Rapid Microbiologic and Pharmacologic Evaluation of Experimental Compounds against Mycobacterium tuberculosis

Abstract

The assessment of physiochemical and pharmacological properties at early stages of drug discovery can accelerate the conversion of hits and leads into candidates for further development. A strategy for streamlined evaluation of compounds against Mycobacterium tuberculosis in the early preclinical stage is presented in this report. As a primary assay to rapidly select experimental compounds with sufficient in vitro activity, the growth inhibition microtiter plate assay was devised as an alternative to current methods. This microdilution plate assay is a liquid culture method based on spectrophotometric readings of the bacillary growth. The performance of this method was compared to the performance of two established susceptibility methods using clinical available tuberculosis (TB) drugs. Data generated from all three assays were similar for all of the tested compounds. A second simple bioassay was devised to assess the oral bioavailability of compounds prior to extensive in vivo efficacy testing. The bioassay estimates drug concentrations in collected serum samples by a microdilution MIC plate method using M. tuberculosis. In the same assay, the MIC of the compound is also determined in the presence of 10% mouse serum as an indication of protein binding. The method was validated using different clinically available TB drugs, and results are discussed in this report. With these methodological advances, screening of compounds against tuberculosis in the preclinical phase will be rapid, can be adapted to semi-high-throughput screening, and will add relevant physicochemical and basic pharmacological criteria to the decision process of drug discovery.The pharmaceutical process to develop a therapeutically useful drug requires an enormous budget and amount of time, due to high attrition rates of experimental compounds in preclinical and clinical development (2, 10). Pharmaceutical companies nowadays are focusing on reducing these preclinicaldevelopment attrition rates by attempting to accurately evaluate efficacy, safety, and drug manufacturing costs much earlier in the drug discovery process. A published survey on the causes of failure in drug development indicated that inappropriate pharmacokinetics were a major cause (19). This observation has led to an increased emphasis on pharmacokinetic input to the drug discovery process throughout the pharmaceutical industry (9,20,21).Traditional assays and models for early preclinical screening of experimental compounds against Mycobacterium tuberculosis are lengthy due to the slow-growing nature of the bacteria. In addition, many of the current in vitro and in vivo assays for drug testing against tuberculosis (TB) are not well adapted for a higher throughput approach, which is necessary for rapid screening of compound series from different drug classes. Generally, compounds are first evaluated in vitro for activity against M. tuberculosis by determination of their MICs with either the BACTEC system (4, 16), the agar proportion method (7, 13, 25), or a microdilution assay mic...

Importance of Confirming Data on the In Vivo Efficacy of Novel Antibacterial Drug Regimens against Various Strains of Mycobacterium tuberculosis

“…Drug susceptibility testing by agar proportion or Bactec MGIT 960 system and pncA sequencing were performed by the CDC. Additional MIC testing was performed in our laboratory as previously described (16) to obtain exact MIC endpoints. Spoligotyping revealed the following spoligotypes: HN878, 000000000003771; CSU H37Rv, 777777475760771; JHU H37Rv, 777777475760771; Erdman, 777777774020771; and CDC 1551, 700076757760771.…”

Comparative Studies Evaluating Mouse Models Used for Efficacy Testing of Experimental Drugs against Mycobacterium tuberculosis

“…Drug susceptibility testing of the M. tuberculosis Erdman strain used was recently performed for PZA and moxifloxacin at the Mycobacteriology Laboratory at the National Jewish Health, Denver, CO, by Bactec (17,18). MIC testing for isoniazid and rifampin was performed by the microdilution plate method (16). The pncA gene was sequenced by the U.S. Centers for Disease Control and Prevention (Atlanta, GA).…”

Allelochemicals produced by Caribbean macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms

“…Growth was classified as inhibited/stimu- bacteria using a microdilution plate assay, a liquid culture method based on spectrophotometric readings of cellular growth developed by Gruppo et al (2006). First, bacteria were re-streaked onto fresh FSWA 48 h prior to experimentation.…”