D Att. 1_Statement of Work.pdf

Version 11-30-2016-1 FAR Part 11—Describing Agency Needs

STATEMENT OF WORK

GENERAL INFORMATION

Title of Project:

Testing of compounds in CYP inhibition, hERG inhibition, PPB, and Caco-2 permeability assays.

Statement of Need and Purpose:

The National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH) requires the testing of compounds in CYP inhibition, hERG inhibition, PPB, and Caco- 2 permeability assays for the LATS project.

Background Information and Objective:

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 therapeutic agents.

Prior work at NCATS has produced a series of small molecule LATS kinase inhibitors, a key enzyme of the Hippo signaling pathway with potential use in wound healing. Our compounds shave shown promising results in biochemical, and functional assays . Further optimization of the ADME (Absorption, Distribution, Metabolism, and Excretion) and pharmacokinetic properties of these compounds is required and may eventually produce a molecular probe that could be administered in animal models of diseases.

Period of Performance:

The period of performance is six (6) weeks from receipt of compounds.

Solicitation No. 75N95022Q00438

SCOPE OF WORK

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 tasks listed below.

In order to assess our top analogs’ ADME properties, we need to test them in assays that measure inhibition of CYP450 enzymes, inhibition of hERG channels, cell permeability and plasma protein binding. This will be conducted through the four (4) tasks listed below.

Task 1: Test ten (10) compounds in four CYP450 Inhibition Assays

CYP450 Inhibition Assay: 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.

The contractor shall:

a. Test each of the ten (10) compounds for inhibition of CYP3A4 (midazolam substrate), CYP2C9, and CYP2D6.

i. Compounds will be tested in 7 different doses (n = 1). Doses will be determined by the Government at the time the batch is submitted.

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

iii. The Government anticipates sending 10 compounds in one batch.

b. Provide one comprehensive report with the results of testing each of the 10 compounds in four CYP inhibition assays.

Task 2: Test ten (10) compounds in the hERG inhibition assay hERG (the human Ether-a-go-go-Related Gene) codes for the protein known as Kv11.1, which contributes to the electrical activity of the heart. As such it is a critical potential off-target that small molecule compounds should avoid having activity against. In order to assess the safety of small molecules in the discovery process, testing against hERG has become increasingly important, in order to avoid this target.

The contractor shall:

a. Test ten (10) compounds in the hERG inhibition assay.

i. The assay will have an N= 2

ii. The compounds will be tested at 3 different concentrations

iii. An automated patch-clamp type system will be used

b. Provide one comprehensive report including the mean percentage hERG inhibition at each test concentration as well as the IC50 when possible. The report should include any relevant comments by the contractor.

Task 3: Test ten (10) compounds for bi-directional membrane permeability in a Caco-2 assay

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).

The contractor shall:

a. Test ten (10) compounds for bi-directional membrane permeability in a Caco-2 assay.

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

b. Provide one comprehensive report with the results of each of the 10 compounds' bi-directional membrane permeability. Data will be expressed as permeability (Papp): Papp = (dQ/dt)/(C0*A), where dQ/dt is rate of permeation, C0 is initial concentration of test article, and A is the area of monolayer. In bi-directional permeability studies, the Efflux Ratio (Re) will also be calculated: Re = PApp(B to A)/Papp(A to B); Re > 2 indicates a potential substrate for P-gp or other active efflux transporters.

Task 4: Test ten (10) compounds in Plasma Protein Binding Assay

Plasma Protein Binding Assay: Drug distribution describes the movement of a drug from the blood to various tissues. It depends on numvrous factors, one of each is the ability of the drug to bind plasma proteins: if the compound is highly bound, it will remain in the plasma, resulting in a low volume of distribution. This may affect the therapeutic effect of the compound by limiting the amount of free compound available to act on the target molecule. Extensive plasma protein binding also limits the amount of free compounds available for metabolism, reducing compound clearance.

This assay utilizes equilibrium dialysis as the method for evaluating plasma protein binding because non-specific binding effects are minimized compared to other methods (such as ultrafiltration). A semipermeable membrane separates the protein-containing compartment from the protein-free compartment. The system is equilibrated at 37°C, and then LC-MS/MS quantifies the test compound present in each compartment.

The contractor shall:

a. Test ten (10) compounds for plasma protein binding. Compounds will be tested in duplicate at a single concentration, typically 5 µM in 100% plasma in the equilibrium dialysis assay.

b. Provide one comprehensive report with the results of each of the 10 compounds' fraction unbound in 100% plasma recovery.

GOVERNMENT RESPONSIBILITIES

The Government will provide the compounds to be tested. The Government will send the ten (10) compounds in multiple batches or as one batch. The Government will coordinate shipment of the compounds with the contractor.

DELIVERABLES

1. One comprehensive report with the results of testing each of the 10 compounds in four CYP inhibition assays (Task 1).

2. One comprehensive report with the mean percentage hERG inhibition at each test concentration as well as the IC50 when possible. The report should include any relevant comments by the contractor (Task 2).

3. One comprehensive report with the results of each of the 10 compounds' bi-directional membrane permeability (Task 3). Data will be expressed as permeability (Papp): Papp = (dQ/dt)/(C0*A), where dQ/dt is rate of permeation, C0 is initial concentration of test article, and A is the area of monolayer. In bi-directional permeability studies, the Efflux Ratio (Re) will also be calculated: Re = PApp(B to A)/Papp(A to B); Re > 2 indicates a potential substrate for P-gp or other active efflux transporters.

4. One comprehensive report with the results of each of the 10 compounds' fraction unbound in 100% plasma recovery (Task 4).