Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein.
If tuberculosis therapy is to be shortened it is imperative that the sterilising activity of current and future anti-tuberculosis drugs is enhanced. Intracellular Mycobacterium tuberculosis (MTB) phagocytosed by macrophages may be a key subpopulation of bacteria that are less readily eliminated by therapy. Here we investigate whether macrophages provide MTB with a pharmacological sanctuary site, making them less susceptible to chemotherapy than extracellular bacilli. Intracellular drug activity was determined by a novel colorimetric method that measures the ability of a drug to protect A-THP1 cells from infection-mediated cell death by H37Rv. Extracellular bactericidal activity was determined by the microplate alamar blue assay (MABA). Further, the effect of P-glycoprotein (P-gp) expressed on macrophages on the intracellular kill of H37Rv was assessed. To screen the anti-tuberculosis drugs for P-gp substrate specificity, their toxicity and cellular accumulation were determined in CEM and CEM(VBL100) cells. Intracellular and extracellular anti-tuberculosis drug activity following 7-day treatment with isoniazid (mean EC(50)+/-SD: 36.7+/-2.2 and 57.2+/-2.5 ng/mL, respectively) and ethambutol (243+/-95 and 263+/-12 ng/mL, respectively) were similar. However, for rifampicin a higher concentration was required to kill intracellular (148+/-32 ng/mL) versus extracellular (1.27+/-0.02 ng/mL) bacilli. The P-gp inhibitor tariquidar, significantly increased intracellular kill of H37Rv by ethambutol and rifampicin and both of these drugs were shown to be substrates for P-gp using the P-gp overexpressing CEM(VBL100) cells. We observed a large discrepancy between intracellular and extracellular activity of rifampicin (but not with isoniazid or ethambutol). Several factors could have accounted for this including inoculum size, media and cell-mediated metabolism. These factors make the comparison of intracellular and extracellular drug activity complex. However, the intracellular assay described here has potential for studying the impact of host proteins (such as drug transporters) on the intracellular activity of drugs, and has been used successfully here to demonstrate that both rifampicin and ethambutol are substrates for P-gp.
Hartkoorn
Ruben C
RC
Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK. rchart@liverpool.ac.uk <rchart@liverpool.ac.uk>
Chandler
Becky
B
Owen
Andrew
A
Ward
Stephen A
SA
Bertel Squire
S
S
Back
David J
DJ
Khoo
Saye H
SH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000995:Antitubercular Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003124:Colorimetry; D024881:Drug Resistance, Bacterial; D024901:Drug Resistance, Multiple, Bacterial; D004977:Ethambutol / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D007538:Isoniazid / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D010078:Oxazines; D010587:Phagocytosis; D011804:Quinolines / Q000494:pharmacology; D012293:Rifampin / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014376:Tuberculosis / Q000188:drug therapy; D014966:Xanthenes
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000995:Antitubercular Agents; D007202:Indicators and Reagents; D010078:Oxazines; D011804:Quinolines; D014966:Xanthenes; C005843:resazurin; D004977:Ethambutol; C402343:tariquidar; D007538:Isoniazid; D012293:Rifampin