Pharmacodynamics of Aerosolized Fosfomycin and Amikacin against Resistant Clinical Isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae in a Hollow-Fiber Infection Model: Experimental Basis for Combination Therapy

There has been a resurgence of interest in aerosolization of antibiotics for treatment of patients with severe pneumonia caused by multidrug-resistant pathogens. A combination formulation of amikacin-fosfomycin is currently undergoing clinical testing although the exposure-response relationships of these drugs have not been fully characterized. The aim of this study was to describe the individual and combined antibacterial effects of simulated epithelial lining fluid exposures of aerosolized amikacin and fosfomycin against resistant clinical isolates of Pseudomonas aeruginosa (MICs of 16 mg/liter and 64 mg/liter) and Klebsiella pneumoniae (MICs of 2 mg/liter and 64 mg/liter) using a dynamic hollow-fiber infection model over 7 days. Targeted peak concentrations of 300 mg/liter amikacin and/or 1,200 mg/liter fosfomycin as a 12-hourly dosing regimens were used. Quantitative cultures were performed to describe changes in concentrations of the total and resistant bacterial populations. The targeted starting inoculum was 108 CFU/ml for both strains. We observed that neither amikacin nor fosfomycin monotherapy was bactericidal against P. aeruginosa while both were associated with rapid amplification of resistant P. aeruginosa strains (about 108 to 109 CFU/ml within 24 to 48 h). For K. pneumoniae, amikacin but not fosfomycin was bactericidal. When both drugs were combined, a rapid killing was observed for P. aeruginosa and K. pneumoniae (6-log kill within 24 h). Furthermore, the combination of amikacin and fosfomycin effectively suppressed growth of resistant strains of P. aeruginosa and K. pneumoniae. In conclusion, the combination of amikacin and fosfomycin was effective at maximizing bacterial killing and suppressing emergence of resistance against these clinical isolates

Population Pharmacokinetics and Cerebrospinal Fluid Penetration of Fluconazole in Adults with Cryptococcal Meningitis

Robust population pharmacokinetic (PK) data for fluconazole are scarce. The variability of fluconazole penetration into the CNS is not known. A fluconazole PK study was conducted in 43 patients receiving oral fluconazole (usually 800 mg q24h) in combination with amphotericin B deoxycholate (1 mg/kg q24h) for cryptococcal meningitis (CM). A 4-compartment PK model was developed and Monte Carlo simulations performed for a range of fluconazole dosages. A meta-analysis of trials reporting outcomes of CM patients treated with fluconazole monotherapy was performed. Adjusted for bioavailability, the PK parameter means (standard deviation) were: clearance, 0.72 (0.24) litres/hour; volume of the central compartment, 18.07 (6.31) litres; volume of central nervous system (CNS) compartment, 32.07 (17.60) litres; first-order rate constant from central to peripheral compartment, 12.20 (11.17) hours-1; from peripheral to central compartment, 18.10 (8.25) hours-1; from central to CNS compartment 35.43 (13.74) hours-1; from CNS to central compartment 28.63 (10.03) hours-1 Simulations of area under concentration-time curve resulted in median (interquartile range) values 1143.2 mg.h/litre (988.4 - 1378.0) in plasma and 982.9 (781.0 - 1185.9) in CSF after a dosage of 1200mg q24h. The mean simulated ratio of AUCCSF:AUCplasma was 0.89 (SD 0.44). The recommended dosage of fluconazole for CM induction therapy fails to attain the PD target in respect to the wild-type MIC distribution of C. neoformans The meta-analysis suggested modest improvements in both CSF sterility and mortality outcomes with escalating dosage. This study provides the pharmacodynamic rationale for the long-recognised fact that fluconazole monotherapy is an inadequate induction regimen for CM

Comprehensive folate profiling in plants using LC-MS/MS

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Software for Dosage Individualization of Voriconazole: a Prospective Clinical Study.

Voriconazole is a first-line antifungal agent. Therapeutic drug monitoring is a standard of care. The best way to adjust dosages to achieve desired drug exposure endpoints is unclear due to nonlinear and variable pharmacokinetics. Previously described software was used to prospectively adjust voriconazole dosages. CYP2C19 and CYP3A4 and CYP3A5 genotype was determined. The primary endpoint was the proportion of patients with a Cmin at 120 hours in the range 1-3 mg/L using software to adjust voriconazole dosages. A total of 19 patients were enrolled and 14 were evaluable. Of these, 12/14 (85.7%, 95% CI 57.2 - 98.2%) had a Cmin at 120 hours post-treatment initiation of 1-3 mg/L, which was higher than the a priori expected proportion of 33%. There was no association of CYP genotype-derived metaboliser phenotype with voriconazole AUC. Software can be used to adjust the dosages of voriconazole to achieve drug exposures that are safe and effective

Oxidative Bioactivation of Abacavir in Subcellular Fractions of Human Antigen Presenting Cells

Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir’s electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir <i>in vitro</i> was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug’s protein-reactive aldehyde metabolites <i>in vivo</i>

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development.

International audienceCellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis