D.SOW_ADME Assays_01-04-2021.pdf

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9.0.0.2.20101008.1.734229 Statement of Work for Services (SOW) General Information:

Title of Project:

Statement of Need and Purpose:

Background Information and Objective:

Period of Performance:

Scope of Work:

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

Specific Requirements:

Level of Effort:

Government Responsibilities:

Delivery or Deliverables:

Reporting Requirements:

Other Considerations:

Travel:

Key Personnel:

Information System Security Plan:

Data Rights:

Section 508:

Publications and Publicity:

Confidentiality of Information:

Testing of compounds in ADME assays NCATS requires the testing of novel small molecule compounds in a variety of ADME assays. This testing is for four (4) separate studies and will be performed in four separate assays:

1. The testing of eighty (80) novel small molecule compounds in four CYP450 enzyme inhibition assays: CYP3A4 (midazolam substrate), CYP3A4 (testosterone substrate), CYP2C9, and CYP2D6. This testing will inform on CYP450 inhibitory activity in our compound series, and will facilitate further optimization and development of these IRAK4 / FLT3 inhibitor compounds as potential treatments for cancers such as AML and MDS.

2. The testing of forty-five (45) novel small molecule compounds for binding to mouse and human plasma protein. This testing will inform on plasma protein binding in our compound series, and will facilitate further optimization and development of these IRAK4 / FLT3 inhibitor compounds as potential treatments for cancers such as AML and MDS.

3. The testing of forty-five (45) novel small molecule compounds for bi-directional membrane permeability in a Caco-2 assay. This testing will inform on membrane permeability in our compound series, and will facilitate further optimization and development of these IRAK4 / FLT3 inhibitor compounds as potential treatments for cancers such as AML and MDS.

4. The testing of thirty (30) novel small molecule compounds for stability in mouse and rat hepatocytes. This testing will inform on the metabolic stabilty of our compounds, and will facilitate further optimization and development of these IRAK4 / FLT3 inhibitor compounds as potential treatments for cancers such as AML and MDS.

The National Institutes of Health (NIH) is the nation’s leading medical research agency and the primary Federal agency whose mission is to seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability, conducting, supporting and making medical discoveries that improve people’s health and save lives. The National Center for Advancing Translational Sciences (NCATS) is a translational science center that prides itself on its tremendously productive pipeline and is innovative in a number of ways.

NCATS brings together a diverse range of scientists, including medicinal chemists, biologists, toxicologists, and engineers, in order to ultimately translate basic science into real products and services that help improve people’s lives. Included in this process is the development of unique small molecules as potential anticancer agents.

Prior work at NCATS has produced a series of small molecule IRAK4 / FLT3 kinase dual inhibitors, including NCGC00371481 and its structural analogs. Compounds such as ‘1481 have shown promising results in cellular and mouse models of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Further optimization of the ADME (Absorption, Distribution, Metabolism, and Excretion) and pharmacokinetic properties of these compounds is required, and may eventually produce a clinical candidate that could be used to treat cancers such as MDS and / or AML.

We are currently synthesizing structurally novel analogs of NCGC00371481 that may show improved properties relative to previous analogs. In order to assess these analogs’ ADME properties, we need to test them in four separate assays: 1) inhibition of CYP450 enzymes; 2) binding to mouse and human plasma protein; 3) bi-directional membrane permeability as measured in a Caco-2 assay; 4) metabolic stability as measured in mouse and rat hepatocyte stability assays.

CYP450 Inhibition Assays: In order for a compound to advance into clinical trials, it must display a wide range of acceptable properties. For instance, compounds must not potently inhibit major CYP450 enzymes such as CYP2C9, CYP2D6 and CYP3A4. These enzymes are responsible for oxidizing and metabolizing a majority of the toxins the body is exposed to, as well as for metabolizing many of the drug medications a patient may be taking. If a compound causes unwanted inhibition of these CYP450 enzymes, it could interfere with the metabolism and elimination of toxins from the body. The compound could also disrupt the metabolism of other drug medications a patient is taking, causing an undesired and potentially dangerous buildup of the unmetabolized drug in the body. This situation is referred to as a drug-drug interaction. We thus need to test our novel small molecule compounds in CYP450 inhibition assays, to ensure that they will not cause unwanted drug-drug interactions, or interfere with the elimination of toxins from the body.

Binding to Mouse and Human Plasma Protein: When drugs are dosed in preclinical species (mouse, rat, dog or monkey) or in humans, they enter into systemic blood circulation. There, blood plasma proteins such as albumin are often effective at binding to drugs, and in many cases, a significant percentage of the drug that is in circulation is plama protein-bound. Importantly, the portion of drug that is bound to plasma protein is not available to act at its desired target. Only the unbound portion is free to act at the target to produce the desired effect. Because drugs have widely varying propensities for binding to plasma protein, the percentage that binds to such protein (and the corresponding free fraction) must be determined experimentally. In order for doses to be properly calculated, it is essential to know the unbound fraction of a drug, as a drug given at a certain dose (for example, 100 mg) would yield only a low level of unbound drug (the active species) if plasma protein binding is high (for example, 99%), but a much higher level of unbound drug if plasma protein binding is moderate (for example, 20%).

For a given compound, binding to plasma protein may vary across species. That is, a compound's binding to mouse, rat, dog, monkey or human plasma proteins may vary. For that reason, preclinical drug discovery programs that test in a variety of preclinical species, and that ultimately want to advance compounds into human clinical trials, need to test for plasma protein binding against mouse, rat, dog, monkey and human plasma proteins.

Testing of our novel small molecule compounds for binding to mouse and human plasma protein will allow a determination of the compounds’ free (unbound) fraction in the presence of such proteins, and will provide essential information that will aid in dosing calculations for important preclinical in vivo experiments run in mice, and ultimately for clinical experiments run in human patients.

Bi-directional Membrane Permeability in a Caco-2 Assay: Compounds dosed in preclinical or clinical experiments must display good cell permeability, so that they can be effectively absorbed across cell membranes into systemic circulation, and ultimately be delivered to the target tissue of interest. Caco-2 cells are widely used as a model cell line for determining a compound’s cell permeability. In these experiments, a test compound can be applied to either the apical (A) or basolateral (B) side of the cell membrane. If a compound is applied to the apical (A) side, one then takes samples from the basolateral (B) side to see how well the compound permeates the cell membrane. The experiment can also be run in reverse, wherein compound is applied first to the basolateral (B) side, with samples then being taken from the apical (A) side. If the experiment is run in two parts – first applying compound to side A and sampling from side B, and then vice versa – one can determine if a compound is being preferentially pumped into or out of the cell. This bidirectional Caco-2 cell experiment provides information not only on passive cell permeability, but also on whether the test compound is a substrate for active transport into or out of the cell (uptake or efflux transport).

Testing of our novel small molecule compounds for permeability in bidirectional Caco-2 cell assays will allow a determination of the compounds’ passive cell permeability, as well as a determination of whether they are substrates for active uptake or efflux transporters. This will allow us to identify compounds with optimal transport properties, and to preferentially select those for subsequent, more advanced experiments.

Stability in Mouse and Rat Hepatocytes: To advance into clinical trials, a compound must display suitable pharmacokinetic properties in a range of preclinical species, including at least one rodent species (mouse or rat), as well as at least one higher preclinical species (dog or monkey). Good pharmacokinetic profiles in these species are required 1) in order to build models that predict good pharmacokinetic properties in humans, and 2) so that compounds can be given at high doses and high plasma exposure levels in preclinical safety studies.

Of the pharmacokinetic properties that must be optimized, drug clearance is perhaps the most important one. Clearance can be measured in vivo, in pharmacokinetic studies run in a preclinical species (mouse, rat, dog, or monkey), but such studies are expensive. A more cost-effective way to measure clearance involves in vitro studies, by incubating the test compound with either liver microsomes or hepatocytes. Of those two, incubation with hepatocytes is preferred, as it is typically more predictive of in vivo outcomes.

For a given compound, clearance in hepatocytes may vary considerably across species. That is, clearance in mouse, rat, dog, monkey or human hepatocytes may vary. For that reason, preclinical drug discovery programs that run experiments in a variety of preclinical species, and that want to advance compounds into human clinical trials, will ultimately need to test for clearance in mouse, rat, dog, monkey and human hepatocytes.

Testing of our novel small molecule compounds for clearance in mouse and rat hepatocytes will allow a determination of the compounds’ clearance rates. This will allow us to identify compounds with optimal clearance rates, and to preferentially select those for subsequent, more expensive pharmacokinetic studies in mouse and rat.

The period of performance is 12 months from the date of contract award.

1. Test eighty (80) novel small molecule compounds in four CYP inhibition assays. The contractor will:

a. Test each of the 80 small molecule compounds for inhibition of CYP3A4 (midazolam substrate), CYP3A4 (testosterone substrate), CYP2C9, and CYP2D6.

i. Compounds will be tested in 7 different doses, which will be determined by the Government at the time each batch is submitted.

ii. The 7 different doses will be used to generate an IC50 curve.

iii. The Government anticipates sending the 80 compounds in four batches of 20 compounds per batch.

b. Provide four comprehensive reports with the results of testing each of the 80 compounds in four CYP inhibition assays; one report for each of the four batches of compounds.

2. Test forty-five (45) novel small molecule compounds for binding to mouse and human plasma protein.

The contractor will:

a. Test each of the 45 small molecule compounds for binding to mouse and human plasma protein.

i. Compounds will be tested in duplicate at a single concentration, which will be determined by the Government at the time each batch is submitted.

ii. The Government anticipates sending the 45 compounds in three batches of 15 compounds per batch.

b. Provide three comprehensive reports with the results of each of the 45 compounds' binding to mouse and human plasma protein; one report for each of the three batches of compounds.

3. Test forty-five (45) novel small molecule compounds for bi-directional membrane permeability in a Caco-2 assay.

The contractor will:

a. Test each of the 45 small molecule compounds for bi-directional membrane permeability in a Caco-2 assay.

i. Compounds will be tested in duplicate at a single concentration, which will be determined by the Government at the time each batch is submitted.

ii. The Government anticipates sending the 45 compounds in three batches of 15 compounds per batch.

b. Provide three comprehensive reports with the results of each of the 45 compounds' bi-directional membrane permeability; one report for each of the three batches of compounds.

4. Test thirty (30) novel small molecule compounds for stability in mouse and rat hepatocyte assays.

The contractor will:

a. Test each of the 30 small molecule compounds for stability in mouse and rat hepatocyte assays.

i. Compounds will be tested at a single concentration, which will be determined by the Government at the time each batch is submitted.

ii. Compound stability (% parent compound remaining) will be determined at 6 different time points.

iii. Data from the % parent compound remaining at each of 6 time points will be used to calculate the half-life and intrinsic clearance of each compound.

iv. The Government anticipates sending the 30 compounds in three batches of 10 compounds per batch.

b. Provide three comprehensive reports with the results (half-life and intrinsic clearance) of each of the 30 compounds' stability in mouse and rat hepatocytes; one report for each of the three batches of compounds.

Estimated personnel: 1 PhD level-Manager. 1 Ph.D. level scientist. 1 - 2 months total.

The Government will not furnish property, facilities, workspace, computers, or other equipment. The contractor will not use federal facilities. The Government will provide sufficient quantities of the lead compounds in order for the contractor to accomplish the requirements described above. The Government will have the responsibility to review and approve the comprehensive reports.

As noted in the Requirements section above, the contractor will provide the data analysis and report for the contents of this statement of work. The data analysis and report shall be submitted within 60 days after receipt of the compounds, and shall be submitted electronically to Scott Hoyt at scott.hoyt@nih.gov.

The contractor will provide monthly updates on the progress of the project, or more often as appropriate. The updates shall be provided electronically to Scott Hoyt at scott.hoyt@nih.gov.

No travel is required.

None specified.

Not applicable.

FAR 52.227-17, Rights in Data – Special Works The contractor will not be involved in EIT Standards.

The contractor will not publish results of the work.

The contractor will keep the results of the acquisition confidential.