Screening Assays

in vitro TB	in vivo TB	M. avium
% Inhibition MIC Cytotoxicity Macrophage SDR-MIC MBC	MTD GKO murine aerosolized TB model	Preliminary MIC Expanded MIC Macrophage in vivo

in vitro Evaluation of Anti-Mycobacterium tuberculosis Activity

% Inhibition

Primary screening is conducted at 6.25 µg/ml (or molar equivalent of highest molecular weight compound in a series of congeners) against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA).1 Compounds exhibiting fluorescence are tested in the BACTEC 460-radiometric system.1 Compounds effecting <90% inhibition in the primary screen (MIC >6.25 µg/ml) are not generally evaluated further.  Minimum compound requirements: 1.0 mg.

MIC

Compounds demonstrating at least 90% inhibition in the primary screen are re-tested at lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration (MIC) in the MABA. The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to controls.  Minimum compound requirements: 1.0 mg.

Cytotoxicity

Concurrent with the determination of MICs, compounds are tested for cytotoxicity (IC50) in VERO cells at concentrations less than or equal to 62.5 mg/ml or 10 times the MIC for M. tuberculosis H37Rv.  After 72 hours exposure, viability is assessed on the basis of cellular conversion of MTT into a formazan product using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay.  Minimum compound requirements: 2.0 mg.

Macrophage

Compounds for which the IC50:MIC (SI) ratio is ³10 will have in vitro activity confirmed in the BACTEC 460 at 6.25 mg/ml.  Compounds are then tested for killing of M. tuberculosis Erdman (ATCC 35801) in monolayers of mouse bone marrow macrophages.3 (EC99 and EC90; lowest concentration effecting a 90% and 99% reduction in colony forming units at 7 days compared to drug-free controls) at 4-fold concentrations equivalent to 0.25, 1, 4 and 16 times the MIC. Minimum compound requirements: 3.0 mg.

Single Drug Resistant Strain MIC & MBC

Concurrent with the testing of compounds in macrophages, MICs are determined in the MABA for three strains of drug-resistant M. tuberculosis, (each strain resistant to a single TB drug) as well as M. avium. Typically, all compounds progressing to this stage of screening will be tested against M. avium (ATCC 25291), M. tuberculosis strains resistant to isoniazid (ATCC 35822), rifampin (ATCC 35838), and one other drug resistant strain (the latter determined by the compound type) as well as the drug-sensitive strains H37Rv and Erdman. Minimum bactericidal concentration (MBC) is then determined for M. tuberculosis H37Rv and Erdman (and the corresponding drug-resistant strain for analogs of known antitubercular drugs) by subculturing onto drug-free solid media following exposure in supplemented Middlebrook 7H9 media to drug concentrations equivalent to and higher than the previously determined MICs of the respective strains. Minimum compound requirements: 7 mg.

in vivo Evaluation of Anti-Mycobacterium tuberculosis Activity

Maximum Tolerated Dose (MTD) Determination

The screen is designed to determine the maximally tolerated dose of experimental compounds.

C57BL/6 female mice (6-8 weeks in age) are administered a one-time dose (oral gavage) of the compound at concentrations of 100, 300 or 1000 mg/Kg. The compounds are dissolved in an appropriate vehicle (ETOH, DMSO or methylcellulose), administered in a solution if necessary. There are 3 animals per dose and they are observed post-administration for 4 hours again 6 hours later then twice daily for the duration of the study (1 week). If an animal exhibits obvious signs of distress (hunched posture, ruffled fur etc.), it is euthanized. The surviving mice are sacrificed day 7 post-administration and the critical organs are observed for evidence of drug toxicity. If abnormalities exist or there were other animals in the same group which did not survive to day 7, the tissues from the liver, heart, and kidneys are extracted and placed into 10% formalin solution. These fixed tissues are sectioned and examined for abnormalities resulting from drug toxicity. The MTD (mg/Kg) is the highest dose that results in no lethality/tissue abnormality.

Gamma Knock-out (GKO) Model

Interferon gamma knockout mice (unable to control an infection of MTB) from a C57BL/6 background are infected in the standard manner (aerosol infection (Erdman strain) utilizing the Glas-Col Inhalation Exposure System). Five mice are sacrificed day 1 post-infection to determine bacterial uptake. Day 20 post-infection 5 mice are sacrificed to determine bacterial load in the lungs and spleens. Therapy, administered via oral gavage, i.p. injection or alzet osmotic pumps, begins day 20 post-infection and continues for 10 days until day 30. Day 30 post-infection the mice are sacrificed and bacterial loads are determined. An INH control group, administered via oral gavage at 25 mg/Kg/day is included in each study. Log 10 protection values >0.30 indicate activity. INH has demonstrated values around 2.75.

Murine Aerosolized TB Model

The purpose of this program is to provide a resource whereby new experimental compounds can be tested for their capacity to inhibit the growth of virulent M. tuberculosis in a realistic in vivo aerosol mouse function model.2

Mice are exposed to an aerosol of M. tuberculosis Erdman, which deposits approximately 50 bacilli into the lungs of the animal. The course of this infection is then followed in the lungs and spleen for 50 days by plating homogenates of harvested organs [n=5] on nutrient agar and determining bacterial numbers. As the growing infection is slowly controlled and contained, a peak number of about log 5.0 can be observed in the infected lungs.

Test compounds are administered to groups of mice starting on day 20 post-inoculation.  Typically are administered either i.p. or by oral gavage.  At this point in the screening program the supplier is consulted regarding available information on compound formulation and/or animal bioavailability and toxicity data.   Depending on the route of administration, three dose levels of drug are given twice a day; an additional group is given isoniazid as a positive control. Any effect the compound may be having on bacterial numbers is then assessed on days 35 and 50, and compared to untreated control values on these days. The data is expressed as the log 10 protection provided by a given dose of the compound against the growth of the organism in the untreated control group. Statistical tests are also applied to the raw data to determine levels of significance. Minimum compound requirements: 120 mg.

Evaluation of Anti-Mycobacterium avium Activity

Higher level evaluation of compounds against M. avium is available for compounds showing a MIC £ 6.25 µg/ml against M. avium (described in "Concurrent with the testing of..." above). The available screens and compound requirements are outlined below:

M. avium Preliminary Assay

For compounds demonstrating our criteria for Level III TB testing, MICs are determined in the MABA against a single strain of

 M. avium (ATCC 25291).

M. avium Primary Assay

Primary screening is conducted at a range of 1

 µg/ml to 64 µg/ml against 5 clinical isolates of Mycobacterium avium (100, 101, 108, 109, 116) in Middlebrook 7H9 broth using the Microplate Alamar Blue Assay and in the BACTEC 460-radiometric system. Minimum compound requirements: 5.0 mg.

M. avium Expanded Assay

Compounds demonstrating our criteria for expanded testing (MIC

 <8 µg/ml for at least 3 of the 5 strains tested) are re-tested at lower concentrations against 30 strains of M. avium, including 5 strains that are resistant to Clarithromycin (MIC>32µg/ml). Minimum compound requirements: 10 mg.

M. avium Macrophage Assay

Compounds demonstrating primary

 in vitro screening activity are tested for their anti- M.avium activity in an intracellular model using a human monocyte cell line, U937, against 3 M.avium strains (100, 101, 109) representing the three serotypes encountered in AIDS patients (8, 1, 4 respectively). In cases where activity is shown against any of the strains, ethambutol (4µg/ml) is added in combination with the lowest concentration of the evaluated compound that showed activity in order to examine if there is increased activity compared to either compound alone. Minimum compound requirements: 10 mg.

M. avium in vivo Assay

In vivo

 activity is studied in a mouse model for M.avium infection. Beige-C57BL/J bg female mice are infected I.V. with 3x107 cfu of M. avium . After one week, therapy is initiated and continued for four weeks. The mice are then harvested. Liver and spleen are aseptically dissected, weighed and homogenized. Serial dilutions are plated onto 7H11 agar for quantitative culture. Minimum compound requirements: 2 g.

Biological Data Returned and Reviewed

When each stage of testing is completed, the laboratory facilities return the test results to the TAACF repository data center. Returned results are checked and loaded to appropriate databases. A report to the supplier is generated.

Once the quality of the testing data is verified, the TAACF team, in conjunction with the supplier and NIAID, determines whether further testing is warranted. The original supplier is asked to send additional amounts of compound to the TAACF for specific testing and either verification in the in vitro screen or further screening at the in vivo test facility.

 

Reference List

1. Collins, L. and S. G. Franzblau. 1997. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41:1004-9.

2. Kelly, B. P., S. K. Furney, M. T. Jessen, and I. M. Orme. 1996. Low-dose aerosol infection model for testing drugs for efficacy against Mycobacterium tuberculosis. Antimicrob Agents Chemother 40:2809-2812.

3. Skinner, P. S., S. K. Furney, M. R. Jacobs, G. Klopman, J. J. Ellner, and I. M. Orme. 1994. A bone marrow-derived murine macrophage model for evaluating efficacy of antimycobacterial drugs under relevant physiological conditions. Antimicrob Agents Chemother 38:2557-63.