Combinations of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol Dihydrochloride (All Sigma-Aldrich) were prepared according to study schedules. Drug doses were prepared specifically for each subject based on body weight, collected not more than three days before dose delivery. Each animal’s specific drug dose was weighed in its solid state and mixed with fruit puree, either directly or after being dissolved into solution using the diluents appropriate for each drug: Rifampicin: water containing 20% Encapsin (Sigma-Aldrich); Pyrazinamide: water containing 1% Methyl Cellulose (Sigma-Aldrich). Four milliliters of fruit puree (‘Ella’s Kitchen’, apple and banana, UK) was drawn up into a sterile 5 mL luer lock syringe; this was then transferred via a syringe connector to the separate 5 mL luer lock syringe containing the measured drug. A uniform suspension of drug in puree was obtained by gently passing the suspension between the two syringes, after which the drug formulation syringe was capped and stored at 4–8 °C until use. Each formulation was used within three days of preparation. During each treatment block, animals received a daily dose of the appropriate test substance/regimen delivered via the oral route.
Pyrazinamide
Manufactured by Merck Group
Sourced in United States, United Kingdom
Pyrazinamide is a chemical compound used in laboratory settings. It functions as an antibiotic agent.
Automatically generated - may contain errors
Lab products found in correlation
Isoniazid, by Merck Group (18 mentions)
Rifampicin, by Merck Group (14 mentions)
Ethambutol, by Merck Group (10 mentions)
Clofazimine, by Merck Group (5 mentions)
Streptomycin, by Merck Group (5 mentions)
Levofloxacin, by Merck Group (4 mentions)
Rifampin, by Merck Group (4 mentions)
Pyrazinoic acid, by Merck Group (3 mentions)
Moxifloxacin, by Merck Group (3 mentions)
Rifabutin, by Merck Group (3 mentions)
Rifapentine, by Merck Group (3 mentions)
Amikacin, by Merck Group (2 mentions)
Cycloserine, by Merck Group (2 mentions)
Linezolid, by Merck Group (2 mentions)
P aminosalicylic acid, by Merck Group (2 mentions)
Nicotinic acid, by Merck Group (2 mentions)
Glycerol, by Thermo Fisher Scientific (2 mentions)
Tween 80, by Merck Group (2 mentions)
Kanamycin sulfate, by Thermo Fisher Scientific (2 mentions)
Bedaquiline, by Johnson & Johnson (2 mentions)
31 protocols using pyrazinamide
1
Oral Delivery of Anti-Tuberculosis Drugs
2
Mycobacterium tuberculosis H37Ra Drug Susceptibility
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Mycobacterium tuberculosis H37Ra was grown at 37°C in Middlebrook 7H9 liquid medium or on 7H11 agar plates supplemented with 10% (v/v) albumin–dextrose–catalase (ADC, Becton Dickinson, Sparks, MD, United States) plus 0.5% (v/v) glycerol, 0.25% (v/v) Tween 80. Plasmids p2NIL and pGOAL19 used in this study were obtained from Addgene (Cambridge, MA, United States). isoniazid (INH), rifampicin (RIF), streptomycin (SM), ethambutol, pyrazinamide (PZA), levofloxacin, amikacin, cycloserine, p-aminosalicylic acid (PAS), clofazimine (CFZ), tetracycline, linezolid, clarithromycin, and piperine were purchased from Sigma-Aldrich (St Louis, MO, United States). The drugs were dissolved in DMSO and further diluted to obtain the desired concentrations in culture media for drug susceptibility testing (see below).
3
Quantitative Analysis of Anti-TB Drugs
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Certified reference materials (>97% purity) of the drug analytes isoniazid, rifampicin, ethambutol, pyrazinamide, and bedaquiline were obtained from Sigma Aldrich. All solvents (methanol (MeOH), ethanol (EtOH), water (H2O), isopropanol (IPA), acetonitrile (ACN) and formic acid (FA)) were Optima™ LC-MS grade, obtained from Fischer Scientific.
The culture media for the cell culture procedure was prepared as previously described in Wishart et al.43 Dulbecco's modified Eagle's medium (DMEM) with high glucose (Sigma-Aldrich, Merck, UK) was supplemented with 10% fetal bovine serum (Fisher Scientific, Loughborough, UK), 1% penicillin/streptomycin (Fisher Scientific, UK), and 2 mMl -glutamine (Sigma-Aldrich, Merck UK).
The culture media for the cell culture procedure was prepared as previously described in Wishart et al.43 Dulbecco's modified Eagle's medium (DMEM) with high glucose (Sigma-Aldrich, Merck, UK) was supplemented with 10% fetal bovine serum (Fisher Scientific, Loughborough, UK), 1% penicillin/streptomycin (Fisher Scientific, UK), and 2 mM
4
LCMS Analysis of Pyrazinamide and Metabolite
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We purchased Liquid chromatography-mass spectrometry (LCMS) grade Pyrazinamide and Pyrazinoic acid (pure form drug), methanol, water, and acetonitrile from Sigma Aldrich, ltd.
5
Evaluating Tuberculosis Treatment in HIS-NSG Mice
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Cord blood-engrafted HIS-NSG mice were purchased from Jackson Laboratory. Briefly, Jackson laboratory injected human CD34+ stem cells i.v. into myeloablated NSG mice and 12 weeks later humanization was confirmed by presence of >25% huCD45+ in the peripheral blood. These HIS-NSG mice were Mtb infected at 12 weeks of age. Isoniazid (H: 30 mg/kg), rifampicin (R: 10 mg/kg) and pyrazinamide (Z: 125 mg/kg) (HRZ), and moxifloxacin (M: 100 mg/kg) were purchased from Sigma or ChemPacific. The regimens were formulated in 200 μl 0.4% methyl cellulose and administered 6 days per week by oral gavage starting at day 28 p.i. At 2 and 4 weeks after treatment start, serial dilutions of tissue homogenates were plated onto Middlebrook 7H11 agar supplemented with 10% OADC Enrichment (BD Biosciences). To reduce the effect of drug-carry over 0.4% w/v activated charcoal was added to Middlebrook 7H11 agar. CFUs were determined after 3–4 weeks of culture at 37°C.
6
M. bovis BCG and M. tuberculosis Cultivation
7
Cultivation and Drug Testing of Mycobacterium tuberculosis
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M. tuberculosis H37Ra (ATCC 25177) was procured from American Type Culture Collection (ATCC), Manassas, VA. Prior to each experiment M. tuberculosis stock was thawed and grown in Middlebrook 7H9 broth supplemented with 10% oleic acid-dextrose-catalase (OADC) (here termed “broth”) at 37°C under 5% CO2 and shaking conditions for 4 days to achieve log-phase growth (LPG). Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 33591) was purchased from ATCC. Prior to each experiment, MRSA was grown from stock, and a single colony was picked from the Mueller-Hinton agar plate and grown in Mueller-Hinton broth with rapid shaking at 37°C for 3 h. This log-phase MRSA culture was used in subsequent experiments.
Hollow-fiber cartridges were purchased from FiberCell (Frederick, MD). Amikacin was purchased from the Baylor University Medical Center pharmacy. Streptomycin and pyrazinamide were purchased from Sigma-Aldrich (USA). The drugs were serially diluted using broth to the drug concentrations required for study.
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Hollow-fiber cartridges were purchased from FiberCell (Frederick, MD). Amikacin was purchased from the Baylor University Medical Center pharmacy. Streptomycin and pyrazinamide were purchased from Sigma-Aldrich (USA). The drugs were serially diluted using broth to the drug concentrations required for study.
8
Preparation of First-Line Tuberculosis Drugs
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Ethambutol and pyrazinamide were obtained from Sigma‐Aldrich (Zwijndrecht, The Netherlands) and isoniazid was obtained from the hospital pharmacy (Rotterdam, The Netherlands). Compounds were dissolved in distilled water. Rifampicin was obtained from Sanofi‐Aventis (Gouda, The Netherlands), dissolved in the supplemented dissolvent, and further diluted in water.
9
Preparation of TB Drug Compounds
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Moxifloxacin and linezolid were purchased from Sequoia Research Products (Pangbourne, United Kingdom), while rifapentine, rifabutin, pyrazinamide, clofazimine, and isoniazid were purchased from Sigma-Aldrich (MO, USA). Rifampin was purchased from Gold Biotechnology (MO, USA). Kanamycin sulfate was purchased from Fisher Scientific (NH, USA). Stock solutions for all compounds except kanamycin were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C. Kanamycin solution was prepared in water. Rifalazil was synthetized by Bioduro (CA, USA). Bedaquiline was provided by Janssen Research and Development (NJ, USA).
10
Antimicrobial Compounds for Research
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Isoniazid (INH), rifampicin (RIF), streptomycin (STR), ethambutol (EMB), pyrazinamide (PZA), vancomycin (VAN), and methicillin (METH) were purchased from Sigma-Aldrich (St Louis, MO, USA).
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