In vitro and In vivo Evaluation of anti-Mycobacterium avium Activity

Higher level evaluation of compounds against M. avium is available for compounds showing Level II TB activity, (i.e., a Selectivity Index (SI) ≥ 10 (in MIC and cytotoxicity assays)). The available screens and compound requirements are outlined below:

M. avium Primary Screen

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 MIC 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 ≥ 32 µg/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.