M. marinum isolates (1704 and 1705; kindly provided by Dr. Julian Davies, University of British Columbia) were routinely propagated at 30°C in Middlebrook 7H9 broth (Difco) supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC)(Difco), 0.2% glycerol and 0.05% (vol/vol) Tyloxapol or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC)(Difco). M. ulcerans strains S1012, S1013 and S1047 (isolated in 2010 and 2011 from Cameroonian BU patients) were routinely grown in BacT/Alert culture bottles using enrichment medium (bioMérieux) or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) OADC. Macrocyclic lactones were purchased from the following providers: abamectin and doramectin (Sigma), emamectin and eprinomectin (LKT Labs), ivermectin (Alpha Diagnostic), milbemycin oxime (US Pharmacopeia), moxidectin and selamectin (European Pharmacopoeia).
Doramectin
Manufactured by Merck Group
Sourced in United States, Germany, Belgium
Doramectin is a pharmaceutical product manufactured by Merck Group. It is a veterinary antiparasitic agent used to treat various parasitic infestations in livestock and companion animals. The core function of Doramectin is to provide effective control and treatment of parasites.
Automatically generated - may contain errors
Lab products found in correlation
Moxidectin, by Merck Group (4 mentions)
Abamectin, by Merck Group (3 mentions)
Selamectin, by Merck Group (3 mentions)
Ivermectin, by Merck Group (3 mentions)
Ivermectin, by Alpha Diagnostic (2 mentions)
Bact alert culture bottles, by bioMérieux (1 mentions)
Niclosamide, by Merck Group (1 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
Poroshell 120 ec c18 column, by Agilent Technologies (1 mentions)
Milli q sp reagent water system, by Merck Group (1 mentions)
Methanol, by Biosolve (1 mentions)
Formic acid, by Biosolve (1 mentions)
Acetic acid, by Biosolve (1 mentions)
Acetonitrile uplc ms grade, by Biosolve (1 mentions)
Pimozide, by Merck Group (1 mentions)
Nicardipine hydrochloride, by Merck Group (1 mentions)
Oxethazaine, by Merck Group (1 mentions)
Menadione, by Merck Group (1 mentions)
Clofazimine, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
10 protocols using doramectin
1
Mycobacterium spp. Cultivation and Macrocyclic Lactones
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M. marinum isolates (1704 and 1705; kindly provided by Dr. Julian Davies, University of British Columbia) were routinely propagated at 30°C in Middlebrook 7H9 broth (Difco) supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC)(Difco), 0.2% glycerol and 0.05% (vol/vol) Tyloxapol or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC)(Difco). M. ulcerans strains S1012, S1013 and S1047 (isolated in 2010 and 2011 from Cameroonian BU patients) were routinely grown in BacT/Alert culture bottles using enrichment medium (bioMérieux) or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) OADC. Macrocyclic lactones were purchased from the following providers: abamectin and doramectin (Sigma), emamectin and eprinomectin (LKT Labs), ivermectin (Alpha Diagnostic), milbemycin oxime (US Pharmacopeia), moxidectin and selamectin (European Pharmacopoeia).
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M. marinum isolates (1704 and 1705; kindly provided by Dr. Julian Davies, University of British Columbia) were routinely propagated at 30°C in Middlebrook 7H9 broth (Difco) supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC)(Difco), 0.2% glycerol and 0.05% (vol/vol) Tyloxapol or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC)(Difco). M. ulcerans strains S1012, S1013 and S1047 (isolated in 2010 and 2011 from Cameroonian BU patients) were routinely grown in BacT/Alert culture bottles using enrichment medium (bioMérieux) or on Middlebrook 7H10 agar plates (Difco) supplemented with 10% (vol/vol) OADC. Macrocyclic lactones were purchased from the following providers: abamectin and doramectin (Sigma), emamectin and eprinomectin (LKT Labs), ivermectin (Alpha Diagnostic), milbemycin oxime (US Pharmacopeia), moxidectin and selamectin (European Pharmacopoeia).
2
Ivermectin and Doramectin Analysis by HPLC
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All reagents had known analytical purity. The water used was deionized and ultrafiltrated water.
Methanol for analysis from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy) was used for the extraction, while Aluminium oxide 90 (neutral alumina) from Merck (Merck KGaA, Darmstadt, Germany) was used for the preliminary chromatography phase. The following solvents were used for SPE column activation and elution as well as components of the HPLC mobile phase: methanol for HPLC from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy), acetonitrile for HPLC from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy), and dichloromethane for analysis from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy). Ivermectin with declared purity from Dr Ehrenstorfer (LGC Labor GmbH Augsburg, Germany) and doramectin with declared purity from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) were used, respectively, for the fortified specimen and as an internal standard.
For each new experiment, a mixture of solvents for dilution made of acetonitrile (56%), methanol (37%), and water (7%) (v/v) and the activated neutral alumina (100 g of neutral alumina moisturized with 6 mL of deionized water) were prepared before use.
All solvents and solutions used for the HPLC system were filtered on a 0.2 μm filter.
Methanol for analysis from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy) was used for the extraction, while Aluminium oxide 90 (neutral alumina) from Merck (Merck KGaA, Darmstadt, Germany) was used for the preliminary chromatography phase. The following solvents were used for SPE column activation and elution as well as components of the HPLC mobile phase: methanol for HPLC from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy), acetonitrile for HPLC from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy), and dichloromethane for analysis from Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy). Ivermectin with declared purity from Dr Ehrenstorfer (LGC Labor GmbH Augsburg, Germany) and doramectin with declared purity from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) were used, respectively, for the fortified specimen and as an internal standard.
For each new experiment, a mixture of solvents for dilution made of acetonitrile (56%), methanol (37%), and water (7%) (v/v) and the activated neutral alumina (100 g of neutral alumina moisturized with 6 mL of deionized water) were prepared before use.
All solvents and solutions used for the HPLC system were filtered on a 0.2 μm filter.
3
Compound Preparation for Biological Assays
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Niclosamide, doramectin, moxidectin and selamectin were purchased by Sigma-Aldrich (St. Louis, MI, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) at the final concentration of 1 mg/mL and stored at −20 °C until use (Table 2 ).
4
Doramectin Quantification in Fermentation
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Thirteen-day fermentation cultures were extracted with 9 volumes of methanol, followed by centrifugation at 16,000× g for 10 min. The liquid phase was passed through a 0.2 μm membrane filter and analyzed with an Agilent 1260 HPLC system (Agilent Inc., Santa Clara, CA, USA) using an Agilent Poroshell 120 Ec-C18 column (2.7 μm, 4.6 × 50 mm). The column was eluted at a flow rate of 1 mL/min for 10 min with acetonitrile:methanol:H2O (40:40:20). Metabolites were monitored at a wavelength of 245 nm. The production was calibrated with standard doramectin from Sigma-Aldrich Inc. (Indianapolis, IN, USA).
5
Dissolution of Anthelmintic Compounds
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Doramectin, moxidectin, and selamectin were bought by Sigma-Aldrich (Burlington, MA, USA). The compounds were dissolved in DMSO at a concentration of 1 mg/mL.
6
Optimized UPLC-MS Quantification of Anthelmintics
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All the standards (ivermectin (IVER), doramectin (DORA), moxidectin (MOXI), ivermectin-d2 (IVER-d2), and moxidectin-d3 (MOXI-d3)) were obtained from Sigma-Aldrich (Bornem, Belgium). All the standards were stored at ≤−15 °C. UPLC-MS grade acetonitrile, methanol, acetic acid, and formic acid obtained from Biosolve BV (Valkenswaard, The Netherlands) were used for the preparation of mobile phases. While, the ultrapure water used in the mobile phase was from a Milli-Q-SP reagent water system (Merck Millipore, Overijse, Belgium). Oasis Ostro® Protein Precipitation & Phospholipid Removal 96-well plates used for sample clean-up were purchased from Waters (Zellik, Belgium).
7
Screening FDA Pharmakon Library for Anthelmintic Compounds
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The FDA Pharmakon compound library was purchased from MicroSource Discovery Systems, Inc. (USA). Compounds were delivered in microplates (10 mM, dissolved in DMSO) and kept at -80°C until use. For in vivo studies, flunarizine hydrochloride, pimozide, nicardipine hydrochloride, oxethazaine, menadione, clofazimine, doramectin and metitepine mesylate were purchased from Sigma-Aldrich (Buchs, Switzerland) and fendiline hydrochloride, manidipine hydrochloride and lomerizine hydrochloride were purchased from Santa-Cruz Biotechnology (California, USA). Hanks Balanced Salt Solution (HBSS) was obtained from Gibco (Lucerne, Switzerland). Culture medium components for NTS and adult worms were obtained as follows: Medium 199 RPMI 1640 and penicillin (100 U/ml) and streptomycin (100 μg/ml) were purchased from Lubioscience (Lucerne, Switzerland) whereas inactivated fetal calf serum (iFCS) was purchased from Connectorate AG (Dietikon, Switzerland).
8
Mycobacterial Growth and Compound Screening
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Strains were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin, dextrose, and catalase (ADC) (Difco), and 0.5% glycerol. Middlebrook 7H10 agar plates (Difco) supplemented with 10% oleic acid, albumin, dextrose, and catalase (OADC) (Difco) and 0.2% glycerol were used for bacterial CFU enumeration. All strains were incubated at 35–37 °C. Compounds (and suppliers) were: abamectin, doramectin, clarithromycin, amikacin, and linezolid (Sigma-Aldrich, Steinheim, Germany); emamectin and eprinomectin (LKT Labs, Minnesota, MN, USA); ivermectin (Alpha Diagnostic, Texas, TX, USA); and milbemycin oxime, moxidectin, and selamectin (European Pharmacopeia). Stocks solutions (10 mg/mL) were dissolved in DMSO (except amikacin, which was dissolved in sterile water), aliquoted, and stored at −80 °C until use.
9
Optimized Endectocide Bioassay Protocol
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Ivermectin, doramectin and moxidectin chemical reference standard powders were obtained from Sigma-Aldrich (Bornem, Belgium). All the standards were stored at ≤ − 15 °C. A fresh working solution (WS) of 40, 20, 4, 2, 0.4, 0.2, and 0.04 µg/mL of the analyte’s concentration were prepared in phosphate buffer solution by serial dilution from the stock solution (1 mg/mL in dimethyl sulfoxide).
For the in-vitro study, 1000, 500, 100, 50, 25, 10, 5, and 1 ng/mL concentrations of the endectocide were prepared by transferring 25 µL of 40, 20, 4, 2, 1, 0.4, 0.2, and 0.04 µg/mL of analyte’s working solution, respectively into a tube containing 975 µL cattle whole blood (see Additional file1 : Table S1). Control blood meals consisted of dimethyl sulfoxide diluted (DMSO) in phosphate buffer solution (PBS) to match the concentration found in the highest drug group.
For the in-vitro study, 1000, 500, 100, 50, 25, 10, 5, and 1 ng/mL concentrations of the endectocide were prepared by transferring 25 µL of 40, 20, 4, 2, 1, 0.4, 0.2, and 0.04 µg/mL of analyte’s working solution, respectively into a tube containing 975 µL cattle whole blood (see Additional file
10
Macrocyclic Lactone Preparation Protocol
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Glutamate , piperazine and the macrocyclic lactones (selamectin, ivermectin, doramectin, emamectin, eprinomectin, abamectin and moxidectin) were purchased from Sigma‐Aldrich (Saint‐Quentin Fallavier, France). These macrocyclic lactones were first dissolved in DMSO at 10 mM and then diluted in Ringer solution to the required concentration with a final concentration of DMSO which did not exceed 1%. Glutamate was directly prepared in recording solution.
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