Antimicrobial Agents and Chemotherapy, July 2001, p. 1943-1946, Vol. 45, No. 7
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).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.1943-1946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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FIG. 1.
Progress of the in vitro tuberculosis drug screening
program. Level 1 testing determines the percent inhibition (PI) for
compounds; drugs having a PI of >90 advance to level 2. An initial
cutoff of 12.5 µg/ml was amended to 6.25 µg/ml to reduce the number
of compounds that could be screened further. Level 2 compounds are then
further selected on the basis of the SI (SI equals
IC50/MIC) for further testing in the macrophage infection
and mouse therapy assays.
Each compound is first screened for activity at 6.25 µg/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).
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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. Compounds that
provide this level of reduction are defined as active in this in vivo model.
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Additional in vivo models are under development in this program. These include use of gamma interferon gene knockout mice, in which the bacterial load in the lungs cannot be prevented from reaching very high levels (3). In current evaluation studies, these mice are given a short course of treatment for 10 days to determine if such treatment is sufficient to detect positive drug activity and thus can be used as a new model with a much shorter duration than the standard assay.
To date, 11% of the compounds tested (5,251 compounds), have been highly active in vitro and 453 have an SI of >10. Of these, 340 have undergone evaluations in the macrophage infection model, and 53 have been tested in vivo. Of these, nine, from various compound classes, have been found to significantly reduce the bacterial load in the lungs of infected mice. While these results are encouraging, it is clear that much more work needs to be done, including the acquisition and testing of many more test compounds if the ultimate goal is to be achieved. Institutions or organizations possessing compounds that may have antimycobacterial properties are encouraged to visit www.taacf.org.
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ACKNOWLEDGMENTS |
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This work was supported by NIH programs AI-95384, AI-95385, and AI-95364 and interagency agreement AI-5106-04 (supervised by Barbara Laughon, NIAID).
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FOOTNOTES |
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* 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{at}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).
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.
The members of the Tuberculosis Drug Screening
Program 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. Krahenbuhl, 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 and I. Orme (at Colorado State University, ).
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Antimicrobial Agents and Chemotherapy, July 2001, p. 1943-1946, Vol. 45, No. 7
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.1943-1946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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