Addition of probenecid to oral ß-lactam antibiotics: a systematic review and meta-analysis.

OBJECTIVES: To explore the literature comparing the pharmacokinetic and clinical outcomes from adding probenecid to oral ß-lactams. METHODS: Medline and EMBASE were searched from inception to December 2021 for all English language studies comparing the addition of probenecid (intervention) with an oral ß-lactam [flucloxacillin, penicillin V, amoxicillin (±â€Šclavulanate), cefalexin, cefuroxime axetil] alone (comparator). ROBINS-I and ROB-2 tools were used. Data on antibiotic therapy, infection diagnosis, primary and secondary outcomes relating to pharmacokinetics and clinical outcomes, plus adverse events were extracted and reported descriptively. For a subset of studies comparing treatment failure between probenecid and control groups, meta-analysis was performed. RESULTS: Overall, 18/295 (6%) screened abstracts were included. Populations, methodology and outcome data were heterogeneous. Common populations included healthy volunteers (9/18; 50%) and those with gonococcal infection (6/18; 33%). Most studies were crossover trials (11/18; 61%) or parallel-arm randomized trials (4/18; 22%). Where pharmacokinetic analyses were performed, addition of probenecid to oral ß-lactams increased total AUC (7/7; 100%), Cmax (5/8; 63%) and serum t½ (6/8; 75%). Probenecid improved PTA (2/2; 100%). Meta-analysis of 3105 (2258 intervention, 847 control) patients treated for gonococcal disease demonstrated a relative risk of treatment failure in the random-effects model of 0.33 (95% CI 0.20-0.55; I2 = 7%), favouring probenecid. CONCLUSIONS: Probenecid-boosted ß-lactam therapy is associated with improved outcomes in gonococcal disease. Pharmacokinetic data suggest that probenecid-boosted oral ß-lactam therapy may have a broader application, but appropriately powered mechanistic and efficacy studies are required.

Activity of Chitosan and Its Derivatives against Leishmania major and Leishmania mexicana In Vitro.

There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages.

Activity of Amphotericin B-Loaded Chitosan Nanoparticles against Experimental Cutaneous Leishmaniasis.

Chitosan nanoparticles have gained attention as drug delivery systems (DDS) in the medical field as they are both biodegradable and biocompatible with reported antimicrobial and anti-leishmanial activities. We investigated the application of chitosan nanoparticles as a DDS for the treatment of cutaneous leishmaniasis (CL) by preparing two types of chitosan nanoparticles: positively charged with tripolyphosphate sodium (TPP) and negatively charged with dextran sulphate. Amphotericin B (AmB) was incorporated into these nanoparticles. Both types of AmB-loaded nanoparticles demonstrated in vitro activity against Leishmania major intracellular amastigotes, with similar activity to unencapsulated AmB, but with a significant lower toxicity to KB-cells and red blood cells. In murine models of CL caused by L. major, intravenous administration of AmB-loaded chitosan-TPP nanoparticles (Size = 69 ± 8 nm, Zeta potential = 25.5 ± 1 mV, 5 mg/kg/for 10 days on alternate days) showed a significantly higher efficacy than AmBisome® (10 mg/kg/for 10 days on alternate days) in terms of reduction of lesion size and parasite load (measured by both bioluminescence and qPCR). Poor drug permeation into and through mouse skin, using Franz diffusion cells, showed that AmB-loaded chitosan nanoparticles are not appropriate candidates for topical treatment of CL.

A low-volume LC/MS method for highly sensitive monitoring of phenoxymethylpenicillin, benzylpenicillin, and probenecid in human serum.

Background: The optimization of antimicrobial dosing plays a crucial role in improving the likelihood of achieving therapeutic success while reducing the risks associated with toxicity and antimicrobial resistance. Probenecid has shown significant potential in enhancing the serum exposure of phenoxymethylpenicillin, thereby allowing for lower doses of phenoxymethylpenicillin to achieve similar pharmacokinetic/pharmacodynamic (PK/PD) targets. We developed a triple quadrupole liquid chromatography mass spectrometry (TQ LC/MS) analysis of, phenoxymethylpenicillin, benzylpenicillin and probenecid using benzylpenicillin-d7 and probenecid-d14 as IS in single low-volumes of human serum, with improved limit of quantification to support therapeutic drug monitoring. Methods: Sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using TQ LC/MS. The mobile phase consisted of 55% methanol in water + 0.1% formic acid, with a flow rate of 0.4 mL min-1. Antibiotic stability was assessed at different temperatures. Results: Chromatographic separation was achieved within 2 minutes, allowing simultaneous measurement of phenoxymethylpenicillin, benzylpenicillin and probenecid in a single 15 µL blood sample. Validation indicated linearity over the range 0.0015-10 mg L-1, with accuracy of 96-102% and a LLOQ of 0.01 mg L-1. All drugs demonstrated good stability under different storage conditions. Conclusion: The developed method is simple, rapid, accurate and clinically applicable for the quantification of phenoxymethylpenicillin, benzylpenicillin and probenecid in tandem.

Triple quadrupole LC/MS method for the simultaneous quantitative measurement of cefiderocol and meropenem in serum.

Background: therapeutic drug monitoring is a crucial aspect of the management of hospitalized patients. The correct dosage of antibiotics is imperative to ensure their adequate exposure specially in critically ill patients. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC/MS) method for the simultaneous quantification of two important antibiotics in critically ill patients, cefiderocol and meropenem in human plasma. Methods: sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using triple quadrupole LC/MS. The mobile phase was consisted of 55% methanol in water +0.1% formic acid, with flow rate of 0.4 ml min-1. Antibiotics stability was assessed at different temperatures. Serum protein binding was assessed using ultrafiltration devices. Results: chromatographic separation was achieved within 1.5 minutes for all analytes. Validation has demonstrated the method to be linear over the range 0.0025-50 mg L-1 for cefiderocol and 0.00028-50 mg L-1 for meropenem, with accuracy of 94-101% and highly sensitive, with LLOQ ≈ 0.02 mg L-1 and 0.003 mg L-1 for cefiderocol and meropenem, respectively. Both cefiderocol and meropenem showed a good stability at room temperature over 6 h, and at (4 °C) over 24 h. Cefiderocol and meropenem demonstrated a protein binding of 49-60% and 98%, respectively in human plasma. Conclusion: the developed method is simple, rapid, accurate and clinically applicable for the quantification of cefiderocol and meropenem.

A rapid, simple, high-performance liquid chromatography method for the clinical measurement of beta-lactam antibiotics in serum and interstitial fluid.

Background: enhanced methods of therapeutic drug monitoring are required to support the individualisation of antibiotic dosing based on pharmacokinetics (PK) parameters. PK studies can be hampered by limited total serum volume, especially in neonates, or by sensitivity in the case of critically ill patients. We aimed to develop a liquid chromatography-mass spectrometry (LC/MS) analysis of benzylpenicillin, phenoxymethylpenicillin and amoxicillin in single low volumes of human serum and interstitial fluid (ISF) samples, with an improved limit of detection (LOD) and limit of quantification (LOQ), compared with previously published assays. Methods: sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using triple quadrupole LC/MS. The mobile phase consisted of 55% methanol in water + 0.1% formic acid, with a flow rate of 0.4 mL min-1. Antibiotics stability was assessed at different temperatures. Results: chromatographic separation was achieved within 3 minutes for all analytes. Three common penicillins can now be measured in a single low-volume blood and ISF sample (15 µL) for the first time. Validation has demonstrated the method to be linear over the range 0.0015-10 mg L-1, with an accuracy of 93-104% and high sensitivity, with LOD ≈ 0.003 mg L-1 and LOQ ≈ 0.01 mg L-1 for all three analytes, which is critical for use in dose optimisation/individualisation. All evaluated penicillins indicated good stability at room temperature over 4 h, at (4 °C) over 24 h and at -80 °C for 6 months. Conclusion: the developed method is simple, rapid, accurate and clinically applicable for the quantification of three penicillin classes.

Rapid Measurement of Lactate in the Exhaled Breath Condensate: Biosensor Optimization and In-Human Proof of Concept.

Lactate concentration is of increasing interest as a diagnostic for sepsis, septic shock, and trauma. Compared with the traditional blood sample media, the exhaled breath condensate (EBC) has the advantages of non-invasiveness and higher user acceptance. An amperometric biosensor was developed and its application in EBC lactate detection was investigated in this paper. The sensor was modified with PEDOT:PSS-PB, and two different lactate oxidases (LODs). A rotating disk electrode and Koutecky-Levich analysis were applied for the kinetics analysis and gel optimization. The optimized gel formulation was then tested on disposable screen-printed sensors. The disposable sensors exhibited good performance and presented a high stability for both LOD modifications. Finally, human EBC analysis was conducted from a healthy subject at rest and after 30 min of intense aerobic cycling exercise. The sensor coulometric measurements showed good agreement with fluorometric and triple quadrupole liquid chromatography mass spectrometry reference methods. The EBC lactate concentration increased from 22.5 µM (at rest) to 28.0 µM (after 30 min of cycling) and dropped back to 5.3 µM after 60 min of rest.

Development of an in vitro media perfusion model of Leishmania major macrophage infection.

BACKGROUND: In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. METHODS: Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360µl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. RESULTS: Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10-9 m/s and 1.23 x 10-7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. CONCLUSIONS: Perfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.