Search for New Drugs for Treatment of Tuberculosis

The purpose of this brief commentary is to describe the growing effort to discover new drugs that can be used to treat disease caused by Mycobacterium tuberculosis. In this regard, it is estimated that over 8 million people contract tuberculosis each year, and approximately 2 to 3 million people die of this disease (6). In addition, it is thought that as many as 2 billion people have been exposed to the tuberculosis bacillus and are therefore at risk of developing active disease. In certain areas of the world, such as countries within the former Soviet Union, the case rates are climbing alarmingly. This problem is further compounded by a dramatic increase in multidrug-resistant strains of M. tuberculosis (10). An additional factor is human immunodeficiency virus, which has significantly increased the incidence of tuberculosis in sub-Saharan Africa and elsewhere (5). While the long-term solution is a better vaccine (14), for the next several decades there will have to be continued reliance on chemotherapy. In this regard, however, there have been few additions to the existing “main-line” drugs for a considerable time. While there are some promising new agents, such as the longer-acting rifamycins, fluoroquinolones, oxazolidinones, and nitroimidazopyrans (1, 4, 7, 8, 11–13, 15, 17–19), there are as yet no extensive clinical trial data to determine the efficacy of these compounds. Finding new drugs represents a challenge. Therapy currently takes a considerable time, and it is hoped that new compounds can be found that will reduce the total duration of treatment regimens. Drugs are also needed that will be effective against the growing number of drug-resistant strains and against bacilli that may be in a state of latency. As part of the overall research effort at the National Institute of Allergy and Infectious Diseases, the National Institutes of Health has established a screening program at several institutions to efficiently screen large numbers of compounds for possible activity against M. tuberculosis. An acquisitions program receives from both academic and commercial sources compounds which are initially screened for in vitro activity. To date, over 50,000 compounds have been provided to the program (Fig. 1). Each compound is first screened for activity at 6.25 mg/ml (or the molar equivalent of the highest-molecular-weight compounds in a series of congeners) against cultures of M. tuberculosis H37Rv in BACTEC 12B medium using a microplate alamar blue assay (2). Compounds that are active in this assay are reconfirmed using a BACTEC 460 radiometric system. In the next stage of the procedure, the MIC of the compound is determined. In addition, the compound evaluated for cytotoxicity in cultures of Vero cells to determine a 50% inhibitory concentration (IC50). A selectivity index (SI) can then be calculated by dividing the IC50 by the MIC; if the SI is . 10, the compound is then evaluated further. The next stage of screening uses the bone marrow macrophage infection assay (16) to determine if the compound can inhibit the growth of M. tuberculosis in its intracellular environment. Cultures of bone marrow-derived macrophages are infected with M. tuberculosis and then cultured for 7 to 8 days in the presence of a range of concentrations of the test compound. The monolayers are then lysed and plated on 7H11 agar to determine the numbers of surviving bacteria. By plotting the bacterial load in each culture against the drug concentration, one can determine whether a compound is bacteriostatic or bactericidal under these relevant physiological conditions. The concentration of drug reducing the bacterial load by 1 log unit (90% reduction [EC90]) or by 2 log units (99% reduction [EC99]) is recorded as a numerical readout (Fig. 2). Compounds that perform well in the macrophage infection assay are then tested in a mouse model if sufficient amounts of the compounds are available. After the maximum tolerated dose for a compound is determined, mice are exposed to a low-dose aerosol infection with M. tuberculosis (9). Twenty days later, as the bacterial load reaches its peak in control animals, the test compound is administered daily at appropriate dosages for 45 days. The route of administration is usually gavage, although subcutaneous or intraperitoneal injection can sometimes be given for a limited time. At 15, 30, and 45 days of therapy, groups of five mice are removed and euthanatized. The lungs and spleen are homogenized and plated on agar to determine the bacterial load. A positive control group of mice is given isoniazid (25 mg/kg of body weight/day) for comparison. The bacterial load for each test group is then plotted against time and compared to the values for untreated control mice (Fig. 3). Under these conditions, a reduction of 0.7 log unit or greater is considered statistically significant, as determined by analysis of variance. * Corresponding author, Ian Orme. Mailing address: Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-5777. Fax: (970) 491-5125. E-mail: iorme@lamar.colostate. edu. Other members are J. Secrist, S. Anathan, C. Kwong, J. Maddry, and R. Reynolds (all at Southern Research Institute, Birmingham, Ala.); A. Poffenberger, M. Michael, and L. Miller (all at Southern Research Institute, Frederick, Md.); J. Krahenbuh, L. Adams, and A. Biswas (all at National Hansen’s Disease Laboratory Research Branch, Baton Rouge, La.); S. Franzblau (at Institute for Tuberculosis Research, University of Illinois); D. Rouse and D. Winfield (at Research Triangle Institute, Research Triangle Park, N.C.); and J. Brooks (at Colorado State University).