Data Interpretation: Data Report Reference Page

As a service to our suppliers, this page aids interpretation of assay data.  Please note that specific notes and column references are made in reference to official TAACF data reports mailed to suppliers upon testing completion.  Browse to Screening Assays for compound requirements and test descriptions.

Dose Response Assay Data (Level 1)

All compounds are initially screened against Mycobacterium tuberculosis strain H37Rv in the Dose Response assay. This assay is the TAACF's primary screen. The assay returns IC90, IC50, and all of the %Inh values at the tested concentrations. IC stands for 'inhibitory concentration' - this is the concentration where a drug inhibits the TB strain by 90% or 50%. The report contains two tabs - IC90 Summary and DR Raw Data. Please note the column labeled IC90 on the IC90 Summary tab. The significance of this value depends on several factors such as compound structure, novelty, toxicity, and potential mechanism of action. Compounds are considered active in the dose response screen if IC90 ≤ 10 µg/mL. Activity is designated by the word "Active" in the column labeled Activity. Compounds deemed "Weakly active" or  "Inactive" are considered not active and no further testing is anticipated. In this and all other types of data reports, a column labeled Comments may be present noting any observations, unusual considerations, or resupply needs for more advanced screening.

Cytotoxicity Assay Data (Level 2)

The VERO cell cytotoxicity assay is done in parallel with the TB Dose Response assay. Compounds are screened by serial dilution to assess toxicity to a VERO cell line, if sample solubility in culture media permits. The VERO cell cytotoxicity assay returns a CC50 value, which allows a SI (selectivity index) to be calculated. The report contains three tabs: DR+Cytotox, DR Raw Data and Cytotox Raw Data. Note the column labeled SI on the DR + Cytotox tab. The selectivity index is defined as the ratio of the measured CC50 (mammalian cell toxicity) to the IC90 (H37Rv Mycobacterium tuberculosis) described above. If the SI value is ≥10, then the compound may be considered for further screening. TAACF medicinal chemists will review the data.

Macrophage Assay Data (Level 3)

Selective compounds as determined in Level 2 assays are tested for efficacy in vitro in a TB-infected macrophage model. The columns labeled EC₉₀ and EC₉₉ list the concentrations effecting 90% and 99% reduction in residual mycobacterial growth after seven days, compared to untreated controls. Compounds with EC₉₀ > (16 X MIC) (as reported in the column labeled EC₉₀:MIC) are considered inactive in the model.  Level 3 values for control drugs:  INH (EC₉₀ = 0.03 µg/mL & EC₉₉ = 0.42 µg/mL) & RMP (EC₉₀ = 0.04 to 0.1 µg/mL & EC₉₉ = 0.5 to 1.5 µg/mL)

SDRMIC Assay Data (Level 3)

The column labeled L3 MIC lists MICs against various strains of M. tuberculosis (as specified in column labeled Strain). Confirmatory MICs are typically performed against the strains H₃₇Rv and Erdman.  The MIC is also determined against a panel of singly-drug-resistant strains. Generally, strains resistant to isoniazid (INH), rifampin (RMP), and thiacetazone (TAC) are used, unless the structure of the compound suggests another specific strain. The corresponding MIC values serve as further confirmation of activity. Note the column labeled Resistant Strain: L3 H₃₇Rv MIC; these values are the ratios of MICs in the resistant and non-resistant strains, and should generally be approximately 1, though there is considerable experimental variability in MIC determinations. A ratio greater than eight between the MICs in the resistant to the non-resistant strains indicates that the strain is cross-resistant to the sample.  Level 3 values for control drugs: INH (MIC INH-R >0.2 mg/mL).

Tuberculosis Animal Model (in vivo) Assay Data

Prior to animal screening the maximally tolerated dose (MTD) of a compound is determined using C₅₇BL/6 female mice by administration of a one-time dose/animal of the compound at concentrations of 100, 300 or 1,000 mg/Kg. The 9 mice (3 mice/dose) in each study are observed for a total of 1 week. The MTD is reported in the column labeled Maximum Tolerated Dose. Surviving mice are sacrificed and organs are examined for signs of overt toxicity as reported in the column labeled Toxicity Issues. The column labeled Comments reports observations made during the experiment. The MTD is used as a guide for compound dosing in the murine TB model.

Next is determined in vivo bioavailability of compounds after oral administration to mice via a 96-well microtiter plate assay. This assay measures approximate compound levels in the serum of mice at specific time points after oral dosing, using the growth of Mycobacterium tuberculosis H37Rv as the indicator strain for drug activity.  Inhibition of bacterial growth in the bioassay indicates sufficiently high concentrations of bioactive compound in the bloodstream. Wells with serum from dosed mice are arbitrarily scored as “bioactive” when the OD₆₀₀ values are less than 50% of the OD₆₀₀ value of the untreated control wells with serum. As reported in the column Bioavailability, scoring occurs as follows. Low: when activity of drug is observed in 1-2 wells of the bioassay; Medium: activity of drug is observed in 3 or 4 wells of the bioassay; High: activity of drug is observed in 5 or 6 wells.

Subsequently, there are two animal models available, the GKO [IFN gamma KO mice on a C₅₇BL/6 background (Jackson)] model and a standard (C₅₇BL/6 mouse) TB model. In both models, drug treatment begins 20 days after inoculation of the animal with M. tuberculosis. Bacterial counts are measured on day 30 (GKO only) and days 35 and 50 (standard model) in two tissues (lung and spleen), and compared with counts from negative (untreated) controls. Compounds are considered active if they yield at least a 0.75 log₁₀ reduction in bacterial counts, and moderately active if they afford somewhat less (approximately 0.5 log₁₀) protection, as noted in the column labeled Activity. Log₁₀ protection data for both tissues are reported in the column labeled Log protection (lung/spleen), and any unusual details of the experiment are summarized in the column labeled Observations. Data from a positive control (i.e., treated with a known TB drug, typically INH) are also reported in the column labeled Control protection (lung/spleen).