Linda A. Bester

Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis

ABSTRACT The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 μg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 μg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.

Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry suppliers in KwaZulu-Natal, South Africa.

OBJECTIVES Campylobacter jejuni isolated from broiler and layer chickens from registered abattoirs in KwaZulu-Natal, South Africa, were tested for their susceptibility to eight antibiotics. METHODS Using agar dilution, susceptibility to eight antibiotics was determined for C. jejuni recovered from the caeca. RESULTS A total of 155 isolates were collected of which 77 were identified as C. jejuni (broilers n = 56 and layers n = 21). Resistance was highest to tetracycline (broilers 98.2% and layers 100%) and ceftriaxone (broilers 96.4% and layers 100%). High susceptibility was found to ciprofloxacin (broilers 91% and layers 76%) and gentamicin (broilers 98% and layers 81%). Susceptibilities to each of the antibiotics for the broilers and layers, respectively, were: 50% and 57% for erythromycin, 45% and 24% for clarithromycin, 68% and 43% for ampicillin and 64% and 48% for nalidixic acid. Statistically significant differences were detected for the MIC(50) of gentamicin, ciprofloxacin and tetracycline between broilers and layers (P < 0.001) with the MIC(90) of gentamicin also of significant difference (P = 0.01). Multiresistance was detected in 23% and 43% of the isolates from broiler and layer chickens, respectively. CONCLUSIONS Mass therapy procedures used in animal husbandry have a potential impact on antibiotic resistance development in C. jejuni.

Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods.

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase‐producing Gram‐negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans‐complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT‐PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC‐FGH‐IJK, mexAB‐XY‐oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr‐1 gene conferring resistance to colistin was identified via WGS, trans‐complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI‐TOF MS, micro‐array and real‐time multiplex PCR hold much promise for the future as new detection tools.

NOTA: a potent metallo-β-lactamase inhibitor

Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa; Antimicrobial Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa; Biomedical Resource Unit, Westville Campus, University of KwaZulu-Natal, Durban, South Africa; Department of Virology, National Health Laboratory Service/University of KwaZulu-Natal, c/o Inkosi Albert Luthuli Central Hospital, Durban, South Africa; Science for Life Laboratory, Drug Discovery and Development Platform, and Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

Tissue distribution of pretomanid in rat brain via mass spectrometry imaging

Abstract 1. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) combines the sensitivity and selectivity of mass spectrometry with spatial analysis to provide a new dimension for histological analyses of the distribution of drugs in tissue. Pretomanid is a pro-drug belonging to a class of antibiotics known as nitroimidizoles, which have been proven to be active under hypoxic conditions and to the best of our knowledge there have been no studies investigating the distribution and localisation of this class of compounds in the brain using MALDI MSI. 2. Herein, we report on the distribution of pretomanid in the healthy rat brain after intraperitoneal administration (20 mg/kg) using MALDI MSI. Our findings showed that the drug localises in specific compartments of the rat brain viz. the corpus callosum, a dense network of neurons connecting left and right cerebral hemispheres. 3. This study proves that MALDI MSI technique has great potential for mapping the pretomanid distribution in uninfected tissue samples, without the need for molecular labelling.

Observational study of the prevalence and antibiotic resistance of Campylobacter spp. from different poultry production systems in KwaZulu-Natal, South Africa.

Campylobacter bacteria are important foodborne pathogens that cause acute diarrheal illness, and infection is often associated with contaminated poultry. In a blind observational study, the prevalence and resistance profiles of thermophilic Campylobacter strains collected from different poultry production systems were tested against the clinically used antibiotics ciprofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Campylobacter strains were isolated from chickens in rural production systems, a free-range commercial facility, and industrially raised broiler and egg-laying chickens all situated in KwaZulu-Natal, South Africa. Isolates were collected from the chicken cecae and were identified with conventional methods and tested for antibiotic resistance with the Clinical and Laboratory Standards Institute agar dilution method. The prevalence of Campylobacter spp. isolates in chickens was 68% (56 samples) in rural production, 47% (140 samples) in commercial free-range broilers, 47% (133 samples) in industrial broilers, and 94% (34 samples) in industrial layer hens. Isolates from the rurally raised chickens showed significantly (P < 0.01) less resistance against ciprofloxacin (7.9%), erythromycin (0%), and tetracycline (21.6%) than those from commercially produced chickens. Isolates from the commercially raised chickens (free range and industrial) were highly resistant to tetracycline (98.9 to 100%). The incidence of gentamicin and streptomycin resistance was 1.6 and 11.5%, respectively, in commercial free-range broilers, 1.7 and 16.4%, respectively, in industrially raised broilers, and 12.9 and 40%, respectively, in industrially raised layers. It is possible that variations among the poultry production systems, including antimicrobial usage, result in differences in antibiotic resistance profiles in Campylobacter.

MALDI MSI and LC‐MS/MS: Towards preclinical determination of the neurotoxic potential of fluoroquinolones

Fluoroquinolones are broad-spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague-Dawley rats, via Multicenter matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a sensitive LC-MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright

Determination of the antitubercular drug PA-824 in rat plasma, lung and brain tissues by liquid chromatography tandem mass spectrometry: application to a pharmacokinetic study.

A selective, sensitive and high performance liquid chromatography-tandem mass spectrometry (LC-(ESI)MS/MS) method has been developed and validated for the quantification of the potent antitubercular drug candidate, PA-824, in rat plasma, lung and brain tissues. Sample clean-up involved protein precipitation and solid-phase extraction. Chromatographic separation was performed on YMC Triart C18 column (150 mm × 3.0 mm, 3.0 μm). The method was validated over the concentration range of 75-1500 ng/mL for plasma, 50-1200 ng/g for lungs and 100-1500 ng/g for brain tissue. Evaluation of the pharmacokinetic properties of PA-824 utilized Sprague Dawley rats with a dosage of 20mg/kg at various time points. The new method was applied successfully for the determination of PA-824 with liquid desorption followed by liquid chromatography with ultra-high resolution quadrupole time-of-flight mass spectrometry in the different biological samples.

Clofazimine Contributes Sustained Antimicrobial Activity after Treatment Cessation in a Mouse Model of Tuberculosis Chemotherapy

ABSTRACT Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-μg/ml MIC for M. tuberculosis. Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.

Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo

Objectives The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. Methods We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. Results In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. Conclusions Our data suggest that clofazimines delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.