doses. Netilmicin, and still more amikacin and isepamicin, appears safer in this respect. Whereas a relation between aminoglycoside-induced tubular apoptosis and cortical proliferative response seems to be established, no simple correlation with phospholipidosis can be drawn.
Abstract: We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by 31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH <6.5, (iii) an increase in the mean diameter of liposomes for pH <6.0, and (iv) a mixing of liposome membranes for pH <5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5-7.0 to 6.5-6.0 and (ii) a release of approximately 15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellularly after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.
Abstract: Active efflux from procaryotic as well as eucaryotic cells strongly modulates the activity of a large number of antibiotics. Effective antibiotic transport has now been observed for many classes of drug efflux pumps. Thus, within the group of primary active transporters, predominant in eucaryotes, six families belonging to the ATP-binding cassette superfamily, and including the P-glycoprotein in the MDR (Multi Drug Resistance) group and the MRP (Multidrug Resistance Protein), have been recognized as being responsible for antibiotic efflux. Within the class of secondary active transporters (antiports, symports, and uniports), ten families of antibiotic efflux pumps have been described, distributed in five superfamilies [SMR (Small Multidrug Resistance), MET (Multidrug Endosomal Transporter), MAR (Multi Antimicrobial Resistance), RND (Resistance Nodulation Division), and MFS (Major Facilitator Superfamily)]. Nowadays antibiotic efflux pumps are believed to contribute significantly to acquired bacterial resistance because of the very broad variety of substrates they recognize, their expression in important pathogens, and their cooperation with other mechanisms of resistance. Their presence also explains high-level intrinsic resistances found in specific organisms. Stable mutations in regulatory genes can produce phenotypes of irreversible multidrug resistance. In eucaryotes, antibiotic efflux pumps modulate the accumulation of antimicrobials in phagocytic cells and play major roles in their transepithelial transport. The existence of antibiotic efflux pumps, and their impact on therapy, must now be taken fully into account for the selection of novel antimicrobials. The design of specific, potent inhibitors appears to be an important goal for the improved control of infectious diseases in the near future.
Abstract: A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was produced using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template. The model passed critical validation criteria with an accuracy similar to that of the template crystallographic structure and showed that ADP and methylphosphinophosphate were positioned in a large empty pocket at the interface between the central and the C-terminal domains, as in E. coli. It evidenced the residues important for substrate binding and catalytic activity in the active site and demonstrated a large body of conserved interactions between the active sites of the E. faecalis and the E. coli D-Ala:D-Ala ligase, the major differences residing in the balance between the hydrophobic and aromatic environment of the adenine. The model also successfully explained the inactivity of four spontaneous mutants (D295 --> V, which impairs interactions with Mg2+ and R293, which are both essential for binding and catalytic activity; S319 --> I, which perturbs recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in the vicinity of the deletion remains unaltered but phosphate transfer from ATP is perturbed because of lack of K253; T316 --> I, which causes the loss of a hydrogen bond affecting the positioning of S319 and therefore the binding of D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this approach, combining molecular modeling and molecular biology, may help in the design of specific ligands which could inhibit the enzyme and serve as novel antibiotics.
Abstract: Gentamicin, an aminoglycoside antibiotic, induces apoptosis in the proximal tubule epithelium of rats treated at low, therapeutically relevant doses (El Mouedden et al., Antimicrob. Agents Chemother. 44, 665-675, 2000). Renal cell lines (LLC-PK(1) and MDCK-cells) have been used to further characterize and quantitate this process (electron microscopy; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling of fragmented DNA [TUNEL]; and DNA size analysis [oligonucleosomal laddering]). Cells were exposed for up to 4 days to gentamicin concentrations of up to 3 mM. Apoptosis developed, almost linearly, with time and drug concentration, and was (i) preventable within the time-frame of the experiments by overexpression of the anti-apoptotic protein Bcl-2, and by co-incubation with cycloheximide (MDKC but not LLC-PK(1) cells); (ii) associated with an increased activity of caspases (MDCK cells; bcl-2 transfectants showed no increase of caspase activities and Z-VAD.fmk afforded full protection). Gentamicin-induced apoptosis also developed to a similar extent in embryonic fibroblasts cultured under the same conditions. In the 3 cell types, apoptosis (measured after 4 days) was directly correlated with cell gentamicin content (apoptotic index [approximately 10 to 18% of TUNEL (+) cells for a content of 20 microg of gentamicin/mg protein; kidney cortex of rats showing apoptosis in proximal tubule epithelium typically contains approximately 10 microg of gentamicin/mg protein). Thus, gentamicin has an intrinsic capability of inducing apoptosis in eucaryotic cells. Development of apoptosis in proximal tubules of kidney cortex in vivo after gentamicin systemic administration is therefore probably related to its capacity to concentrate in this epithelium after systemic administration.
Abstract: BACKGROUND: Saccharomyces boulardii is a non-pathogenic yeast which exerts trophic effects on human and rat small intestinal mucosa. AIMS: To examine the effects of S boulardii on ileal adaptation after proximal enterectomy in rats. METHODS: Wistar rats, aged eight weeks, underwent 60% proximal resection or transection and received by orogastric intubation either 1 mg/g body wt per day lyophilised S boulardii or the vehicle for seven days. The effects on ileal mucosal adaptation were assessed eight days after surgery. RESULTS: Compared with transection, resection resulted in mucosal hyperplasia with significant decreases in the specific and total activities of sucrase, lactase, and maltase. Treatment of resected animals with S boulardii had no effect on mucosal hyperplasia but did upgrade disaccharidase activities to the levels of the transected group. Enzyme stimulation by S boulardii was associated with significant increases in diamine oxidase activity and mucosal polyamine concentrations. Likewise, sodium dependent D-glucose uptake by brush border membrane vesicles, measured as a function of time and glucose concentration in the incubation medium, was significantly (p<0.05) increased by 81% and three times respectively in the resected group treated with S boulardii. In agreement with this, expression of the sodium/glucose cotransporter-1 in brush border membranes of resected rats treated with S boulardii was enhanced twofold compared with resected controls. CONCLUSION: Oral administration of S boulardii soon after proximal enterectomy improves functional adaptation of the remnant ileum.
Abstract: The influence of interferon (IFN)-gamma and interleukin (IL)-6 on the intracellular growth of Listeria monocytogenes phagocytosed from the apical pole was examined in polarized Caco-2 cells. IFN-gamma (from the apical pole) and IL-6 (from the basolateral pole) considerably reduced the bacterial intracellular growth, an effect largely abolished by l-monomethyl arginine. Both cytokines caused overexpression of inducible nitric oxide synthase. IL-6, but not IFN-gamma, caused a partial restriction of L. monocytogenes in phagosomes and largely prevented the cytosolic forms from being surrounded by actin. Ampicillin was bacteriostatic in unstimulated cells but modestly bactericidal in cells treated with IFN-gamma and IL-6. Azithromycin (a macrolide) was fairly bactericidal and sparfloxacin (a fluoroquinolone) highly bactericidal in all situations. IFN-gamma and IL-6 may therefore be important determinants in the protection of epithelial cells from intracellular multiplication of L. monocytogenes. Ampicillin may fail in their absence, requiring the use of other antibiotics such as the fluoroquinolones.
Abstract: Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of L-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225-1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by L-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of L-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection.
Abstract: The potential of 14/15 membered macrolides to cause phospholipidosis has been prospectively assessed, and structure-effects examined, using combined experimental and conformational approaches. Biochemical studies demonstrated drug binding to phosphatidylinositol-containing liposomes and inhibition of the activity of lysosomal phospholipase A1 toward phosphatidylcholine included in the bilayer, in close correlation with the number of cationic groups carried by the drugs (erythromycin A </= roxithromycin < erythromycylamine </= azithromycin). In cultured cells (fibroblasts), phospholipidosis (affecting all major phospholipids except sphingomyelin) was observed after 3 days with the following ranking: erythromycin A </= roxithromycin < erythromycylamine < azithromycin (roxithromycin could, however, not be studied in detail due to intrinsic toxicity). The difference between erythromycylamine and azithromycin was accounted for by the lower cellular accumulation of erythromycylamine. In parallel, based on a methodology developed and validated to study drug-membrane interactions, the conformational analyses revealed that erythromycin A, roxithromycin, erythromycylamine, and azithromycin penetrate into the hydrophobic domain of a phosphatidylinositol monolayer through their desosamine and cladinose moieties, whereas their macrocycle is found close to the interface. This position allows the aminogroups carried by the macrocycle of the diaminated macrolides (erythromycylamine and azithromycin) to come into close contact with the negatively charged phosphogroup of phosphatidylinositol, whereas the amine located on the C-3 of the desosamine, common to all four drugs, is located at a greater distance from this phosphogroup. Our study suggests that all macrolides have the potential to cause phospholipidosis but that this effect is modulated by toxicodynamic and toxicokinetic parameters related to the drug structure and mainly to their cationic character.
Abstract: Computer-aided simulations suggest that the doses and schedules of administration of azithromycin proposed in treatment and prophylaxis of Mycobacterium avium complex (MAC) in AIDS patients will result in drug concentrations in serum and extracellular fluids remaining for sustained periods of time in the 0.03-0.1 mg/L range. We exposed cultured rat embryo fibroblasts to these concentrations (and multiples up to 20 mg/L) for up to 16 days. Electron microscopy showed that after 7 days' incubation in 0.03 mg/L azithromycin, there was conspicuous accumulation of osmiophilic, lamellar structures (myeloid bodies) in lysosomes, suggesting the onset of a phospholipidosis. Assay of total cell phospholipids and cholesterol showed significant increases in cells exposed to > or = 1 to 5 mg/L of azithromycin in association with hyperactivity of the lysosomal enzyme cathepsin B. The data suggest that azithromycin, at extracellular concentrations pertinent to its use for MAC treatment, and perhaps also prophylaxis, causes limited morphological alterations of the lysosomes in cultured cells which are of the same nature as those developing rapidly and extensively at higher concentrations.
Abstract: The relative in-vivo intracellular concentration of various macrolides in phagocytes cannot be directly extrapolated from in-vitro experiments that use a fixed and constant extracellular concentration for all compounds, since this fails to consider different rates of intracellular penetration, dosage regimens and pharmacokinetic data. In the proposed model, which takes into account the free plasma concentrations and accumulation characteristics of three antibiotics, roxithromycin, azithromycin and erythromycin, we show that roxithromycin and azithromycin may reach similar concentrations in human polymorphonuclear leucocytes when conditions mimic clinical administration of these drugs, while erythromycin concentrations are lower. This approach may be useful to predict the behaviour of other drugs or other cells, and to assist in the design of rational treatment schemes.
Abstract: Renal damage caused by polycationic peptides is well documented, but renal damage caused by polyanionic peptides is not. During our attempts to inhibit the nephrotoxicity of aminoglycoside antibiotics by polyanionic peptides, we discovered that poly-D-glutamic acid (molecular weight, 20 kd; 250 mg/kg/day subcutaneously for 1 to 4 days) produces an acute thesaurismosis in the proximal tubular cells associated with a marked proliferation of peritubular interstitial cells in rat kidney. Thesaurismotic bodies were easily visualized by light microscopy at the basal pole of proximal tubular cells with the cationic stain Giemsa. By electron microscopy, these bodies appeared membrane-limited, frequently distorted, filled with heterogeneous granular material, accessible to injected peroxidase (a tracer of the endocytic pathway), and generally stainable for the lysosomal enzyme arylsulfatase. Specimens obtained 3 hours after injection of poly-D-glutamic acid and horseradish peroxidase suggested an impairment of endosome and/or lysosome fission, but not fusion. By histoautoradiographic examination after 3H-thymidine incorporation, global labeling indices of cortical cells were increased 11- to 18-fold in poly-D-glutamic acid-treated rats as compared with controls, with > 80% of labeled cells localized in the interstitium. Distal tubular and glomerular cells also showed a moderate proliferation, but proximal tubular cells showed no significant necrosis or proliferation. Although tubular thesaurismosis persisted, interstitial cell proliferation resolved within 7 days after cessation of treatment. We suggest that poly-D-glutamic acid is a convenient tool to induce a rapid and sustained lysosomal storage disorder. It could also help clarify the relationship between insults to tubular cells and proliferation of peritubular cells, two features frequently associated in tubulointerstitial disorders. The mechanism of the thesaurismosis and of the interference with the dynamics of fusion-fission of the endocytic apparatus are addressed in the companion paper.
Abstract: In the companion paper, we report that a single injection of poly-D-glutamic acid causes an acute lysosomal storage condition and apparently impairs the lysosomal fission dynamics. The present paper addresses the mechanisms of these two alterations using a combination of in vivo and in vitro biochemical approaches. After a single intravenous injection, 14C-poly-D-glutamic acid was rapidly cleared from the plasma and appeared in the urine. Yet, a small but sizable fraction of the injected polymer was taken up by the kidney cortex through a saturable process (Kuptake, 150 mg/kg body wt; uptakemax 96 micrograms/g cortex). Analytical subcellular fractionation of cortex homogenates demonstrated that at initial stages, the 14C label was predominantly associated with subcellular particles of intermediate size and low equilibrium density, and was therefore slowly transferred to larger particles equilibrating at high density, then codistributing with the lysosomal hydrolases. At a concentration of 10 mg/ml (equivalent to its estimated concentration in lysosomes), poly-D-glutamic acid formed micronic aggregates ( > or = 10 microns) when brought to solution at pH < or = 6 in relation to its decreased ionization (pKa of lateral chains approximately equal to 4.25). Finally, 1 day after the injection of poly-D-glutamic acid, the activities of several lysosomal enzymes (hexosaminidase, cathepsin B, acid sphingomyelinase, and sulfatase B), but not of all of them (eg, acid phosphatase), were increased in the kidney cortex. We propose that poly-D-glutamic acid reaches lysosomes by adsorptive endocytosis and becomes concentrated within these organelles because its withstands hydrolysis until it forms aggregates or precipitates, causing a decrease in the fluidity or the deformability ("gelling") of the lysosomal matrix. This should alter the dynamics of intercommunication of these organelles by impairing their fission without a proportionate effect on their fusion properties. In addition, the data suggest that the presence of poly-D-glutamic acid directly or indirectly slows down the degradation of several lysosomal enzymes.
Abstract: Listeria monocytogenes is a facultative intracellular pathogen which enters cells by endocytosis and reaches phagolysosomes from where it escapes and multiplies in the cytosol of untreated cells. Exposure of macrophages to gamma interferon (IFN-gamma) restricts L. monocytogenes to phagosomes and prevents its intracellular multiplication. We have tested whether IFN-gamma also modulates the susceptibility of L. monocytogenes to antibiotics. We selected drugs from three different classes displaying marked properties concerning their cellular accumulation and subcellular distribution, namely, ampicillin (not accumulated by cells but present in cytosol), azithromycin (largely accumulated by cells but mostly restricted to lysosomes), and sparfloxacin (accumulated to a fair extent but detected only in cytosol). We used a continuous line of myelomonocytic cells (THP-1 macrophages), which display specific surface receptors for IFN-gamma, and examined the activity of these antibiotics against L. monocytogenes Hly+ (virulent variant) and L. monocytogenes Hly- (a nonvirulent variant defective in hemolysin production). Untreated THP-1 and phorbol myristate acetate-differentiated THP-1 were permissive for infection and multiplication of intracellular L. monocytogenes Hly+ (virulent variant). All three antibiotics tested were bactericidal against this Listeria strain when added to an extracellular concentration of 10x their MIC. After preexposure of THP-1 to IFN-gamma, L. monocytogenes Hly+ was still phagocytosed but no longer grew intracellularly. The activity of ampicillin became almost undetectable (antagonistic effect), and that of azithromycin was unchanged (additive effect with that of IFN-gamma), whereas that of sparfloxacin was markedly enhanced (synergy). A similar behavior (lack of bacterial growth, associated with a loss of activity of ampicillin, an enhanced activity of sparfloxacin, and unchanged activity of azithromycin) was observed in cells infected with L. monocytogenes Hly-. This modulation of antibiotic activity, which we ascribe to the change of subcellular localization of L. monocytogenes caused by IFN-gamma or by the lack of virulence factor, could result from a change in bacterial responsiveness to antibiotics, a modification of the drug activity, or differences in drug bioavailabilities between cytosol and phagosomes.
Abstract: Azithromycin accumulates in lysosomes where it causes phospholipidosis. In homogenates prepared by sonication of fibroblasts incubated for 3 days with azithromycin (66 microM), the activities of sulfatase A, phospholipase A1, N-acetyl-beta-hexosaminidase and cathepsin B increased from 180 to 330%, but not those of 3 non-lysosomal enzymes. The level of cathepsin B mRNA was unaffected. The hyperactivity induced by azithromycin is non-reversible upon drug withdrawal, prevented by coincubation with cycloheximide, affects the Vmax but not the Km, and is not reproduced with gentamicin, another drug also causing lysosomal phospholipidosis. The data therefore suggest that azithromycin increases the level of lysosomal enzymes by a mechanism distinct from the stimulation of gene expression but requiring protein synthesis, and is not in direct relation to the lysosomal phospholipidosis.
Abstract: Azithromycin, the first clinically developed dicationic macrolide antibiotic, displays an exceptional accumulation in lysosomes of cultured cells. In fibroblasts incubated with 50 mg/l (66.6 microM), it induces a distinct phospholipidosis as evidenced by biochemical and ultrastructural criteria, which strikingly resembles alterations described previously with gentamicin, a pentacationic aminoglycoside antibiotic which inhibits the lysosomal catabolism of phospholipids. We show that both drugs inhibit, in an equimolar manner, the activity of phospholipase A1 (assayed for phosphatidylcholine, included in negatively charged liposomes), in a way consistent with the model of 'charge neutralization' proposed already for gentamicin (Mingeot-Leclercq et al., 1988, Biochem. Pharmacol. 37, 591). Both drugs bind to negatively charged liposomes. Yet, in spite of this binding, azithromycin does not induce aggregation or fusion of negatively charged vesicles, under conditions in which gentamicin (or spermine, a fully hydrophilic polycation) causes a massive aggregation, and bis(beta-diethylaminoethylether)hexestrol (a dicationic amphiphile) causes fusion. The molecular interactions of azithromycin with acidic phospholipids are further examined in a companion paper.
Abstract: In a comparison paper, we show the azithromycin causes a lysosomal phospholipidosis in cultured cells, binds in vitro to negatively charged bilayers without causing aggregation or fusion, and inhibits lysosomal phospholipase A1. In this paper, we show that azithromycin decreases the mobility of the phospholipids in negatively charged liposomes (using 31P nuclear magnetic resonance) and that it increases the fluidity of the acyl chains close to the hydrophilic/hydrophobic interface, but not deeper into the hydrophobic domain (assessed by measuring the fluorescence polarization of trimethylammonium-diphenylhexatriene and diphenyhexatriene, respectively). Computer-aided conformational analysis of mixed monolayers of azithromycin and phosphatidylinositol shows that the drug can be positioned largely in the hydrophobic domain, but close to the interface, with the macrocycle facing the C1 of the fatty acids (allowing the N9a endocyclic tertiary amine to interact with the phospho-groups), the cladinose located on the hydrophobic side of the lipid/water interface and the desosamine projected into the hydrophobic domain. This position is consistent with the experimental data. Analysis of virtual molecules shows that this unanticipated behavior to the shielding of the ionizable N3' amino-group in the desosamine by methyl-groups, and to the wide dispersion of hydrophobic domains all over the molecule. The interaction of azithromycin with phospholipids may account for some of its unusual pharmacokinetic properties and for its potential to cause lysosomal phospholipidosis.
Abstract: Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.
Abstract: Amino acid and peptide derivatives of aminoglycosides have been obtained by substitution of the 1-N or 6'-N amino functions of kanamycin A and netilmicin via the temporary complexation of vicinal and nonvicinal amino and hydroxy functions by copper ion [1-N kanamycin A derivatives: L-Ala (6a), D-Ala (6b), Gly (6c), L-Asp (6d), L-Ala-L-Ala (6e). 6'-N kanamycin A derivatives: L-Ala (3a), D-Ala (3b), Gly (3c), L-Ala-L-Ala (3e), L-Leu (3f). 6'-N netilmicin derivatives: L-Ala (9a), D-Ala (9b), Gly (9c), L-Asp (9d), L-Ala-L-Ala (9e)]. Characterization was made by FAB-MS, IR, 1H-NMR, and 13C-NMR. All derivatives were essentially inactive. The nephrotoxic potential of the derivatives obtained in sufficient quantities (3b,e and 9a-e) was assessed by measuring their inhibitory potential toward the activity of lysosomal phospholipase A1 acting on phosphatidylcholine embedded in negatively-charged membranes. One compound, 6'-N-L-Ala-netilmicin (9a), showed a 2-fold decrease of inhibitory potency compared to its parent drug. A conformational analysis revealed that it adopts two equally probable conformations and orientations when interacting with phosphatidylinositol. The first in which the drug lies parallel to the hydrophobic-hydrophilic interface, is similar to that of netilmicin. The second, in which the drug inserts itself in the bilayer across the hydrophilic/hydrophobic interface, is similar to that described for streptomycin, an almost non-nephrotoxic aminoglycoside.
Abstract: The binding of aminoglycoside antibiotics to acidic phospholipids of membranes is an essential step in the development of both their renal and auditory toxicities, which could be associated with critical modifications of the membrane properties. This work examines the capacity of aminoglycosides to induce membrane aggregation and fusion. Three techniques were used in parallel: (i) measurement of the dequenching rate of a lipid-soluble fluorescent probe (octadecylrhodamine B) incorporated at self-quenched concentration in membranes; (ii) measurement of the increase in the energy transfer between two fluorescent derivatives of phospholipids; and (iii) electron microscopy of negatively-stained replicas. The results were compared with those obtained with spermine (an aggregating polycation) and melittin (a fusogenic peptide). The three approaches indicate that aminoglycosides induce liposomes aggregation, but not fusion. Aggregation is related to the capacity of each drug studied to bind phosphatidylinositol, as evaluated by its energy of interaction with this acidic phospholipid, and to its toxic potential. Membrane aggregation occurring in vivo could therefore contribute to, or be a determinant of this toxicity, which could rationally be screened for new derivatives by the methods applied here.
Abstract: Aminoglycosides, among the most commonly used antibiotics in neonates, have frequently been implicated in nephrotoxic reaction. Studies in adults have indicated that phospholipiduria (PLU) is rapidly increased during aminoglycoside therapy, in relation to the renal phospholipidosis these drugs are known to induce in renal cortex. We studied the effect of amikacin (AK) on PLU in male prematurely-born neonates (gestational age > 34 weeks; postnatal age < or = 2 days) by assessing the urinary excretion of 4 enzymes (N-acetyl-beta-D-glucosaminidase [NAG], alkaline phosphatase, tau-glutamyltransferase and alanine aminopeptidase) and 4 low-molecular-weight proteins (beta-2-microglobulin, clara cell protein, microalbumin and retinol-binding protein) which are currently used to monitor the development and extent of renal tubular damage. Twenty-two patients and 8 healthy (as control) neonates were enrolled in the study. Patients were treated with AK (15 mg/kg per day) given in one (qd, n = 10) or two equal injections (b.i.d., n = 12) for durations of 7-11 days. PLU and proteinuria were determined in 24-h urine sample collections, and enzymes were assessed in spot urine collected at 9 a.m. We found that in neonates, AK causes a significant increase in PLU, and in enzymuria except for NAG in the qd group. Proteinuria showed no significant change due to AK treatment. No significant differences were observed between qd and b.i.d. administrations of AK for all parameters tested. We conclude that PLU could be used in neonates as well as in adults as a non-invasive method to monitor the development of the renal phospholipidosis during aminoglycoside therapy.
Abstract: Leupeptin is an established, reversible inhibitor of cathepsin B, a lysosomal cysteine proteinase. Yet, in rat fibroblasts as well as in foetal mouse calvaria, we observed an increase of the activity of cathepsin B in homogenates of cells and tissue harvested after culture in the presence of leupeptin. This effect was also seen for other lysosomal hydrolases, namely sphingomyelinase, N-acetyl-beta-glucosaminidase, arylsulphatase A and phospholipase A1 in fibroblasts, and beta-glucuronidase in mouse calvaria. In calvaria, antipain, another reversible cysteine proteinase inhibitor, caused a similar effect, whereas E-64, an irreversible inhibitor, was consistently inhibitory of the cathepsin B activity; yet it also caused an increase of beta-glucuronidase activity. The effect of leupeptin in fibroblasts was dose and time-dependent, required the continuous presence of the inhibitor, and was not dependent from protein synthesis. Actually, addition of cycloheximide caused a severe loss of activity of cathepsin B and of sphingomyelinase. In the presence of both cycloheximide and leupeptin, however, these two activities were retained to a value corresponding to that found in excess in cells cultivated with leupeptin alone. The data therefore suggests that leupeptin exerts the effects described in this paper by preventing the degradation of cathepsin B, sphingomyelinase and probably several other lysosomal hydrolases by cysteine proteinases. We therefore propose that cysteine proteinases play a key role in the control of the steady-state levels of these enzymes in normal conditions.
Abstract: Aminoglycoside antibiotics, such as gentamicin, cause an early lysosomal phospholipidosis in the renal cortex, which is considered as a key event in the onset of acute tubular necrosis induced by these drugs. In a model of primary cultures of embryonic rat fibroblasts which develop typical lysosomal phospholipidosis when incubated with gentamicin (decrease of sphingomyelinase activity; increase in total cells lipid phosphorus; appearance of so-called 'myeloid bodies' in lysosomes), we observed a protective effect exerted by inhibitors of cysteine proteinases (leupeptin, E-64) against this alteration on the basis of both biochemical and morphological criteria. Actually leupeptin and E-64 caused a marked stimulation of sphingomyelinase activity both in control and in gentamicin-treated cells, which we suggest to be the cause of protection.
Abstract: The authors have examined the pharmacokinetic parameters of azithromycin in phagocytic (J774 macrophages) and non-phagocytic (rat embryo fibroblasts and NRK-cells) cultured cells. Azithromycin demonstrates an exceptionally large accumulation in all the cell types tested (perhaps in two functionally and structurally distinct compartments) and a slow release of the cell-associated drug. Azithromycin probably accumulates in cells by a non-specific transport process following the model of diffusion/segregation. The cell-associated drug distributes mostly in the lysosomal compartment (50-70%) and the remaining part is freely soluble in the cytosol. In fibroblasts, and to a lesser extent in NRK-cells, azithromycin (10mg/l) induces a decrease of the buoyant density of the lysosomes which may be brought about by the drug itself together with osmotically-bound water and/or by the accumulation of low-density materials within these organelles. These observations open important questions with respect to the potential toxicity of azithromycin. The significance of such alterations and of their biological consequences are at present under investigation.
Abstract: Aminoglycoside antibiotics bind to negatively-charged membranes in vitro as well as in vivo. We have examined if this binding could be associated with a change in the properties of membrane permeability. We have used a series of aminoglycoside derivatives and two independent test systems, namely (i) the release of calcein and of Mn2+ from phosphatidylinositol-containing large unilamellar vesicles, and (ii) the influx of Ca2+ into cultured macrophages. We found that certain aminoglycosides (e.g., streptomycin, isepamicin) markedly increase the membrane permeability whereas others (e.g., gentamicin) barely or do not influence it. This increase, when it occurs, is slower or less extensive than observed with pore-forming agents (mellitin, nystatin) or a Ca(2+)-ionophore (ionomycin). It is not observed with an agent [bis(beta-diethylaminoethylether)hexestrol] known to cause membrane fusion, and is not associated with any detectable change in membrane fluidity. In computer-aided conformational analysis of mixed monolayers between phosphatidylinositol and the aminoglycosides studied, it was found that those derivatives inducing an increase in membrane permeability in our experiments adopted an orientation rather perpendicular to the interface, whereas those with no or only a moderate effect were placed in a parallel orientation to this interface. The perpendicular orientation might cause a local condition of disorder which could explain the effects observed.
Abstract: Neonates, especially preterms, are known to have low glomerular filtration rates (GFR). This may result in elevated trough concentrations during multiple administration of aminoglycosides (AGs), potentially leading to nephro- and ototoxic reactions. The once-daily administration (q.d.) of AGs has been shown to be equally or better tolerated in adults and children than the conventional schedules (twice daily, b.i.d.; thrice daily, t.i.d.), while offering potential pharmacodynamic and nursing advantages. No data, however, are available for neonates. As a consequence, this pilot study was conducted in order to assess the tolerance of the once-a-day administration of amikacin in comparison with the twice daily dose regimen, in relation to the pharmacokinetics of the drug under these two schedules. 22 Male neonates (gestational age > or = 34 weeks; postnatal age < or = 2 days) were randomized to receive amikacin (AK) (15 mg/kg/day) q.d. (n = 10) or b.i.d. (n = 12) together with ampicillin (50 mg/kg/12 h). AK plasma levels were measured at days 1, 3, 5 and 7 of treatment just before the next dose (trough level) and 1 h after completion of infusion (peak level) and after 3 and 6 h only at day 1. Due to the small size of the samples, no difference in efficacy could be assessed and was not the aim per se. Glomerular dysfunction was assessed by creatinine clearance, and tubular injuries by the urinary excretion of proteins (retinol binding protein, beta 2-microglobulin, clara cell protein (P1) and microalbumin), enzymes (N-acetyl-beta-D-glucosaminidase, alkaline phosphatase, alanine aminopeptidase, and gamma-glutamyltransferase), and total phospholipids (TPL) in urine. Ototoxicity was assessed by brainstem auditory evoked potentials (BAEPs) at days 0, 3 and 9 of therapy. Eight healthy neonates served as controls. All patients showed a normal and similar increase of GFR during the first postnatal days. Proteinuria did not increase, but enzymuria and TPL increased significantly during the treatment in both AK groups without significant difference between groups. BAEPs at day 9 were not significantly different between treated and untreated patients. We conclude from this pilot study that, in the absence of more toxicity, the q.d. administration of AK in neonates of > or = 34 weeks of gestational age may be recommended over its bid schedule in view of its potential advantages.
Abstract: The effect of several aminoglycoside (AG) antibiotics on aqueous multilamellar dispersions of mixtures of phosphatidylinositol (PI) and deuterated phosphatidylcholine (PC) has been studied by deuterium (2H) NMR. Isepamicin and amikacin gave rise to no significant changes in 2H-NMR lineshape relative to that of the lipid mixture without antibiotic. Both kanamycin A and B, which have a greater affinity for PI than the other two antibiotics examined in this study, induced temperature-dependent changes in 2H-NMR lineshapes and associated spectral moments. The results are consistent with an antibiotic-induced lateral phase separation giving rise to PC-enriched domains free of drug and PI-AG domains. These effects are correlated with the inhibitory potency of aminoglycosides towards PC degradation.
Abstract: The mode of interaction between aminoglycosides and negatively charged phospholipids plays a critical role in the inhibition of lysosomal phospholipases induced by these antibiotics and therefore in their nephrotoxicity. Previous works suggested that accessibility of the drug interacting with phospholipids to water could be crucial in this respect. We have used the concept of molecular hydrophobicity potential described by Brasseur [J Med Chem 266: 16120-16127, 1991] to visualize the hydrophobic and hydrophilic envelopes around aminoglycosides assembled with phosphatidylinositol molecules, and to obtain a three-dimensional representation of the complex formed. Using a series of different aminoglycosides, we showed that molecules with a lower inhibitory potential (gentamicin B, amikacin and isepamicin) are surrounded by both hydrophobic and hydrophilic envelopes whereas aminoglycosides which are more inhibitory are enveloped primarily by either hydrophilic (kanamycin A or B) or hydrophobic (gentamicin C1a) envelopes. This approach, which is here for the first time applied to the study of drug-lipid complexes, could help in the better understanding of the molecular mechanism of lysosomal phospholipase inhibition induced by aminoglycosides.
Abstract: Aminoglycoside antibiotics, such as gentamicin, induce a lysosomal phospholipidosis in the kidney cortex of experimental animals and humans. In vitro, gentamicin binds to negatively charged phospholipids, such as phosphatidylinositol, and decreases the activity of lysosomal phospholipases towards a neutral phospholipid (phosphatidylcholine) included in lipid vesicles. The mechanism of such an inhibition was not unequivocally established. On one hand Mingeot-Leclercq et al. (Biochem Pharmacol 37: 591-599, 1988) observed that the activity of phospholipase A1 is modulated by the negative charges of the bilayer and that the inhibitory potency of gentamicin is inversely related to the phosphatidylinositol content of the vesicles, and therefore proposed that inhibition is due to charge neutralization. On the other hand, Hostetler and Jellison (J Pharmacol Exp Ther 254: 188-191, 1990) observed that the activity of phospholipase A1 is not modulated by the negative charges of the vesicles and that the inhibitory potency of gentamicin is directly related to the phosphatidylinositol content of the bilayer, and therefore proposed that inhibition is due to substrate depletion. However, the experimental designs of these two models differed in several respects such as the source (liver versus kidney) and nature of the enzyme (native lysosomal extract versus purified delipidated phospholipase A1), and the composition of lipid vesicles (those containing constant amounts of phosphatidylcholine and cholesterol, and inversely varying amounts of phosphatidylinositol and sphingomyelin versus those containing inversely related amounts of phosphatidylcholine and phosphatidylinositol only). In order to assess the nature of the differences between these models, we compared the activity of phospholipase A1 and its inhibition by gentamicin using only one source of enzyme, the rat liver lysosomal extract, and the two types of lipid vesicles as used in the above models. Our results showed that both models are true within the frame work of their respective experimental designs. However, since the composition of the lipid vesicles as well as the nature of the enzyme preparation (whole lysosomal extract) in the "charge neutralization" model is closer to in vivo conditions, we suggest that this model may be more relevant to the in vivo situation.
Abstract: Coadministration of poly-L-aspartic acid (poly-L-Asp) protects rats against all measured signs of aminoglycoside nephrotoxicity. Based on in vitro and acute in vivo models, previously we hypothesized that poly-L-Asp protects by forming complexes with the drug in lysomes of proximal tubular cells. However, another closely related peptide, poly-L-glutamic acid (poly-L-Glu), could not protect against gentamicin-induced phospholipidosis and nephrotoxicity, presumably because it is susceptible to rapid hydrolysis in sysosomes in vivo. The present study expands the in vivo comparison between these two polyanions to a subacute model of rats and examines in detail the influence of these polymers on the qualitative and quantitative morphological alterations of lysosomes, phospholipiduria and proliferation of cortical cells induced by gentamicin. Our results not only demonstrated that despite a significantly higher drug cortical accumulation, the coadministration of poly-L-Asp almost completely protects against the development of all these early renal alteration but also pointed to the possibility of a mild, albeit apparently nonlethal, lysosomal thesaurismosis to develop under these conditions. In contrast, poly-L-Glu could not protect against these early renal alterations, though cortical drug accumulation was not significantly higher; however, it induced a conspicuous proliferation of peritubular interstitial cells. Therefore, the present work, taken together with the earlier results of ours as well as that of others, tends to strengthen the hypothesis that the site of action of poly-L-Asp must be in lysosomes, which are also the organelles that sequester and accumulate the drug.
Abstract: The distribution of epidermal growth factor (EGF) was examined by immunocytochemistry in the kidneys of rats exposed to amikacin, an aminoglycoside antibiotic causing tubular necrosis at high dose. Five-animal groups were treated for 4 or 10 days with amikacin at daily doses of 15, 40, 80 or 200 mg/kg. The drug was delivered i.p. twice a day. One hour before termination, each rat received an i.p. injection of [3H] thymidine to evaluate DNA synthesis in renal tissue. After sacrifice, the kidneys were processed for morphological (semithin and paraffin sections) and biochemical analysis (measurement of DNA synthesis by [3H] thymidine incorporation in vivo). Amikacin induced in proximal tubules a dose-related lysosomal phospholipidosis, which was assessed by the morphometric evaluation of altered lysosomes ("myeloid bodies") on semithin section. However, frank evidence of acute tubular necrosis was only observed in rats receiving amikacin at a daily dose of 200 mg/kg. Concomitantly with the development of tubular necrosis, there was a rise in the rate of cell turnover, reflected by an increase of DNA synthesis in renal tissue. This sign of tubular regeneration was accompanied by a redistribution of EGF immunoreactivity, as revealed by immunocytochemical staining. Within renal cortex of control rats, EGF immunoreactivity predominantly appeared in distal tubules and collecting ducts (97% of examined tubular sections). In contrast, in treated animals where the renal cortex displayed evidence of tubular necrosis/regeneration, EGF immunoreactivity was frequently associated with proximal tubules (more than 30% of examined tubular sections, as compared to 3% in controls).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The safety and pharmacokinetics of netilmicin (6.6 mg/kg) and amikacin (14.5 mg/kg) once daily (od) have been compared to their corresponding conventional schedules thrice daily (tid), and twice daily (bd), in patients (20 per group) suffering from pelvic inflammatory disease. Sensitive criteria of early renal and auditory alterations, namely urinary excretion of phospholipids and audiometry over a wide frequency range (0.25-18 kHz), respectively, were used. The first criterion (phospholipiduria) was validated by an animal study which demonstrated that rats receiving poly-L-aspartic acid, which protects against gentamicin-induced nephrotoxicity, are also protected against renal phospholipidosis and phospholipiduria caused by this antibiotic. On that basis, netilmicin od was better tolerated than netilmicin tid. Amikacin caused less phospholipiduria than netilmicin, and, given od, resulted in little increase over baseline (95% CI, 95-147% increase). Reduction in threshold by greater than or equal to 15 dB for frequencies between 10-18 kHz occurred in nine of 19 patients receiving netilmicin tid compared with three or four of 19 or 20 patients treated with netilmicin od or amikacin (od or bd). However, changes at lower frequencies (0.25-8 kHz) were infrequent with all regimens (from 0/19 to 2/20). In conclusion, these very sensitive tests of nephro- and oto-toxicity suggest that od dosing of amikacin or netilmicin is, if anything, safer than bd or tid dosing.
Abstract: Intracellular penetration, accumulation and disposition are important parameters governing the activity of antibiotics against intracellular bacteria. Beta-lactams diffuse into but do not accumulate in phagocytes, probably because of their acidic character. Aminoglycosides are too polar to pass across membranes and are therefore only taken up slowly by endocytosis, which results in an exclusively lysosomal localization. Lincosaminides, macrolides and fluoroquinolones all accumulate in phagocytes, the two former classes of drugs showing both a cytosolic and a lysosomal localization. Fluoroquinolones appear to be entirely soluble in cells. Analysis of their activity in a model of Staphylococcus aureus-infected J774 macrophages has revealed low activity of clindamycin, whereas macrolides, and even more so fluoroquinolones, easily reduce the original inoculum.
Abstract: The optimal management of fever in granulocytopenic patients remains controversial. This pilot study investigated the potential value of single daily doses of amikacin administered empirically with ceftriaxone in febrile granulocytopenic patients. None of the patients died as a result of infection or toxicity from the prescribed regimen. Serum concentrations failed to show drug accumulation. Modifications of empirical antimicrobial therapy were made at a similar rate to other conventional regimens. Vancomycin seemed to increase the incidence of nephrotoxicity. Overall, this pilot study suggests that empirical therapy with single daily doses of amikacin plus ceftriaxone is safe and effective and should be further investigated in a larger number of patients.
Abstract: The pharmacologic parameters and toxicity of netilmicin (6 mg/kg/day) given once daily (qd) or thrice daily (tid) for the treatment of urinary tract infections were studied in a randomized prospective study of 60 cancer patients. The overall efficacy was 96%. Nephrotoxicity, assessed by the measure of urinary excretion of phospholipids, was lower for the patients receiving the qd regimen than for those receiving the tid regimen. Elevation of serum creatinine (20% over baseline) occurred in one patient receiving the qd regimen and in three receiving the tid regimen. Cochleotoxicity, assessed by pure-tone audiometry (250 to 18,000 Hz) occurred in one patient receiving the qd regimen and none receiving the tid regimen. Concentrations in sera were measured on days 1 and 5. No significant accumulation was observed in either group. Median serum bactericidal titers, expressed as reciprocal values (percentage of the sera with a titer greater than or equal to 8), were measured against 25 test organisms in samples collected 6 h after the administration of netilmicin and were, for the qd group, 16 (82%) against members of the family Enterobacteriaceae and less than 2 (8%) against Pseudomonas aeruginosa, and for the tid group, 4 (57%) against members of the Enterobacteriaceae and less than 2 (0%) against P. aeruginosa. The rate of killing in serum was rapid (2 to 3 log in 2 h against P. aeruginosa; 3 to 5 log in 2 h against members of the Enterobacteriaceae) and correlated with the sampling time and hence the concentration in serum of netilmicin. The duration of the postantibiotic effect in serum depended also on the strain and the sampling time of the serum.
Abstract: Substitution of the C-1 atom in the 2-deoxystreptamine moiety of gentamicin C2, a broad-spectrum aminoglycoside antibiotic, by an axial hydroxymethyl group has been reported to confer protection against most clinically important bacterial enzymes inactivating aminoglycosides, while simultaneously reducing the nephrotoxic potential of this drug. We report here on a similar modification of kanamycin B. Microbiological evaluation, however, revealed no useful protection, as established by the almost complete lack of activity of 1-C-(hydroxymethyl)kanamycin B against an array of organisms producing defined types of aminoglycoside-inactivating enzymes and against which 1-C-(hydroxymethyl)gentamicin C2 and amikacin (1-N-[(S)-2-hydroxy-4-aminobutyryl]kanamycin A) are active. Moreover, toxicological evaluation, based on the in vitro measurement of the drug inhibitory potential toward lysosomal phospholipases, a predictive test of the intrinsic nephrotoxic potential of aminoglycosides, showed not decreased but rather increased toxicity. Comparative conformational analysis of the interactions of the drug with a phosphatidylinositol monolayer explained the lack of protective effect, since no significant change of the mode of insertion of the derivative in this monolayer was detected compared to that of kanamycin B. Combination of a 1-C-(hydroxymethyl) substituent with a 6"-chloro, 6"-acetamido substituent resulted in a partial improvement of the toxicological behavior with no loss of activity for the 6"-chloro and the 6"-azido derivatives, but not to the extent of obtaining better derivatives than kanamycin B itself. We, therefore, suggest that the advantages of an axial hydroxymethyl substituent at C-1 are probably restricted to the gentamicin family and do not extend to kanamycins. It might be concluded that the structural differences between gentamicins and kanamycins play an important, still undescribed role both in their effective recognition by aminoglycoside-inactivating enzymes, which are responsible for most of the clinically important cases of resistance to aminoglycosides, and also in the interactions with phospholipids, which in turn cause nephrotoxicity.
Abstract: In a companion paper (previous paper in this issue), we report on the synthesis and microbiological evaluation of new derivatives of the aminoglycoside antibiotic kanamycin B carrying substitutions in 6" (halogeno, or amino, amido, thioalkyl, and alkoxy groups, each series with increasingly bulkier chains). These modifications were intended to potentially modulate the interactions of kanamycin B with phospholipids since these are related to inhibition of lysosomal phospholipase activities and lysosomal phospholipidosis, an early and predictive index of the nephrotoxic potential of aminoglycosides. The new derivatives were therefore examined for inhibitory potency in vitro toward lysosomal phospholipase A1 acting on phosphatidylcholine included in negatively charged liposomes. No simple correlation was observed between the nature or the size of the 6''-substituent and the inhibitory potencies of the corresponding derivatives, although certain groups (diethylamino, isopropylthio) caused a significant increase in inhibitory potency, whereas an N-acetyl-N-methylamino substituent had the opposite effect. 6''-Deoxy-6''-chlorokanamycin B, however, was the only derivative showing both a decrease (albeit limited) of inhibitory potency toward phospholipase A1 associated with the maintenance of a satisfactory microbiological activity (actually equal or slightly better than that of kanamycin B). Computer-aided conformational analysis showed that this chloro substituent did not allow the molecule to insert itself very differently compared to kanamycin B or 6''-deoxykanamycin B in a monolayer of phosphatidylinositol, all three drugs adopting an orientation largely parallel to the hydrophobic-hydrophilic interface and being largely "embedded" in the bilayer at that level. In contrast, the N-acetyl-N-methylamino and isopropylthio substituents caused the corresponding derivatives to adopt an orientation largely perpendicular to the interface, because of the attraction of this substituent, and therefore of the 3''-amino sugar moiety of kanamycin B into the hydrophobic domain of the monolayer, whereas the opposite part of the drug (2',6'-diamino sugar) protruded into the aqueous phase. No simple correlation, however, could be drawn between these changes of conformation and the relative inhibitory potencies of the derivatives.
Abstract: The clinical use of the potent, wide-spectrum aminoglycoside antibiotics is limited by oto- and nephrotoxicities. The latter is related to the binding of these polycationic drugs to negatively charged phospholipids and to the subsequent inhibition of lysosomal phospholipases. In order to explore the influence of a modification of the hydrophobic/hydrophilic balance at a specific site of an aminoglycoside, kanamycin B has been chemically modified in position 6" by substitution of the hydroxyl group with a halogen atom (or a pseudohalogen group), or an amino, an amido, a thioalkyl, or an alkoxy group, each series containing increasingly bulkier chains. Examination of the antibacterial activity of the synthesized compounds revealed a negative correlation between the size of the 6"-substituent and the antibacterial activity against kanamycin B sensitive Gram-positive and -negative organisms. Only derivatives with small substituents in position 6", namely chloro, bromo, azido, amino, methylcarbamido, acetamido, methylthio, methylsulfinyl, O-methyl, O-ethyl, and O-isopropyl, showed acceptable activity (geometric mean of minimum inhibitory concentrations for Gram-negative strains less than or equal to 2.5 mg/L; value for kanamycin B, 0.5 mg/L). In vitro toxicological evaluation of all derivatives and computer-aided conformational analysis of selected compounds inserted in a phosphatidylinositol monolayer are presented in the following paper in this issue.
Abstract: Coadministration of polyaspartic acid protects rats against aminoglycoside-induced nephrotoxicity, with respect to functional and pathological changes as well as to early signs of renal alterations (lysosomal phospholipidosis of proximal tubular cells, increased proliferation of proximal tubular and peritubular cells), without reduction, but actually by increasing the drug cortical content (Williams et al., J. Pharmacol. Exp. Ther. 237: 919, 1986; Gilbert et al., J. Infect. Dis. 159: 945, 1989; Beauchamp et al., 1990). Because aminoglycoside accumulation in kidney cortex involves their segregation in lysosomes, we have examined the possibility of formation of intracellular aminoglycoside-polyaspartic acid complexes that would render the drug less toxic. We found that in vitro polyaspartic acid (MW 9-15,000) 1) binds gentamicin with an optimum at acidic pH (5.4), 2) displaces it from negatively charged liposomes and 3) restores the activity of gentamicin-inhibited lysosomal phospholipase A1 toward phosphatidylcholine included in negatively charged liposomes. In parallel, we also observed that at pH 7.0, polyaspartic acid binds and displaces gentamicin from purified brush-border membrane vesicles, causing an apparent decrease of affinity of gentamicin for these membranes, which was falsely interpreted by Williams et al., J. Pharmacol. Exp. Ther. 237: 919, 1986 as "competition for a common membrane binding site." Assuming that, after its administration in vivo, polyaspartic acid gains access to lysosomes of proximal tubular cells, as many low molecular weight proteins and polypeptides do, our results suggest that protection against gentamicin-induced nephrotoxicity is obtained by the binding of the aminoglycoside to the polyanion in lysosomes, preventing thereby the development of phospholipidosis and therefore interfering with the cascade of events leading from drug accumulation to nephrotoxicity.
Abstract: Coadministration of polyaspartic acid protects against functional and pathological signs of gentamicin-induced nephrotoxicity in rats without reduction of drug accumulation in renal cortex (Williams et al., J. Pharmacol. Exp. Ther. 237: 919-925, 1986; Gilbert et al., J. Infect. Dis. 159: 945-953, 1989). We have assessed the influence of polyaspartic acid on the early alterations induced in kidney cortex by gentamicin, namely the lysosomal phospholipidosis and the increase in cell turnover. We used an infused rat model in which animals received a total dose of 100 mg/kg of gentamicin over 12 hr. Renal cortex was examined 2 hr (day 0) and 48 hr (day 2) after treatment. All animals received an injection of [3H]thymidine (200 microCi i.p.) before sacrifice. Coadministration of polyaspartic acid (drug-polypeptide mass ratio 1:2.5) did not modify the drug serum levels, as recorded during or shortly after the infusion. Yet, it was associated with 1) an increased (approximately 35%) drug cortical content at day 0; 2) a significant protection against both biochemical (decrease of sphingomyelinase activity at day 0; increase of lipid phosphorus content at day 2) and morphological (enlargement of lysosomes and deposition of myeloid bodies at day 2) signs of lysosomal phospholipidosis in proximal tubular cells; and 3) an almost complete protection against increased cell turnover (mostly in proximal tubules) in cortex at day 2, as assessed by the measurement of [3H]thymidine incorporation into DNA and the enumeration of S-phase cells after histoautoradiography. In addition, morphological studies revealed a larger number of apical vacuoles in proximal tubular cells of animals receiving polyaspartic acid alone (but not in combination with gentamicin), and the deposition of osmiophilic, homogenous material in the lysosomes of animals receiving the combination of gentamicin and polyaspartic acid. Together with the results reported in two companion papers (Kishore et al., J. Pharmacol. Exp. Ther. 867-874, and 875-885, 1990), these results provide evidence that protection afforded by polyaspartic acid extends to the earliest cellular alterations described in kidney for gentamicin, namely the lysosomal phospholipidosis, suggesting that this protecting agent exerts blocking effect from this step in the cascade of events relating drug cortical accumulation to renal toxicity.
Abstract: Poly-L-aspartic acid (poly-L-Asp) protects rats against gentamicin (GM)-induced nephrotoxicity (functional and pathological changes) and early cortical alterations (phospholipidosis and increase in cell turnover) without decreasing, but actually increasing, the renal accumulation of the drug. We suggested that this protection occurs through the complexation of GM by poly-L-Asp, after their pinocytosis and accumulation in the lysosomes of the renal cortex (Kishore et al., J. Pharmacol. Exp. Ther. 867-874, 1990). Here we examine further our proposal by comparatively assessing poly-L-Asp (as provided by the Sigma Chemical Co., St. Louis, MO; MW 9-11,000), with two other polyanionic peptides, viz, poly-L-glutamic (poly-L-Glu; MW 14,300) and poly-D-glutamic (poly-D-Glu; MW 20,000) acids obtained from the same supplier. In vitro, all three polyanions showed a similar capacity to bind GM, to displace it from anionic phospholipids at acid pH and thereby to decrease the inhibitory potency of GM toward lysosomal phospholipase A1. In vivo, however, only poly-L-Asp and poly-D-Glu were able to prevent the development of GM-induced renal lysosomal phospholipidosis as assessed by key biochemical criteria (increase in lipid phosphorus and decrease of acid sphingomyelinase activity) and by examination of the lysosomal content in the electron microscope (accumulation of myeloid bodies). Based on these criteria, poly-L-Glu completely failed to protect. In vitro, poly-L-Glu was 13- to 17-fold more susceptible to hydrolysis by liver lysosomal extracts at pH 5.4 after 48 hr incubation, as compared to poly-L-Asp and poly-D-Glu, respectively. Assuming that all three polyanions tested are transported and accumulated in lysosomes of renal cortex to the same extent and that their respective rates of hydrolysis therein compare to that measured in vitro, these results suggest that stability of polyanions in lysosomes is an essential requisite for protection against GM-induced phospholipidosis and thus strengthens our earlier proposal that the site of action of poly-L-Asp must be in lysosomes. Although protecting from phospholipidosis, poly-D-Glu, however, caused a so far undescribed lysosomal storage disorder consisting of the accumulation of osmiophilic, nonlamellar material. This study, therefore, also demonstrates that not all polyanions resistant to lysosomal enzymes can be used as nephroprotectants, inasmuch as these, as is the case for poly-D-Glu, may cause renal alterations on their own.
Abstract: The safety, pharmacokinetics and efficacy of one daily injection (qd) of amikacin (AK) and netilmicin (NT) was compared with the recommended schedules (cs), i.e. twice-daily or thrice-daily, respectively. Women (17-43 years, n = 78) suffering from pelvic inflammatory disease were randomly assigned to qd or cs of either AK (14 mg/kg per day) or NT (6.6 mg/kg per day). Biometric parameters were similar in the 4 groups and all patients received ampicillin (4 g/day) and tinidazole (0.8 g/day). The Repeated Measures Analysis of Variance was used to distinguish the effects of the schedules and of the drugs choice on critical parameters. Efficacy was similar in the 4 groups and not influenced by the schedule of administration. No significant differences in nephro- and oto-toxicity were observed as assessed by serum creatinine and losses of hearing at low frequencies, but early phospholipiduria and auditory losses at high frequencies were significantly reduced with the qd administration compared to cs and by AK compared to NT. These data suggest that the qd schedule of AK and of NT is as efficacious as their cs schedules, and causes less renal and auditory alterations.
Abstract: In vitro and animal data show that the efficacy of an aminoglycoside is primarily related to its serum peak levels and AUC, whereas its toxicity is critically dependent upon the schedule of the administration of the daily dose as well as the duration of the treatment and the total amount of drug administered. The reduction of toxicity by intermittent dosing, e.g. once-a-day dosing (q.d.) versus splitting the daily dose in multiple administrations is connected with the saturable character of the aminoglycoside transport within inner ear and renal tissues. Clinical trials have been conducted in various types of infections in order to investigate the efficacy and tolerance of aminoglycosides q.d. Using conventional criteria of evaluation, this mode of administration was found to be equally efficacious and marginally less toxic than aminoglycosides in conventional dosing regimens. We have studied the pharmacokinetics and the early signs of renal (phospolipiduria) and auditory (high frequency audiometry) alterations of aminoglycosides given q.d. and by conventional dosage schedules to patients with pelvic inflammatory disease. It was shown that netilmicin and amikacin were better tolerated q.d. than after t.i.d. or b.i.d. administration. In addition, amikacin induced less alterations than netilmicin.
Abstract: Administration of the aminoglycoside antibiotic, gentamicin, even at therapeutic doses, causes renal lysosomal phospholipidosis. We now report that protein- and lipid-bound sialic acid levels are increased significantly in a time-dependent fashion in the renal cortex of rats injected with gentamicin (10 mg/kg body wt. per day) for 4-10 days and a significant relationship could be observed between these two parameters. This elevation was not due to tissue regeneration, since it was not observed in cisplatin-treated animals.
Abstract: Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al., Biochem Pharmacol 37: 591-599, 1988). We now extend these observations to phosphatidylserine and phosphatidic acid, and compare the inhibition caused by gentamicin, amikacin and streptomycin towards lysosomal phospholipases on the hydrolysis of phosphatidylcholine in the presence of each of these acidic phospholipids. Inclusion of phosphatidic acid in liposomes, and, to a lesser extent, phosphatidylserine, caused a larger increase in phospholipases activity than phosphatidylinositol. In parallel, the three aminoglycosides tested were found less inhibitory towards phospholipases activity measured on phosphatidic acid-or phosphatidylserine-containing liposomes than was previously observed with phosphatidylinositol, even though equilibrium dialysis experiments failed to demonstrate significant difference in binding parameters of the drug towards each of these liposomes populations. Yet, as for phosphatidylinositol-containing liposomes, the inhibition was inversely related to the amount of phosphatidic acid or phosphatidylserine included in the bilayer and the inhibitory potency of the three drugs was consistently gentamicin greater than amikacin greater than streptomycin with the three types of negatively-charged liposomes used. We conclude that impairment of lysosomal phospholipases activity towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is dependent upon drug binding to the bilayer, but that it is modulated by the nature of the acidic phospholipid that binds the drug as well as by that of the drug itself. A companion paper (Mingeot-Leclercq et al., Biochem Pharmacol 40: 499-506, 1990) will examine by computer-aided conformational analysis the parameters (drug-phospholipid energy of interaction, position of the drug in a monolayer and its accessibility to the aqueous phase) which may be important for these effects.
Abstract: Pefloxacin, like other fluoroquinolones, accumulates in macrophages and several other types of nucleated cells (but not in erythrocytes). Upon fractionation of macrophage homogenates by isopycnic centrifugation in sucrose gradients, fluoroquinolones are not found associated with any specific cellular structure. We have compared the activities of pefloxacin and roxithromycin against intracellular Staphylococcus aureus in mouse J774 macrophages. Pefloxacin was significantly more active for equivalent intracellular drug concentrations (i.e. expressed by reference to the respective MICs of the drugs as determined in broth), suggesting differences in intracellular availability and/or capacity of the drugs to express their activity in the intracellular environment. The difference was enhanced by incubating the cells in acidic medium. We have also examined the cellular pharmacokinetics and intracellular distribution of pefloxacin in uninfected and Legionella pneumophila infected guinea pig macrophages. In contrast to uninfected cells from which pefloxacin was quickly released, macrophages infected with legionella retained approximately 20-30% of the accumulated pefloxacin after a 60-min wash-out. Cell fractionation studies indicated that the drug remaining in cells was associated with components of high buoyant density. These fractions also contained [3H] if cells had been incubated with [3H] labelled legionella (by in-vitro exposure to [3H]-thymidine, before phagocytosis). These results suggest that part of the intracellular pefloxacin becomes associated with legionella, or with legionella-containing cytoplasmic structures.
Abstract: In a companion paper (Mingeot-Leclercq et al. Biochem Pharmacol 40: 489-497, 1990), we showed that the inhibitory potency of gentamicin on the activity of lysosomal phospholipases, measured towards phosphatidylcholine included in negatively-charged liposomes, is markedly influenced by the nature of the acidic phospholipid used (phosphatidylinositol, phosphatidylserine, phosphatidic acid), whereas the binding of the drug to the three types of liposomes is similar. This result challenged previous conclusions pointing to a key role exerted by drug binding to phospholipid membranes and presumably charge neutralization, for phospholipases inhibition (Carlier et al. Antimicrob Agents Chemother, 23: 440-449, 1983; Mingeot-Leclercq et al., Biochem Pharmacol 37:591-599, 1988). Conformational analysis of mixed monolayers of gentamicin and each of the three acid phospholipids shows that gentamicin systematically adopts an orientation largely parallel to the hydrophobic-hydrophilic interface, but that (i) the energies of interaction are largely different (phosphatidylinositol greater than phosphatidylserine greater than phosphatidic acid), and (ii) the apparent accessibility of the bound drug to water varies in an inverse relation with the energies of interaction. Amikacin, a semisynthetic derivative of kanamycin A with a lower inhibitory potential towards phospholipases than gentamicin in the three types of liposomes used, also showed similar differences in energies of interaction and accessibility to water, but constantly exhibited an orientation perpendicular to the hydrophobic-hydrophilic interface. We conclude that impairment of lysosomal phospholipase activities towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is indeed dependent upon drug binding to the bilayer, but is also modulated by (i) the nature of the acidic phospholipid, which influences the energy of interaction and the accessibility of the drug with respect to the hydrophilic phase, and (ii) the orientation of the drug, which it itself related to its chemical structure. Inasmuch as phospholipases inhibition is related to aminoglycoside nephrotoxicity, these findings may help in better defining the molecular determinants and mechanisms responsible for this adverse effect.
Abstract: To be effective against intracellular bacteria, antibiotics must not only reach and preferably be retained in the infected subcellular compartments, but also be able to express their activity therein. beta-lactams are most often ineffective because they fail to concentrate in phagocytes. Aminoglycosides are taken up at a very slow rate and localize almost exclusively in lysosomes where their activity is largely defeated by the acid pH. Lincosamides and macrolides accumulate rapidly by phagocytes, and distribute both in lysosomes and in cytosol. Yet, most surprisingly, macrolides are active, whereas lincosamides are not, or only weakly active against sensitive organisms. Fluoroquinolones are also accumulated by phagocytes, but are not associated with a specific organelle. They show good activity against most sensitive organisms. A model of Staphylococcus aureus-infected macrophages is presented to determine the intrinsic intracellular activity of antibiotics, i.e. to distinguish the influence of drug uptake from the other factors that modulate drug activity such as drug disposition and inactivation. This approach confirms the superiority of the fluoroquinolones as compared to presently available macrolides or to lincosamides. Thus, analysis of the pharmacokinetic and pharmacodynamic behavior of antibiotics in appropriate models of infected cells may help in directing future research towards improved derivatives, and may rationalize their use in vivo.
Abstract: The administration of anticancer platinum derivatives such as cisplatin, or aminoglycoside antibiotics is frequently associated with tubular necrosis which can eventually lead to acute renal failure. Previously, we have shown that renal tissue injury induced by these drugs elicits a process of tissue repair involving the stimulation of cell proliferation. The present study was undertaken to examine the morphological alterations and the proliferative response resulting from tobramycin administration to animals previously challenged with the platinum derivatives cisplatin and carboplatin. Female Sprague-Dawley rats were treated i.p. with cisplatin (8 mg/kg delivered in four daily injections) or carboplatin (40 mg/kg given in one injection) and sacrificed 21 or 60 days after drug administration. Tobramycin was administered i.p. twice a day at a daily dose of 10 mg/kg over the ten days preceding sacrifice. At 1 h before sacrifice, each animal received i.p. 200 microCi of [3H] thymidine for the measurement of DNA synthesis and cell proliferation (determined by histoautoradiography). Successive treatments with cisplatin and tobramycin appeared to produce an increase in the severity of histopathological alterations such as tubular necrosis and cystic degeneration. Moreover, cisplatin pretreatment dramatically increased the severity of tobramycin-induced lysosomal phospholipidosis. Histopathological alterations were followed by an important proliferative response partly associated with tubular regeneration but also due to fibroblastic proliferation which led to peritubular fibrosis. Surprisingly, the additive effect of cisplatin and tobramycin on renal injury became particularly striking with increasing time intervals between treatments. In contrast, successive treatments with carboplatin and tobramycin did not cause significative changes of the degree of renal injury, compared with either drug given alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: In the present study, we compared poly-L-Asp with poly-L-Glu and poly-D-Glu in vitro and in vivo for their ability to inhibit the GM-induced nephrotoxicity. In vitro, all three polyanions (i) bound GM over a wide range of pH; (ii) displaced GM previously bound to negatively charged phospholipid bilayers at acid pH (i.e. under the conditions prevailing in lysosomes in vivo), and thereby (iii) decreased the inhibitory potency of GM towards lysosomal phospholipase A1. Thus, one was tempted to predict that all three polyanions would have the potential of protecting against AG-induced nephrotoxicity. However, when co-administered to rats with GM, poly-L-Asp and poly-D-Glu completely suppressed the development of lysosomal phospholipidosis, as assessed by biochemical criteria and increased drug accumulation, whereas poly-L-Glu did not offer such protection despite a relatively lower increase in drug accumulation levels. Histoautoradiography also confirmed that poly-L-Asp, but not poly-L-Glu, was a nephroprotectant against the tissue proliferative response induced by GM. Morphologically, poly-L-Asp almost completely and poly-D-Glu totally prevented the accumulation of myeloid bodies in lysosomes. In vitro incubation in the presence of purified lysosomal extracts revealed marked differences in the hydrolysis rate of these peptides (poly-D-Glu:poly-L-Asp:poly-L-Glu = 1:1.2:16.9). Assuming that all three polyanionic peptides are transported and accumulated in lysosomes to the same extent, these results not only suggest that their stability in lysosomes is an essential requisite for protection against lysosomal phospholipidosis, but also strengthen our hypothesis that the site of action of poly-L-Asp is inside the lysosomes but not at the level of the renal membranes. In addition, poly-D-Glu alone or combined with GM induced another type of morphological lesion, not related to AG-induced nephrotoxicity which, to our knowledge, has not yet been described.
Abstract: "Uraemia" literally means "urine in blood". With the advancement of basic medical sciences, it is being better understood. The clinical syndrome of uraemia is due to the failure of not only the excretory but also the metabolic, regulatory and endocrine functions of the kidney. Apart from the "retained toxic metabolites", a number of guanidine derivatives had been found which are now considered to be more important in the causation of the uraemic syndrome. Cohen had hypothesised that nitrogen retention in uraemia causes an aberration in the urea cycle that in turn leads to the production of guanidinosuccinic acid (GSA) in large amounts. However, it appears that methylguanidine (MG) is produced from the degradation of creatinine by the gut flora in uraemics. Both GSA and MG are proved to be toxic. The role of GSA in uraemic neurotoxicity and coma is still controversial and needs further investigation. It is possible that the combined effects of a number of compounds are responsible for the development of the uraemic syndrome.
Abstract: Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.
Abstract: Aminoglycoside antibiotics cause transient, usually nonoliguric, renal failure in up to 10-30% of patients treated with these drugs, and are the cause of the largest proportion of drug-induced acute nephrotoxicities. The toxic mechanism includes (i) uptake of the drug by proximal tubular cells, where it is first sequestered within lysosomes and (ii) development of a lysosomal phospholipidosis, which is rapidly associated with cell necrosis and various alterations to subcellular structure and function. Tubular necrosis is often accompanied by (and probably triggers) tubular regeneration and peritubular proliferation. The means whereby such tubular alterations eventually cause a decline in glomerular filtration and hypo-osmotic polyuria has not been established. Various in-vitro and acellular models have been designed to assess and screen for the nephrotoxic potential of aminoglycosides; of these, methods based on the analysis of aminoglycoside-phospholipid interactions appear to be the most meaningful. A number of patient- and drug-related risk factors have been identified, and their avoidance could significantly reduce the risk of nephrotoxic reactions. Because the uptake of aminoglycosides by the kidney is saturable, administration of daily doses of these drugs as one or two injections, rather than as multiple injections or by continuous infusion, may also decrease the risk for toxicity.
Abstract: Cisplatin (cis-diamminedichloroplatinum II) has emerged as an anticancer drug of considerable value for the chemotherapy of several human neoplasms. However, this agent often causes renal toxicity, which appears to be the dose-limiting untoward effect. The present animal study was undertaken to compare, with regard to kidney injury and renal tissue repair, cisplatin and carboplatin (cis-diammine-1,1-cyclobutane dicarboxylate platinum II), a platinum derivative more recently introduced in clinics. Female Sprague-Dawley rats (four animals per group) were treated ip with cisplatin (4 or 8 mg/kg, delivered in four consecutive daily injections) or carboplatin (40 mg/kg given in one injection) and terminated 4, 7, and 21 days after drug administration. One hour prior to sacrifice, each animal received ip 200 microCi of [3H]thymidine for the measurement of DNA synthesis and cell proliferation (frequency of S-phase cells in renal tissue, determined by histoautoradiography). Cisplatin, particularly at 8 mg/kg, caused severe tubular injury (acute tubular necrosis) culminating in a long-lasting cystic tubular dilatation in the outer stripe of outer medulla. Tubular damage was followed by a sharp proliferative response, indicative of tubular regeneration. However, the proliferative activity was still above basal level at the end of the observation period, suggesting that the tissue repair process had not reached completeness 3 weeks after cisplatin administration. In contrast, carboplatin only induced focal tubular necrosis in proximal tubules. Distal and collecting tubules also showed ultrastructural evidence of hydropic degeneration after exposure to the latter drug. Renal tubular injury associated with carboplatin was followed by a mild proliferative response. From this study, we can infer that carboplatin is less nephrotoxic than cisplatin, but still causes histopathological alterations in renal tissue. Furthermore, the lesser nephrotoxicity of carboplatin has a primary origin and is not due to a more efficient tissue repair reaction.
Abstract: Cis-diamminedichloroplatinum (II) (cisplatin), an inorganic platinum salt used in cancer chemotherapy, is characterized by a renal toxicity recognized both in experimental animals and in patients treated with the compound. The purpose of the present study was to explore by both light and electron microscopy the morphological alterations induced in the rat kidney by cisplatin administration and, in particular, to analyse the tissue repair reaction following nephrotoxic injury. Experimental animals (four rats per group) were treated i.p. with 2, 4 or 8 mg/kg cisplatin administered in four consecutive daily injections. The rats were sacrificed 4 days after the last injection. In addition, the persistence of renal lesions and the duration of the repair reaction were determined in rats given 8 mg/kg cisplatin and killed 4, 7, 14 or 21 days after the last injection. The cell proliferation associated with tissue repair was estimated both quantitatively (rate of DNA synthesis) and qualitatively (histoautoradiography and electron microscopy examination) 1 h after in vivo exposure to [3H] thymidine. Renal tissue alterations and the repair reaction were minimal after the administration of 2 or 4 mg/kg cisplatin. In contrast, 8 mg/kg cisplatin caused a spectrum of morphological abnormalities affecting proximal, distal and collecting tubules, and ranging from sublethal cell alterations to tubular necrosis and cystic dilatation. The latter degenerative change primarily involved the straight portion of proximal tubules and seemed to develop over the weeks following cisplatin administration. Concomitantly with the tissue lesions, a burst of cell proliferation, associated with stimulation of DNA synthesis, was apparent in the renal cortex and outer medulla. Whereas a very high incidence of S-phase cells was encountered in seemingly undifferentiated tubules, they also appeared in differentiated proximal, distal and collecting tubules, but were infrequent in cystic tubules. Proliferation of fibroblasts was also stimulated in the renal interstitium. The proliferative response persisted for the whole duration of the experiment, indicating incomplete tissue repair. The long-lasting tubular injury and the slowness of repair are consistent with the chronic renal dysfunction (polyuria and hypomagnesemia) that cisplatin is known to induce in both man and experimental animals.
Abstract: Aminoglycosides such as gentamicin are hydrophilic, polycationic drugs which bind to negatively-charged phospholipid bilayers, inhibit the activities of the lysosomal enzymes involved in the degradation of the major phospholipids and cause, in kidney in vivo or in cultured cells, a lysosomal phospholipidosis. In the present study, we show that the hydrolysis of phosphatidylcholine induced in liposomes by lysosomal extracts at pH 5.4 in vitro is critically dependent on the negative charges carried by the bilayer. This hydrolysis, which is predominantly carried on by phospholipases A1 and A2, markedly increases when the phosphatidylinositol content is raised from 10 to 30% of the total phospholipids, i.e. in a range found in natural membranes. Addition of gentamicin decreases the activity of these enzymes in a non-competitive fashion, but the effect is inversely proportional to the amount of phosphatidylinositol present in the bilayer. Gentamicin and bis(beta-diethylaminoethylether)hexestrol (DEH), a cationic amphiphile which also binds to phospholipid bilayers, are equipotent inhibitors when added to negatively-charged liposomes at equinormal concentrations. Although direct aminoglycoside-enzyme interactions cannot be excluded, these results strongly suggest that gentamicin impairs the activities of the lysosomal phospholipases towards phosphatidylcholine by decreasing the available negative charges required for optimal activity.
Abstract: Amino acids have been coupled to the carboxyl group of penicillin V and cephalothin by methods that keep the beta-lactam ring intact. Derivatives were successfully obtained with both neutral (Leu, Val, Ala, Ile, Trp, Tyr, Gly) and one acidic (Glu) amino acids. The new compounds were inactive in vitro against Staphylococcus aureus or Micrococcus luteus. Incubation in the presence of purified carboxypeptidases (A, B), soluble lysosomal fractions from liver, or cellular homogenates from liver, kidney, fibroblasts, and macrophages did not allow recovery of the antibacterial activity. Injection in mice also failed to cause liberation of microbiologically active compounds. HPLC studies confirmed that the amide linkage between the antibiotic and the amino acid was not hydrolyzed in the presence of soluble lysosomal fractions from liver. However, conversion of cephalothin and cephalothin-leucine to desacetyl derivatives was observed in the presence of soluble lysosomal fractions and extracts from liver and semipurified orange peel acetylesterase(s). It is concluded that amino acid derivatives of beta-lactam antibiotics do not offer potential chemotherapeutic use as prodrugs.
Abstract: Administration of aminoglycoside antibiotics is frequently associated with tubular necrosis which can eventually lead to renal dysfunction. Previously, we have shown that renal tissue injury due to aminoglycoside nephrotoxicity elicits a process of tissue repair characterized by stimulation of cell proliferation. The present study was undertaken to examine both quantitatively and qualitatively the cell proliferation associated with renal tissue repair. Female Sprague-Dawley rats (180-200 g body weight) were treated ip for 10 days with various doses of tobramycin (10, 20, or 50 mg/kg twice daily). Each animal received 200 microCi [3H]thymidine 1 hr before sacrifice to evaluate the extent of cell proliferation in renal cortex. The rate of DNA synthesis in renal cortex was estimated by measuring the specific radioactivity of the nucleic acid. The frequency and localization of S-phase cells in cortex tissue were determined on paraffin and plastic tissue sections processed for histoautoradiography. In addition, the ultrastructure of proliferating cells was characterized by electron microscopic examination of consecutive ultrathin sections. An excellent correlation (r = 0.993) was found between the rate of DNA synthesis and the frequency of S-phase cells evaluated in rats receiving various doses of tobramycin. The stimulation of cell proliferation involved mostly proximal tubular cells and interstitial cells. The latter cells had the ultrastructural appearance of fibroblasts at various stages of differentiation. Similarly, S-phase cells in proximal tubules were either fully differentiated epithelial cells or immature elements. Taken together, the present experimental data illustrate the capacity of the kidney to trigger complex tissue reactions in response to nephrotoxic injury.
Abstract: The intracellular accumulation and subcellular distribution of 14C-labelled roxithromycin and erythromycin has been studied in macrophages and polymorphonuclear neutrophils of both human and animal origin. Roxithromycin was consistently and significantly more accumulated than erythromycin, reaching intracellular/extracellular concentration ratios between 14 (in polymorphonuclear neutrophils) and 190 (in alveolar macrophages from smokers). Uptake was reversible, insensitive to anaerobiosis and to the presence of an aminoglycoside, but inhibited by acid pH. Upon subcellular fractionation by isopycnic centrifugation in sucrose gradients., half the roxithromycin or erythromycin recovered in cell homogenates was found associated with the lysosomes in macrophages, and about one third with azurophil granules in polymorphonuclear leucocytes. Inasmuch as cellular uptake is a necessary, albeit not sufficient, condition for antimicrobials to kill or inhibit the growth of intracellular bacteria the properties of roxithromycin may give it a distinct advantage over other antimicrobial agents.
Abstract: beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al. (C. de Duve, T. de Barsy, B. Poole, A. Trovet, P. Tulkens, and A. Van Hoof, Biochem. Pharmacol. 23:2495-2531, 1974), in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.
Abstract: Ultrastructural alterations in the cortical, distal and collecting tubules have been examined in female Sprague-Dawley rats treated with various aminoglycosides in clinical use. Gentamicin, dibekacin (10 mg/kg X day), netilmicin, tobramycin (4 or 10 mg/kg X day) or amikacin (37.5 mg/kg X day) were administered intraperitoneally twice a day over different periods of time, extending from 4 to 14 days. The kidney cortex was examined after 4, 7, 10 or 14 days of aminoglycoside administration by light (semithin sections) and electron microscopy. After 7 or more days of treatment, lysosomes in collecting tubular cells (and to a lesser extent in distal tubular cells) contained concentric lamellar material (myeloid bodies), an ultrastructural alteration typical of drug-induced lysosomal phospholipidosis. Although this alteration appeared qualitatively similar to that observed in proximal tubular cells, it was less conspicuous and occurred later during treatment. In addition, distal tubular cells occasionally showed marked vacuolization and disruption of the basal cell architecture. The possible relationship between these alterations and the urine hypo-osmolality characteristic of aminoglycoside-induced renal dysfunction is discussed.
Abstract: An immunoassay based on fluorescence polarization detection (FPIA) has been recently adapted for dibekacin. This has been compared with a reference method (high-performance liquid chromatography), and two other methods used in clinical laboratories for monitoring this aminoglycoside, namely substrate-labelled fluorescent immunoassay (SLFIA) and radioimmunoassay (RIA). FPIA was fast and more reliable than SLFIA or RIA, and offered therefore superior performance. However, its nominal cost per assay is high.
Abstract: Williams and Hottendorf (1985) recently reported that poly-l-aspartic acid (pAsp) and poly-l-asparagine (pAsn) inhibit gentamicin (G) binding to brush border membrane vesicles in vitro and protect from G-induced nephrotoxicity in vivo. A model of infused rats was used to check for the early tissue alterations induced by G in animals receiving either G alone or the combination G + pAsp or G + pAsn. The cortical tissue was analysed 2 h or 2 days after the end of a 12 h infusion for signs of i) lysosomal phospholipidosis due to interference of G on phospholipids catabolism assessed by both biochemical (measurement of sphingomyelinase activity and of the total phospholipids content in renal cortex) and morphological analysis and ii) tubular regeneration and peritubular cells infiltration subsequent to focal necroses. While no reduction of G cortical levels was detected, significant changes in these parameters showed that G + pAsp and G + pAsn caused less phospholipidosis than G alone. Thus pAsp and pAsn decrease the severity of the early renal alterations induces by G.
Abstract: The nephrotoxicity of aminoglycosides is an important consideration in the clinical use of these agents. The underlying biochemical and cytologic mechanisms have, therefore, been the subject of many extensive studies. Few experimental studies, however, have employed doses relevant to those used in clinical practice. This article focuses on data obtained using such a "low-dose" approach. Aminoglycosides accumulate specifically in the lysosomes of proximal tubular cells by a process of adsorptive pinocytosis; in the lysosomes, they induce a phospholipidosis that has been studied in vivo and in vitro (e.g., by computer-aided conformational analysis). A similar phospholipidosis is also observed in humans. The overall nephrotoxicity of aminoglycosides results from a combination of their intrinsic ability to bind to phospholipids and the extent of their renal uptake. The development of phospholipidosis is accompanied by focal necroses, tubular regeneration, and interstitial proliferation, even at low, therapeutic doses of these agents. These changes are related to the nephrotoxic potential of the aminoglycosides. Of all 2-deoxystreptamine-containing aminoglycosides tested so far, and by all criteria, amikacin is associated with the least dramatic alterations. Together, these approaches may provide a rational basis to study and compare the nephrotoxicity of aminoglycosides at clinically relevant dosages. They may also serve for the screening and design of aminoglycosides with lower toxicity than those currently available.
Abstract: The tissue repair subsequent to aminoglycoside-induced tubular necrosis has been evaluated for five different derivatives given at low doses. Gentamicin, dibekacin, netilmicin, and tobramycin were administered intraperitoneally three times a day at 10 mg/kg per day; amikacin was given intraperitoneally twice a day at 37.5 mg/kg per day. The drugs were administered for 4 or 10 days. Tissue regeneration in kidney cortex was assessed by: the rate of DNA synthesis (i.e., the specific radioactivity of DNA measured 1 hour after intraperitoneal injection of 200 microCi of [3H] thymidine per animal); the occurrence of less differentiated cells estimated by the morphometry of peroxisomes. In mature cells, identified by the presence of drug-induced myelin-like figures, peroxisomes occupied 3.0% of the cytoplasmic area, whereas in less differentiate cells devoid of myelin-like figures, the same organelles made 1.4% of the cytoplasmic area (mean values obtained from 20 treated animals). Gentamicin and amikacin were both shown to induce a dose-related enhancement of DNA synthesis in kidney cortex, except that the latter antibiotic was about 10 times less potent as compared to the former. When the five drugs were compared on the basis of their effect on DNA synthesis after 10 days of treatment, they gave the following ranking: gentamicin (5.6 times the mean control value) greater than or equal to dibekacin greater than netilmicin greater than tobramycin greater than amikacin (1.6 times the mean control value). The differences were less striking after 4 days of treatment. For each antibiotic, sections of proximal tubular cells obtained from four animals treated for 10 days (total surface, 2.10(4) microns2) were scanned for the presence of regenerative tissue (areas poor in peroxisomes). In gentamicin-treated rats, the areas poor in peroxisomes represented more than 18% of the scanned cytoplasmic surface in proximal tubular cells, as compared to about 4.5% in controls. The ranking of the five antibiotics according to the estimated area of undifferentiated tissue in proximal tubules was parallel to that found for DNA synthesis i.e., in gentamicin-treated rats undifferentiated tissue was much more prominent, as compared to amikacin-treated animals (about 6% of the scanned area). Since the relative extent of kidney cortex regeneration induced by each antibiotic at low dose is likely to reflect the degree of tubular necrosis, we conclude that the ranking outlined above indicates a difference in potential nephrotoxicity.
Abstract: To study the effect of gentamicin on the renal uptake of proteins, Sprague-Dawley female rats were intravenously injected with solutions containing unlabeled human beta 2-microglobulin (beta 2-m), retinol-binding protein, and increasing amounts of gentamicin (from 0.063 up to 31.5 mg/kg). The concentrations of human proteins and that of endogenous beta 2-m, albumin, and IgG in the urine collected during the 2 hr following the injection were determined by immunoassays. Gentamicin transiently increased the urinary excretion of rat and human beta 2-m in a dose-dependent manner. The mean relative increase of rat beta 2-m excretion ranged from 2 at a gentamicin dose of 0.06 mg/kg up to 500 at a gentamicin dose of 31.5 mg/kg. By contrast, the urinary excretion of other proteins was only increased by a factor of 2 to 5 at the highest dose of gentamicin. The relative increase of the urinary excretion of proteins was positively correlated with the fractional reabsorption of the proteins by the rat kidney. The inhibitory effect of gentamicin on the renal uptake of protein was very similar to that observed in rats injected with polycationic proteins like lysozyme and cytochrome C. These observations, combined with the fact that gentamicin, like proteins, enters the tubular cell by adsorptive endocytosis, strongly suggest that this drug competes with proteins for common binding sites on the apical tubular membrane and for subsequent endocytosis. Furthermore, the iv injection of large amounts of gentamicin and polycationic proteins induces a lysosomal enzymuria which very likely is a manifestation of an increased exocytosis.
Abstract: Peptidic lysosomotropic prodrugs of antibiotics and antitumoral agents could be of advantage in chemotherapy, providing that free, active drug is released at, or close to, the desired site of action. Thus, aminoacyl derivatives of doxorubicin, e.g., where the drug is attached to the amino acid by a primary amino function, are sensitive to lysosomal hydrolases. We have examined whether a similar approach can be used for drugs carrying a carboxyl group such as beta-lactam antibiotics. Because the C adjacent to the carboxyl group in beta-lactams has the D configuration, we have examined and report here the synthesis and susceptibility of model peptides, namely Boc-D-Pro-L-Ala and Boc-L-Pro-L-Ala to lysosomal hydrolases. Hydrolysis of the D isomer proceeds considerably more slowly than that of the L isomer. Lysosomal carboxypeptidase(s) and/or amidases appear therefore to have a much narrower specificity than aminopeptidase(s), which will severely limit the applicability of the concept of peptidic lysosomotropic prodrugs.
Abstract: Gentamicin, an aminoglycoside antibiotic, is known to accumulate within the kidney cortex and to elicit nephrotoxic reactions due to the necrosis of proximal tubules. Female Sprague-Dawley rats, treated for 9 days with gentamicin at a low dose (10 mg/kg ip, once a day), were used to determine the fate of the antibiotic within the proximal tubular cells and its effects on the functional properties of the lysosomes. The analysis of the lysosomes by isopyknic equilibration in sucrose gradient (density 1.10-1.24 g/ml) revealed that gentamicin remains associated with these organelles (marker enzymes sulfatase B and cathepsin B) throughout the treatment duration. Gentamicin treatment markedly decreased the buoyant density of the lysosomes. As was shown by electron microscopic examination of the subcellular fractions collected from the sucrose gradient, the shift of the lysosomes toward lower densities was a result of overloading with undegraded phospholipids (myelin-like figures). The effect of gentamicin on the functional properties of the lysosomes was examined by using horseradish peroxidase (HRP) as a marker of endocytic activity and of the processing by tubular cells of exogenous proteins. Treatment with gentamicin did not significantly modify the intracortical accumulation of HRP, which was estimated to be 2.2% of the amount injected. HRP was shown by isopyknic equilibration to be mostly associated with the lysosomes. This was confirmed by electron microscopic examination of proximal tubular cells after cytochemical demonstration of HRP with diaminobenzidine and H2O2. In rats not exposed to gentamicin, more than half of the lysosomes contained HRP activity. In animals treated with gentamicin, one-third of the lysosomes that retained a normal appearance exhibited HRP activity. In contrast, lysosomes overloaded with phospholipids (identified by the presence of myelin-like figures) were very seldom labeled with HRP activity. Taken altogether, the present observations suggest that the alterations induced by gentamicin treatment impair their ability to fuse with incoming endocytic vesicles.
Abstract: Aminoglycoside antibiotics induce a lysosomal phospholipidosis in kidney proximal tubules after conventional therapy in animals and man. We have previously demonstrated that these drugs bind to negatively charged phospholipid bilayers at acid pH and inhibit the activity of lysosomal acid phospholipases in vitro and in vivo. A combined biochemical and conformational study [Brasseur et al., Biochem. Pharmac. 33, 629 (1984)] showed major and consistent differences between 6 aminoglycosides in current clinical use with respect to the stability of the complexes they form with phosphatidylinositol, their inhibitory potency towards the activity of lysosomal phospholipases and their current toxicity ranking (e.g. gentamicin greater than amikacin greater than streptomycin). In the present study we have extended this approach to experimental derivatives of streptomycin. The derivatives examined were: dihydrostreptomycin, dideguanyldihydrostreptomycin, streptomycylamine, dideguanylstreptomycylamine, N-butyl- and N-benzyl-dideguanylstreptomycylamine. These compounds were examined for (i) their binding to negatively charged liposomes, measured by gel permeation on Sepharose 4B; (ii) their interactions with phosphatidylinositol assessed by semi-empirical conformational analysis and (iii) their inhibitory effect on the activities of lysosomal phospholipases towards phosphatidylcholine present in negatively charged liposomes. Streptomycin and gentamicin were also used as reference compounds with low and high affinity (and inhibitory potency), respectively. Our observations can be summarized as follows: (i) the replacement of the aldehyde in the streptose ring by a methylamino group strikingly changes the conformation of the molecule, allowing a better interaction with phosphatidylinositol. Thus, streptomycylamine binds much more tightly to phospholipid bilayers and shows a higher inhibitory potency towards phospholipase activity, as compared to streptomycin. The conformational analysis shows, however, that this effect is only partially due to the additional cationic charge carried by streptomycylamine. Other modifications of the streptomycin molecule, such as the replacement of the guanidinium groups by aminogroups or the addition of hydrophobic moieties (butyl or benzyl groups) to the streptose do not markedly further strengthen the interactions of the molecule with phosphatidylinositol. (ii) Even though some derivatives (e.g. dideguanylstreptomycylamine) bind as tightly to phospholipids as gentamicin, they remain much less inhibitory towards lysosomal phospholipases.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: We have performed combined in vivo and in vitro measurements of thymidine uptake into kidney cortex DNA of animals treated with gentamicin for 7 days at 10 or 20 mg/kg daily (BID). Labelled thymidine is taken up by cortex fragments in vitro (90 min incubation) and incorporated into DNA; treatment of the animals with gentamicin results in a significant dose-dependent enhancement of this in vitro thymidine incorporation; labelled cells are found primarily in the proximal tubules and interstitium; there is an excellent correlation (r : 0.983, n = 15) between the changes of incorporation measured in vivo and in vitro as demonstrated by the sequential use of [3H]thymidine (in vivo) and [14C]thymidine (in vitro) within the same animals.
Abstract: The recovery from gentamicin-induced phospholipidosis in the rat kidney cortex was characterized both morphologically and biochemically after a single 12-hr drug infusion. Total dosages administered were 10, 60, or 140 mg/kg, achieving constant serum concentrations of 3, 11, and 27 micrograms/ml, respectively. At the end of the 12-hr infusion, the cortical drug concentrations corresponding to the three dosages were 124, 450, and 993 micrograms/g of wet tissue. At the low dose (10 mg/kg), myeloid bodies were seen inside lysosomes of proximal tubular cells, along with a modest decrease of lysosomal sphingomyelinase activity. The cortical drug level declined steadily following first-order kinetics along with a disappearance of myeloid bodies and return of sphingomyelinase activity to control levels. At the high dose (140 mg/kg), we observed a sustained loss of sphingomyelinase activity (37% of controls), a subsequent increase of phospholipid concentration in the kidney cortex (up to 117% of controls 2 days after) and a prominent accumulation of myeloid bodies inside the lysosomes of proximal tubular cells (up to 4% of cell volume). Tubular regeneration and interstitial infiltration became detectable by histology and the increase of DNA synthesis as from day 1, along with an apparent reduction of the phospholipidosis at days 3 and 4. Drug cortical concentrations showed a sharp decline 2 days after infusion. An intermediate behavior was observed at 60 mg/kg. It is concluded that the proximal tubular cells behave in a fundamentally different way after gentamicin loading with low and high doses. At the low dose there is a regression of the drug-induced changes in the absence of any sign of necrosis-regeneration. Above a threshold in cortical drug concentration there is further development of the alterations leading to cell death-regeneration.
Abstract: The early alterations at the level of the proximal tubule of the human kidney caused by the three most currently used aminoglycosides, gentamicin, tobramycin, and amikacin, were studied. A prospective, randomized, and comparative approach using multidisciplinary methods was used. The patients received either no treatment or one of the three aminoglycosides at a therapeutic dose for 4 days preceding nephrectomy for neoplasia partly involving one kidney. The three aminoglycosides studied induce an early lysosomal phospholipidosis. Gentamicin and tobramycin cannot be distinguished on the basis of drug tissue accumulation, lysosomal overloading, or effect on lysosomal phospholipase A1. Amikacin induces significantly lower lysosomal overloading and no loss of phospholipase A1 activity.
Abstract: Previous studies [Laurent et al., Biochem. Pharmac. 31, 3861 (1982)] have demonstrated that aminoglycoside antibiotics bind to negatively charged phospholipid bilayers and inhibit the activity of lysosomal phospholipases. This inhibition also occurs in vivo in animal and man. It is considered to be an early and significant step in the development of aminoglycoside-induced nephrotoxicity. The binding of 6 aminoglycosides in current clinical use (dibekacin, gentamicin, tobramycin, kanamycin A, amikacin and streptomycin) to phosphatidylinositol has been studied by gel filtration technique and by conformational analysis. Variation of the phosphatidylinositol content from 0 to 27% of total phospholipids causes a cooperative increase in aminoglycoside binding. At fixed phosphatidylinositol concentration, the binding of the different aminoglycosides is related to the number of aminogroups carried by the drug (viz., gentamicin greater than kanamycin A greater than streptomycin) and is largely, but not entirely dependent upon electrostatic interactions. Conformational analysis of the interaction of aminoglycosides with phosphatidylinositol monolayers was made by a step-wise computation approach. We first have taken into account the Vander Waals, torsional and electrostatic energies and we have calculated the hydrophobic and hydrophilic centers of each molecule. Assembly was then computed by successive association of one molecule of drug and up to 4 molecules of phosphatidylinositol. The calculated interaction energies varied from -8.5 kcal/mol (gentamicin) to -4.9 kcal/mol (amikacin) and -3.9 kcal/mol (streptomycin).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Aminoglycosides induce an early and characteristic phospholipidosis in the lysosomes of kidney tubular cells. Inhibition of lysosomal phospholipase activity by gentamicin has been demonstrated in vivo and in vitro. Ten aminoglycosides have been examined for their potency in inhibiting phospholipases A1 and A2. Similar IC50's and IC90's (concentrations causing 50 and 90% inhibition) were found for sisomicin, dibekacin, gentamicin, tobramycin, kanamycin B and netilmicin. Kanamycin A, HABA-dibekacin and amikacin showed increasingly higher IC's. Thus, both the number and the position of amino groups are important. Streptomycin had little effect on phosphatidylcholine hydrolysis. Kinetic studies with gentamicin suggest a competitive type of inhibition. This approach may help for the in vitro screening of less toxic aminoglycosides.
Abstract: The nephrotoxic potential of S86451 (2'guanidyl, 2'deamino gentamicin C1) has been investigated in Sprague-Dawley rats at 10 and 25 mg/kg, in comparison with gentamicin at 4 and 10 mg/kg. The criteria were (i) the severity of the lysosomal phospholipidosis, as assessed by both biochemical and morphological investigations; (ii) the degree of tubular regeneration, a consequence of the aminoglycoside-induced focal necrosis measured by 3H-thymidine incorporation into kidney cortex DNA. S86451 was found at least 2-5 times less toxic than gentamicin. Thus, this compound should be a safer antibiotic than gentamicin in combating organisms with a similar sensitivity to either drug.
Abstract: Tissue injury induced in rat kidney cortex by gentamicin at a low dose (10 mg/kg) has been studied by measuring subsequent regeneration after treatment for 4, 7, and 14 days. Cell proliferation was demonstrated by the incorporation of [3H]-thymidine into kidney cortex DNA. In treated animals, the specific radioactivity of DNA increased up to 4.6 times the mean value found in the controls. Autoradiography showed labelling of nuclei in proximal tubular cells. Electron microscopy showed that a subpopulation of tubular cells, devoid of myeloid bodies, which are characteristic of gentamicin intoxication, is also poorer in peroxisomes, and therefore may belong to less differentiated, regenerating cells. It is concluded that a low dose of gentamicin induces measurable tissue regeneration.
Abstract: Aminoglycoside antibiotics induce an early and characteristic lysosomal phospholipidosis in cultured fibroblasts and in kidney tubular cells. We have recently demonstrated an inhibition of lysosomal phospholipases A1 and A2 by gentamicin and amikacin in vitro. In vivo, gentamicin decreases the activity of phospholipase A1 (Laurent et al., Biochem. Pharmacol. 31:3861-3870, 1982). In the present study, we examined 14 aminoglycosides for in vitro inhibition of phospholipases. To mimic the situation prevailing in lysosomes, the enzymatic activities were assayed with phospholipid vesicles (liposomes) with a composition similar to that of lysosomal phospholipids (phosphatidylcholine, sphingomyelin, phosphatidylinositol, cholesterol; 4:4:3:5.5, molar ratio). We measured the hydrolysis of 1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine contained in the liposomes by a soluble fraction of highly purified lysosomes isolated from rat liver. Similar IC50S (concentrations causing 50% inhibition of enzymatic activity) were observed for dibekacin, gentamicin (with no major difference between C1, C1a, or C2), netilmicin, tobramycin, and kanamycin B. Sisomicin was slightly more inhibitory. Kanamycin A, N1-(L-4-amino-2-hydroxy-1-oxobutyl)dibekacin, and amikacin showed increasing IC50S. Streptomycin caused the least inhibition. Octa- and tetramethylkanamycin A are much less inhibitory than the parent drug. These results point to the number, the nature, and the respective positions of the cationic groups as essential determinants in causing inhibition of phospholipid breakdown. The binding of three aminoglycosides (gentamicin, amikacin, streptomycin) to the liposomes at pH 5.4 was also measured by gel permeation and was found to be related to the respective inhibitory potency of each drug. Insofar as lysosomal phospholipidosis is an early sign of intoxication by aminoglycosides, these results may serve as a basis for the development or screening of less toxic compounds in this class of antimicrobial agents.
Abstract: Kidney cortex DNA synthesis was studied in female rats treated with a low dose of gentamicin (10 mg/kg) up to 14 days. Synthesis was measured by incorporation of [3H]thymidine into DNA 1 h after intraperitoneal injection of the labeled precursor (200 muCi per animal). Gentamicin given in one injection per day resulted in a greater incorporation of [3H]thymidine into DNA after both 7 and 14 days of treatment as compared with control animals. When the daily dose was divided into three equal injections given at 8-h intervals, a statistically significant increase in thymidine incorporation was observed as early as 4 days after starting gentamicin administration. Excellent agreement was found between DNA specific radioactivity and kidney cortex nuclear labeling, as measured by histoautoradiography. The greatest amount of [3H]thymidine incorporation occurred within proximal tubular cells and interstitial cells. We conclude that a finite duration of gentamicin treatment at low dosage induces an increased DNA synthesis in vivo in rat kidney cortex. We suggest that this reaction results from cellular proliferation and could reflect a regenerative process after focal necrosis induced by gentamicin at low doses. The demonstrated early increase in DNA synthesis could be a useful tool to measure kidney cortex alterations caused by various aminoglycosides at low, therapeutic doses.
Abstract: The effect of a previous chronic exposure to cadmium, lead or inorganic mercury on the nephrotoxic potential of gentamicin was investigated in female Sprague-Dawley rats. A daily dose of 10 mg gentamicin/kg body weight/day was administered for 21 days to rats having a renal load of 168 micrograms Cd, 35 micrograms Pb or 129 micrograms Hg/g whole kidney. Urine analysis suggests an attenuation of the nephrotoxic potential of gentamicin while a microscopical examination of kidneys indicates a superimposition of the effects of the metals and the antibiotics. The only clear interaction observed consists in a reduction of gentamicin accumulation in the cortex of cadmium-treated animals. It is concluded that none of the metal pretreatments potentiates the nephrotoxic effects of gentamicin.
Abstract: In 1982, aminoglycosides still are widely prescribed and considered indispensable for the treatment of severe gram-negative infections. All the aminoglycosides are nephrotoxic, but both experimental works and clinical investigations indicate that they do not all have the same nephrotoxic potential. Within the renal tubular cell in several animal species and in man, the initial and the most extensive changes are those that occur in the lysosomes. We compared the effects of gentamicin, tobramycin, netilmicin and, amikacin on (a) lysosomal structural latency, (b) the activity of several enzymes, either lysosomal or those contained in the proximal tubular cell brush border, and (c) the accumulation of myeloid bodies in the lysosomes. From our results, it appears that gentamicin is the aminoglycoside that induces the greatest number of lysosomal changes whereas amikacin induces the least, with the effects of netilmicin and tobramycin quite close to those of amikacin. Other works comparing the nephrotoxicity of aminoglycosides reveal the same high nephrotoxic potential of gentamicin.
Abstract: Kidney cortex of rats and humans treated with low doses of Gentamicin have been examined both morphologically and biochemically. Specific alterations of the lysosomes (loss of activity of phospholipases; accumulation of phospholipid-like material) was detected as early as from 2-4 days of treatment. This finding is consistent with the concept that Gentamicin exerts its toxic action on kidney through lysosomal dysfunction.
Abstract: Gentamicin, a widely used aminoglycoside antibiotic, is concentrated in lysosomes of proximal tubular cells of the kidney, and induces therein an accumulation of myelin-like material. We show that treatment of rats with Gentamicin (10 mg/kg, 7 days) induces a loss of activity of lysosomal sphingomyelinase and phospholipase A1, associated with an increase in the amount of total lipid phosphorus in the kidney cortex. In vitro, Gentamicin is shown by gel permeation to bind to phospholipid bilayers (liposomes) under conditions which mimic the lysosomal environment (acid pH and presence of phosphatidylinositol). The reversal of this binding by an increase in the ionic strength (less than 0.04) suggests electrostatic interaction between the hydrophilic, polycationic aminoglycoside and the negatively charged phospholipids. Binding of Gentamicin impairs the hydrolysis of phosphatidylcholine present in the bilayer, by lysosomal phospholipases A1 and A2 from the liver or kidney. We also show that lysosomal sphingomyelinase is readily and irreversibly inactivated by liposomes in the absence of detergent. The lysosomal phospholipidosis induced by Gentamicin in the kidney, as in cultured cells [Aubert-Tulkens et al., Lab. Invest. 40, 481 (1979)] appears therefore to be a direct consequence of the lysosomotropic character of this drug and its ability to inhibit therein phospholipid breakdown. Amikacin, a semi-synthetic aminoglycoside, binds more loosely to phospholipid bilayers, induces less inhibition of phospholipases in vitro and is less taken up by tubular cells in vivo. Accordingly, Amikacin does not provoke significant lysosomal phospholipidosis or loss of sphingomyelinase and phospholipase A1 activities in vivo at the doses and time investigated (0-40 mg/kg, 7 days). Inasmuch as Amikacin is reported to be less toxic to the kidney, we suggest that lysosomal alterations are an early and significant step in aminoglycoside-induced nephrotoxicity.
Abstract: The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5'-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez-Saura, P., Trouet, A. and Tulkens, P. (1978) Biochim. Biophys. Acta 543, 430-449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4 degrees C and fractionated. Alkaline phosphodiesterase I and IgG showed superimposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome c reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm3, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome c reductase are increased only by 0.03 and 0.015 g/cm3, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome c reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.
Abstract: Cultured rat fibroblasts were used to compare the cellular toxicity of 4 aminoglycosides: accumulation of the antibiotic in the lysosomes, its adverse effect on phospholipases and consequently the intralysosomal accumulation of phospholipids. Gentamicin and Tobramycin are more noxious than Netilmicin and Amikacin. These data, when corrected for the lesser capture of Tobramycin by the kidney proximal tubules, closely match the toxicological evaluation of these aminoglycosides on the basis of animal and clinical studies.
Abstract: Subconjunctival injections of gentamicin induced a lysosomal storage process within the conjunctival fibroblasts in rats, rabbits, and humans. Under electron microscopy, the accumulated substance was composed of a granular material and pleomorphic lamellar structures, corresponding to the presence of complex lipids. In animals, the other ocular tissues as well as the cells reached through the bloodstream remained unaffected, except the proximal convoluted tubules of the kidneys, where important lesions were evident. Although human kidneys were not examined in our study, we believe they might present similar alterations.
Abstract: The cellular uptake and intracellular localization of indomethacin and ketoprofen were studied on rat embryo fibroblasts in vitro. The two drugs are taken up by the cells, but to a lesser extent for ketoprofen than for indomethacin. Inside fibroblasts ketoprofen is rapidly metabolized whereas indomethacin seems accumulated as such inside the cells. After different incubation times, ketoprofen is partly associated to the cytosol and partly to another cellular component, probably the endoplasmic reticulum. Indomethacin on the other hand is almost exclusively distributed inside the cytosol. No clear association of the two drugs can be found with the lysosomes.
Abstract: The uptake and processing by cultured rat embryo fibroblasts of control rabbit immunoglobulins (C IgG) or IgG directed against plasma membrane constituents (anti-PM IgG), and labeled with fluorescein (F) or with radioactive acetate (A), have been investigated by cell fractionation and immunological techniques. Both F and A anti-PM IgGs become bound to the cell surface, by a process that is slow, but largely temperature-independent. In the presence of an excess of high-affinity antibodies, binding reaches an absolute limit which corresponds to extensive coating of the plasma membrane. The anti-PM IgGs remain attached to the membrane for at least several days, even at 37 degrees C, with no significant transfer to lysosomes or degradation. In contrast, C IgGs are handled very differently by the fibroblasts, and their fate is strikingly affected by the type of labeling used. AC IgG is taken up slowly, at a rate proportional to its concentration, and is subsequently broken down in what appears to be lysosomes. Part of the AC IgG also binds to the plasma membrane. FC IgG is taken up many times faster than AC IgG, though with the same strict linearity as a function of concentration. Most of the FC IgG taken up is stored in cytoplasmic granules which behave like lysosomes. For reasons that are not understood, only about half of the stored FC IgG can be broken down. Cells exposed simulatnaously to AC IgG and FC IgG, or to A anti-PM IgG and FC IgG, handle each type of IgG in its characteristic fashion. Kinetic analysis of these results indicates that Ac IgG could be taken up by fluid endocytosis, but that FC IgG must be interiorized by a selective mechanism, presumably adsorptive in nature. That anti-PM antibodies remain stably bound to the plasma membrane and do not interfere with the uptake of FC IgG is interpreted to indicate either that two distinct membrane domains are involved in the two phenomena, or that membrane patches coated with anti-PM IgG participate in endocytosis, and are recycled back to the cell surface after delivering their contents intracellularly.
Abstract: Homogenates of HTC cells have been fractionated by differential centrifugation (in four particulate fractions: N, M, L, P, and a supernatant S) or isopycnic banding in linear sucrose gradients. On this basis, the following subcellular organelles may be characterized: (i) Mitochondria, detected by cytochrome oxidase and succinodehydrogenase, are collected in the M and L fractions, and equilibrate, as a narrow band, at a median buoyant density of 1.18 g/cm3. (ii) Lysosomes, detected by the latent hydrolases beta-glycerophosphatase and N-acetyl-beta-glucosaminidase, are largely sedimented in the M and L fractions, and display a broad density distribution pattern with a median value of 1.17 g/cm3. This density is decreased or increased after cultivation of the cells in presence of Triton WR-1339 or Dextran 500, respectively. The behavior of cathepsin D is somewhat at variance with that of the two other hydrolases. (iii) Plasma membrane is tentatively detected by alkaline phosphodiesterase I. Largely recovered in the P fraction, this enzyme equilibrates at a median density close to that of the lysosomal hydrolases; the bulk of cholesterol and about half of the leucyl-2-naphthylamidase are closely associated with alkaline phosphodiesterase I; HTC cells do not contain typical 5'-nucleotidase. (iv) Catalase-bearing particles, of high buoyant density (1.22 g/cm3) are present, but 30-40% of the catalase is also found readily soluble. NADPH- and NADH: cytochrome c reductase, and RNA show more complex distributions. It is suggested that the former enzyme is associated with the endoplasmic reticulum; as in liver, NADH reductase activity is shared between the endoplasmic reticulum and the mitochondria; half of the RNA is associated with free ribosomes of polysomes. True glucose-6-phosphatase could not be detected.
Abstract: A striking feature of endocytosis is the large amount of surface membrane that is brought into the cells through the formation of endocytic vesicles. Little is known about the fate of this membrane material. It is implausible that it would be destroyed in lysosomes, as the rate of turnover of the constituents of plasma membrane is much too low with respect to the rate of endocytosis in all cells studied so far. Conversely, plasma membrane fragments, internalized by endocytosis cannot merely be incorporated in lysosomes, as these organelles have been shown to maintain their size, despite continuous and active endocytosis. We present evidence that plasma membrane antigens, detected by means of specific antibodies, are internalized during endocytosis and reach lysosomes. They are thereafter returned back to cell surface. These results indicate the existence of a shuttle of membrane elements between the cell surface and lysosomes.
