After receiving a fully signed copy of the Material Evaluation Agreement, the supplier will then submit a confidential list of compound structures, preferably in electronic format. If appropriate, a visit with a TAACF medicinal chemist can be scheduled to discuss compound selection. The structures should be submitted by fax (205-581-2870), e-mail (taacf1@sri.org), or by mail to:

TAACF
Southern Research Institute
PO Box 55305
Birmingham, AL 35255-5305

TAACF medicinal chemists will ensure that candidate compounds are appropriate for screening and have not been previously submitted, and will then recommend to Supplier compounds for submission.

The required information includes the following:

• chemical structure
• solubility
• chemical name (IUPAC preferred)
• chemist's compound ID code
• molecular formula
• molecular weight

The supplier should submit samples of each compound to be screened in vials, preferably of 1 gram size with screw caps.

NOTE:  Weighing of compounds is labor-intensive.  Pre-weighed compounds received in the following manner may receive expedited processing: 

-For in vitro TB screening, submit between 0.98 mg and 1.02 mg of compound in the following vials:

• Fisher Cat# 11-844-10 (case of 1000 vials).
• Fisher Cat# NC9601117 (case of 1000 caps).

*Vials and caps may also be provided to a supplier – send the request to your SRI contact.

Vials should be clearly labeled with a Supplier identification number or code, and appropriate physical data should be submitted for each compound. Use a printed version of the TAACF Compound Submission Form if desired. Please note that if providing < 1 mg, it is necessary to provide exact sample weights on the vial, since such samples will not be re-weighed prior to screening. Compounds and data sheets or diskettes should be shipped by express courier to:

TAACF – FRC
Attention: Ms. Ellen Beattie
Southern Research Institute - Frederick
431 Aviation Way
Frederick, MD 21701-4756

Help in compound submission (e.g., assistance in weighing/packaging samples, shipping expenses) may be available upon request.

TAACF chemists will acknowledge receipt of compounds and register them in the TAACF database. Samples are added to the queue on a first-come-first-serve basis. Approved compounds are scheduled for processing - weighing, solubilizing and plating.  Once plated, they will be shipped to the appropriate screening facility. They are screened using SRI’s High-throughput screening services.  Data will reported to the supplier as received from the screening facility.

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Screening Assays

In vitro TB Assays	In vivo TB Assays	M. Avium	High Throughput Enzyme Screening
IC90 CC50	MTD Bioavailability Murine GKO Model Murine Aerosolized TB Model	Primary Screen Expanded MIC Macrophage In vivo	Dihydrofolate Reductase Enoyl-ACP Reductase Isocitrate Lyase - Malate Synthase Combo Isocitrate Lyase Malate Synthase Pantothenate Synthetase FtsZ

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In Vitro TB Testing

Primary Screen (Dose Response): Determination of a 90% Inhibitory Concentration (IC90)

The initial screen is conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA).¹ Compounds are tested in ten 2-fold dilutions, typically from 100 µg/mL to 0.19 µg/mL. The IC90 is defined as the concentration effecting a reduction in fluorescence of 90% relative to controls. This value is determined from the dose-response curve using a curve-fitting program. Any IC90 value of ≤10µg/mL is considered "Active" for antitubercular activity.

Secondary Screen: Determination of Mammalian Cell Cytotoxicity (CC50)

The VERO cell cytotoxicity assay is done in parallel with the TB Dose Response assay. After 72 hours exposure, viability is assessed using Promega’s Cell Titer Glo Luminescent Cell Viability Assay, a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present. Cytotoxicity is determined from the dose-response curve as the CC50 using a curve fitting program.  Ultimately, the CC50 is divided by the IC90 to calculate an SI (Selectivity Index) value.  SI values of ≥ 10 are considered for further testing.

¹ Collins, L. A. and Franzblau, S. G. 1997. Microplate Alamar Blue Assay versus BACTEC 460 System for High-Throughput Screening of Compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41:1004-1009.

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Colorado State University

In Vivo Evaluation of Anti-Mycobacterium tuberculosis Activity

Colorado State University (CSU) TAACF facilities are located in Fort Collins, CO and are led by Drs. Ian Orme and Anne Lenaerts. CSU performs in vivo and ex vivo testing as described below for compounds it receives from SRI. 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. After each test is complete, results are returned to SRI for ultimate delivery to the compound suppliers.

Determination of Maximum Tolerated Dose (MTD)

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 500 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.
Minimum compound requirements: 54 mg.

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Bioavailability (pK BioAssay)

This bioassay estimates the drug levels in mice at specific time points post oral dosing, using the growth of Mycobacterium tuberculosis H37Rv as an indicator for drug activity.  This assay is conducted in 96-well microtiter plates.  Each sample well on the assay plate contains 100 mL 7H9 broth, with 10% serum and 10⁴ bacteria.  Each assay plate also has control lanes of wells containing drugs of known concentrations, either with or without 10% normal mouse serum.  For each compound tested, three C57BL/6 mice are given drug orally at a dose lower than the MTD (usually 300 mg/kg). At specified time points after dosing (usually 30 minutes and 2.5 hours) the mice are bled by nicking the lateral tail vein. Blood samples are collected aseptically, and centrifuged in serum separator tubes to collect the serum.  Serum samples from mice dosed with drugs are serially diluted using serum from naïve mice as diluent and subsequently added to the 96-well assay plate.

Optical density at 600 nm is measured every 3 – 4 days, up to day 14.  Results are confirmed by visual inspection at 10 days. Inhibition of bacterial growth in the bioassay (<50% of the control without drugs) indicates sufficiently high concentrations of bioactive compound in the bloodstream and bioavailability of the compound.
Minimum compound requirements: 25 mg.

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In vivo efficacy mouse models for M. tuberculosis

Currently, there are two animal models available for efficacy testing of compounds against M. tuberculosis; the Gamma Knock Out (GKO- IFN gamma knock out mice on a C57BL/6 background) model and a standard (C57BL/6 mouse) M. tuberculosis model. The GKO model allows a more rapid, initial assessment of drug efficacy in vivo. In the standard C57BL/6 mouse model, features as Minimal Effective Dose (MED), synergism with other compounds in drug combinations, sterilizing properties can be determined.

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). Three mice are sacrificed day 1 post-infection to determine bacterial uptake. Day 15 post-infection, 5 mice are sacrificed to determine bacterial load in the lungs and spleens. Therapy, administered via oral gavage (or i.p. injection) begins day 15 post-infection and continues for 9 days until day 24. Day 24 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. Log10 protection values >0.30 generally indicate activity. INH has demonstrated Log10 reduction of the bacterial load in the lungs around 2.75.
Minimum compound requirements: 330 mg.

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Murine Aerosolized TB Model³

Wild-type C57BL/6 mice are exposed to an aerosol of M. tuberculosis Erdman, which deposits 50 - 100 bacilli into the lungs of the animal. The course of this infection is then followed in the lungs and spleen for 8-12 weeks 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 6.0 can be observed in the infected lungs.  Test compounds are administered to groups of mice starting on day 21 post-inoculation. Typically these are administered 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. 

An additional group is given isoniazid as a positive control. Any effect the compound may be having on bacterial numbers is then assessed at intermittent time intervals of treatment  (e.g. 2, 4, and 8 weeks of drug treatment) and compared to untreated control values on these days.

The data is expressed as the log₁₀ (and as log₁₀ reduction) provided by a given dose of the compound against the growth of the organism in the untreated control group. Compounds with log₁₀ protection factors > 0.60 are generally considered active in the model. Statistical tests are also applied to the raw data to determine levels of significance.

Minimum compound requirements: 900 mg minimum (varies, depending on dose and duration of experiment.)

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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. 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.
3. 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.
4. Lenaerts, A.J.M., V. Gruppo, J.V. Brooks, and I. M. Orme. 2003. Rapid In Vivo Screening of Experimental Drugs for Tuberculosis Using Gamma Interferon Gene-Disrupted Mice. Antimicrob Agents Chemother 47:783-785.
5. Gruppo, V., C.M. Johnson, K.S. Marietta, H. Scherman, E.E. Zink, D.C. Crick, L.B. Adams, I.M. Orme, and A.J. Lenaerts. 2006. Rapid Microbiologic and Pharmacologic Evaluation of Experimental Compounds against Mycobacterium tuberculosis. Antimicrob Agents Chemother 50:1245-1250.