Probenecid effects on cephalexin pharmacokinetics and pharmacodynamics in healthy volunteers.

OBJECTIVES: We evaluated the effects of probenecid on the Pharmaco Kinetics (PK) and pharmacodynamics (PD) of oral cephalexin in healthy volunteers. METHODS: Cephalexin 1000 mg was administered orally to 11 healthy volunteers following a standardized meal, with and without probenecid 500 mg orally, on two separate days one week apart. Total plasma concentrations of cephalexin and probenecid over a 12 h period were measured by liquid chromatography tandem mass spectrometry. Standard pharmacokinetic measures and contemporary PK/PD targets were compared. RESULTS: Probenecid increased the mean (95% CI) cephalexin area under the concentration-time curve (AUC0-∞) 1.73-fold (1.61-1.85, p < 0.0001), peak concentration 1.37-fold (1.16-1.58, p < 0.01), time to peak concentration 1.45-fold (1.1-1.8, p < 0.01), and half-life 1.33-fold (1.03-1.62, p < 0.05). The effects resulted in clinically meaningful increases in the probability of PK/PD target attainment (PTA). As an example, the PTA of total concentrations above the minimum inhibitory concentration required to inhibit methicillin-susceptible Staphylococcus aureus isolates (MIC ≤ 8 mg/L) for 70% of a 6 h dose interval approached 100% for cephalexin + probenecid while for cephalexin alone it was <15%. CONCLUSIONS: Probenecid prolonged and flattened the plasma concentration-time curve, enhancing the probability of attaining PK/PD targets. Co-administration of probenecid may expand the clinical benefits of oral cephalexin.

Probenecid and food effects on flucloxacillin pharmacokinetics and pharmacodynamics in healthy volunteers.

OBJECTIVES: To measure the effect of probenecid, fasting and fed, on flucloxacillin pharmacokinetic and pharmacodynamic endpoints. METHODS: Flucloxacillin 1000 mg orally was given to 11 volunteers alone while fasting ('flucloxacillin alone'), and with probenecid 500 mg orally while fasting ('probenecid fasting') and with food ('probenecid fed'). Flucloxacillin pharmacokinetic and pharmacodynamic endpoints were compared. RESULTS: Probenecid, fasting and fed, increased free plasma flucloxacillin area under the concentration-time curve (zero to infinity) ∼1.65-fold (p < 0.01) versus flucloxacillin alone. Probenecid fed prolonged time to peak flucloxacillin concentrations ∼2-fold versus the other two regimens (p < 0.01). Probenecid fasting or fed increased free flucloxacillin concentrations exceeding 30%, 50% and 70% of the first 6, 8 and 12 h post-dose by 1.58- to 5.48-fold compared with flucloxacillin alone. As an example of this pharmacodynamic improvement, the probability of target attainment of free concentrations above the minimum inhibitory concentration for Staphylococcus aureus (0.5 mg/L) for 50% of a 6-hour dose interval was > 80% for flucloxacillin plus probenecid (fasting or fed) and < 20% for flucloxacillin alone. CONCLUSIONS: Probenecid increased flucloxacillin exposure, with predicted pharmacodynamic effects greater than pharmacokinetic effects because of the altered shape of the concentration-time curve. Probenecid may improve the applicability of oral flucloxacillin regimens.

In healthy volunteers, taking flucloxacillin with food does not compromise effective plasma concentrations in most circumstances.

It is usually recommended that flucloxacillin is given on an empty stomach. The aim of this study was to compare total and free flucloxacillin concentrations after oral flucloxacillin, given with and without food, based on contemporary pharmacokinetic and pharmacodynamic targets. Flucloxacillin 1000 mg orally was given to 12 volunteers, after a standardised breakfast and while fasting, on two separate occasions. Flucloxacillin concentrations over 12 hours were measured by liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters, and pharmacodynamic endpoints related to target concentration achievement, were compared in the fed and fasting states. For free flucloxacillin, the fed/fasting area under the concentration-time curve from zero to infinity (AUC0-∞) ratio was 0.80 (p<0.01, 90% CI 0.70-0.92), the peak concentraton (Cmax) ratio 0.51 (p<0.001, 0.42-0.62) and the time to peak concentration (Tmax) ratio 2.2 (p<0.001, 1.87-2.55). The ratios for total flucloxacillin concentrations were similar. The mean (90% CI) fed/fasting ratios of free concentrations exceeded for 30%, 50% and 70% of the first 6 hours post-dose were 0.74 (0.63-0.87, fed inferior p<0.01), 0.95 (0.81-1.11, bioequivalent) and 1.15 (0.97-1.36, fed non-inferior), respectively. Results for 8 hours post-dose and those predicted for steady state were similar. Comparison of probability of target attainments for fed versus fasting across a range of minimum inhibitory concentrations (MICs) were in line with these results. Overall, this study shows that food reduced the AUC0-∞ and Cmax, and prolonged the Tmax of both free and total flucloxacillin concentrations compared with the fasting state, but achievement of free concentration targets associated with efficacy was in most circumstances equivalent. These results suggest that taking flucloxacillin with food is unlikely to compromise efficacy in most circumstances.

The Broad-Spectrum Antimicrobial Potential of [Mn(CO)4(S2CNMe(CH2CO2H))], a Water-Soluble CO-Releasing Molecule (CORM-401): Intracellular Accumulation, Transcriptomic and Statistical Analyses, and Membrane Polarization.

AIMS: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. RESULTS: [Mn(CO)4S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. INNOVATION: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. CONCLUSIONS: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration. Rebound Track: This work was rejected during standard peer review and rescued by rebound peer review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Miguel Aon, Giancarlo Biagini, James Imlay, and Nigel Robinson. Antioxid. Redox Signal. 28, 1286-1308.

Carbon Monoxide Gas Is Not Inert, but Global, in Its Consequences for Bacterial Gene Expression, Iron Acquisition, and Antibiotic Resistance.

AIMS: Carbon monoxide is a respiratory poison and gaseous signaling molecule. Although CO-releasing molecules (CORMs) deliver CO with temporal and spatial specificity in mammals, and are proven antimicrobial agents, we do not understand the modes of CO toxicity. Our aim was to explore the impact of CO gas per se, without intervention of CORMs, on bacterial physiology and gene expression. RESULTS: We used tightly controlled chemostat conditions and integrated transcriptomic datasets with statistical modeling to reveal the global effects of CO. CO is known to inhibit bacterial respiration, and we found expression of genes encoding energy-transducing pathways to be significantly affected via the global regulators, Fnr, Arc, and PdhR. Aerobically, ArcA-the response regulator-is transiently phosphorylated and pyruvate accumulates, mimicking anaerobiosis. Genes implicated in iron acquisition, and the metabolism of sulfur amino acids and arginine, are all perturbed. The global iron-related changes, confirmed by modulation of activity of the transcription factor Fur, may underlie enhanced siderophore excretion, diminished intracellular iron pools, and the sensitivity of CO-challenged bacteria to metal chelators. Although CO gas (unlike H2S and NO) offers little protection from antibiotics, a ruthenium CORM is a potent adjuvant of antibiotic activity. INNOVATION: This is the first detailed exploration of global bacterial responses to CO, revealing unexpected targets with implications for employing CORMs therapeutically. CONCLUSION: This work reveals the complexity of bacterial responses to CO and provides a basis for understanding the impacts of CO from CORMs, heme oxygenase activity, or environmental sources. Antioxid. Redox Signal. 24, 1013-1028.

Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)].

This article describes in extended detail the methodology applied for acquisition of transcriptomic data, and subsequent statistical data modelling, published by Wilson et al. (2015) in a study of the effects of carbon monoxide-releasing molecule-3 (CORM-3 [Ru(CO)3Cl(glycinate)]) on heme-deficient bacteria. The objective was to identify non-heme targets of CORM action. Carbon monoxide (CO) interacts with heme-containing proteins, in particular respiratory cytochromes; however, CORMs have been shown to elicit multifaceted effects in bacteria, suggesting that the compounds may have additional targets. We therefore sought to elucidate the activity of CORM-3, the first water-soluble CORM and one of the most characterised CORMs to date, in bacteria devoid of heme synthesis. Importantly, we also tested inactive CORM-3 (iCORM-3), a ruthenium co-ligand fragment that does not release CO, in order to differentiate between CO- and compound-related effects. A well-established hemA mutant of Escherichia coli was used for the study and, for comparison, parallel experiments were performed on the corresponding wild-type strain. Global transcriptomic changes induced by CORM-3 and iCORM-3 were evaluated using a Two-Color Microarray-Based Prokaryote Analysis (FairPlay III Labeling) by Agilent Technologies (Inc. 2009). Data acquisition was carried out using Agilent Feature Extraction software (v6.5) and data normalisation, as well as information about gene products and their function was obtained from GeneSpring GX v7.3 (Agilent Technologies). Functional category lists were created using KEGG (Kyoto Encyclopedia of Genes and Genomes). Relevant regulatory proteins for each gene were identified, where available, using regulonDB and EcoCyc (World Wide Web). Statistical data modelling was performed on the gene expression data to infer transcription factor activities. The transcriptomic data can be accessed through NCBI's Gene Expression Omnibus (GEO): series accession number GSE55097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55097).

CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

AIMS: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools. RESULTS: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3. INNOVATION: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects. CONCLUSION: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

Analysis of the bacterial response to Ru(CO)3Cl(Glycinate) (CORM-3) and the inactivated compound identifies the role played by the ruthenium compound and reveals sulfur-containing species as a major target of CORM-3 action.

AIMS: Carbon monoxide (CO)-releasing molecules (CO-RMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically. One such application is antimicrobial activity; therefore, we aimed to characterize and compare the effects of the CO-RM, CORM-3, and its inactivated counterpart, where all labile CO has been removed, at the transcriptomic and cellular level. RESULTS: We found that both compounds are able to penetrate the cell, but the inactive form is not inhibitory to bacterial growth under conditions where CORM-3 is. Transcriptomic analyses revealed that the bacterial response to inactivated CORM-3 (iCORM-3) is much lower than to the active compound and that a wide range of processes appear to be affected by CORM-3 and to a lesser extent iCORM-3, including energy metabolism, membrane transport, motility, and the metabolism of many sulfur-containing species, including cysteine and methionine. INNOVATION: This work has demonstrated that both CORM-3 and its inactivated counterpart react with cellular functions to yield a complex response at the transcriptomic level. A full understanding of the actions of both compounds is vital to understand the toxic effects of CO-RMs. CONCLUSION: This work has furthered our understanding of how CORM-3 behaves at the cellular level and identifies the responses that occur when the host is exposed to the Ru compound as well as those that result from the released CO. This is a vital step in laying the groundwork for future development of optimized CO-RMs for eventual use in antimicrobial therapy.

On a functional equation model of transient cell growth.

A cell-growth model with applications to modelling the size distribution of diatoms is examined. The analytic solution to the model without dispersion is found and is shown to display periodic exponential growth rather than asynchronous (or balanced) exponential growth. It is shown that a bounding envelope (hull) of the solution to the model without dispersion takes the same shape as the limiting steady-size distribution to the dispersive case as dispersion tends to zero. The effect of variable growth rate on the shape of the hull is also discussed.