BM635

Cell experiment:

The measurement of the minimum inhibitory concentration (MIC) for each tested compounds (including BM635) are performed in 96 wells flat-bottom, polystyrene microtiter plates. Ten two-fold drug dilutions in neat DMSO starting at 200 µM are performed. Five µL of these drug solutions are added to 95 µL of Middlebrook 7H9 medium. Isoniazid is used as a positive control, 8 two-fold dilutions of Isoniazid starting at 160 µg/mL are prepared and 5 µL of this control curve is added to 95 µL of Middlebrook 7H9 medium. Five µL of neat DMSO are added 95 µL of Middlebrook 7H9 medium in row 12 (growth and Blank controls). The inoculum is standardized to approximately 1×107 cfu/mL and diluted 1 in 100 in Middlebrook 7H9 broth to produce the final inoculum of H37Rv strain. One hundred µL of this inoculum is added to the entire plate but G-12 and H-12 wells (Blank controls). All plates are placed in a sealed box to prevent drying out of the peripheral wells and they are incubated at 37°C without shaking for six days. A resazurin solution is prepared by dissolving one tablet of resazurin in 30 mL sterile PBS (phosphate buffered saline). Of this solution, 25 µL are added to each well. Fluorescence is measured after 48 hours to determine the MIC value[1].

Animal experiment:

Mouse: Nine (one per dose) 8-10 week old B6 female mice are infected by intratracheal route with 105CFU H37Rv per mouse suspended in 50 µL phosphate buffer saline. BM635 is administered once a day at nine doses ranging from 40 to 300 mg/kg from day 1 to day 8 after infection, and 24 hours after the last dose the mice are sacrificed. To measure the infection burden in lungs, all lobes are aseptically removed and homogenized. The homogenates are supplemented with 5% glycerol and stored frozen (−80°C) until plating. Plates (10%OADC-7H11 medium) are incubated for 14 days at 37°C[1].

References:

[1]. Poce G, et al. Improved BM212 MmpL3 inhibitor analogue shows efficacy in acute murine model of tuberculosis infection. PLoS One. 2013;8(2):e56980.
[2]. Poce G, et al. Pharmaceutical salt of BM635 with improved bioavailability. Eur J Pharm Sci. 2017 Mar 1;99:17-23.