Attachment 1 Statement of Work_Drug Chemistry Test 7-16-2025.docx

STATEMENT OF WORK

NON-PERSONAL SERVICES

1.0 INTRODUCTION/BACKGROUND:

1.1 The VA is developing new drugs for the treatment of Alzheimer’s disease and is requesting IND testing to be performed on these compounds, which is necessary to determine if the drug is safe for use in human subjects. The IND testing will be the first step to assessing safety and moving toward human clinical trials. These studies are required to move our drug to the next stage of discovery.

1.2 The first phase of testing will be a side-by-side comparison of two compounds.

1.3 A second phase of testing, with one compound, will proceed only if the compound passes the first phase of testing, which will be determined after sponsor’s review of the results from Phase I.

1.4 The VA performed preclinical testing of novel drugs in both rodent and cell culture models to determine that the novel compound shows promise in the treatment of Alzheimer’s disease. The compounds that the VA is requesting IND testing for have been shown to reduce amyloid burden and improve cognitive function in a mouse model of Alzheimer’s disease.

1.5 Further work to take this compound into initial phases of human testing requires the services outlined below to be complete to satisfy regulatory requirements.

2.0 Scope:

2.1 Prior to clinical trial being performed on investigational new drugs, these drugs must undergo testing to demonstrate that they are safe to test in human subjects.

2.2 These services require a contractor that has the expertise to perform these tests in compliance with FDA standards.

2.3 Inhibition studies evaluate the inhibitory effect of a drug candidate on drug-metabolizing enzymes and predict its potential for drug-drug interactions (DDIs). If a drug inhibits enzymes that are responsible for drug metabolism, there is increased toxicity risk with concomitantly administered medications. Knowing the inhibitory potential of a compound can help to better understand its DDI potential and assist with the design of necessary clinical DDI studies. Cytochrome P450 (CYP) enzymes are the major enzymes responsible for drug metabolism and an assessment of CYP inhibition is recommended by regulatory agencies.

2.3.1 The contractor shall offer definitive CYP inhibition studies to evaluate drug candidates as direct, time-, and metabolism-dependent CYP inhibitors and determine IC50 values where possible. The in vitro ADME experimental design features low protein concentrations and short marker substrate incubation times to minimize artifacts caused by protein binding, metabolic instability of drug candidates, and excessive metabolism of marker substrates.

2.4 The work will be broken down into two Phases (tiers), each phase will have multiple tasks.

2.4.1 The tiered system is important, as drugs that do not pass Phase 1 testing will NOT move on to Phase 2 testing.

2.4.2 This approach will result in significant monetary savings, if a drug fails early in testing Phase 1, Phase 2 will not be initiated/funded.

2.5 Tasks. The Contractor shall complete the following:

2.5.1 Phase 1

2.5.1.1 Task 1 Protocol/Work Plan: Standard format document, to delineate objectives, key personnel, test article/test system information, experimental methods, data analysis, project timetable

2.5.1.1.1 Report shall be in PDF format and contain introduction, summary, materials and methods, data tables and figures, results and conclusions, and supporting information

2.5.1.1.2 Report is due within 30 days of task completion. The Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.1.2 Task 2 Test article solubility testing – TA1

2.5.1.2.1 Simple dissolution: Solubility testing in stock solvent and one incubation medium

2.5.1.3 Task 3 Cocktail IC50 Determinations – TA1

2.5.1.3.1 7 Test Article (TA) concentrations will be tested in the following manner; direct, metabolism- and time-dependent inhibition; Direct and metabolism-dependent positive controls (1 concentration); n=2 Cocktail marker substrate incubations: CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), CYP3A4 (midazolam) Analysis using LC-MS/MS methods with duplicate calibration curves; IC50 determination where possible

2.5.1.4 Task 4 Test article solubility testing – TA2

2.5.1.4.1 Simple dissolution: Solubility testing in stock solvent and one incubation medium

2.5.1.5 Task 5 Cocktail IC50 Determinations – TA2

2.5.1.5.1 7 TA concentrations will be tested in the following manner; direct, metabolism- and time-dependent inhibition; Direct and metabolism-dependent positive controls (1 concentration); n=2 Cocktail marker substrate incubations: CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), CYP3A4 (midazolam) Analysis using LC-MS/MS methods with duplicate calibration curves; IC50 determination where possible

2.5.2 Phase 2

2.5.2.1 Item 1: In vitro evaluation of a Test Article as an inhibitor of human CYP enzymes in human liver microsomes 1 Test Article (TA); non-GLP

2.5.2.1.1. Task 1 Protocol / Work Plan: Standard format document, to delineate objectives, key personnel, test article/test system information, experimental methods, data analysis, project timetable

2.5.2.1.1.1 Report shall be in PDF format and contain introduction, summary, materials and methods, data tables and figures, results and conclusions, and supporting information

2.5.2.1.2 Report is due within 30 days of task completion. Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.2.1.2 Task 2: Test article solubility testing

2.5.2.1.2.1 Simple dissolution: Solubility testing in stock solvent and one incubation medium

2.5.2.1.3 Task 3: IC50 Determinations ($3,000 ea)

2.5.2.1.3.1 7 TA concentrations; direct, metabolism- and time-dependent inhibition; enzyme-specific positive controls (direct and metabolism-dependent); n=2 Individual marker substrate incubations: CYP1A2 (phenacetin), CYP2B6 (efavirenz), CYP2C8 (amodiaquine), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), CYP3A4/5 (2 assays: testosterone and midazolam) Analysis using validated LC-MS/MS methods with duplicate calibration curves; IC50 determination where possible

2.5.2.2 Item 2: In vitro evaluation of induction of human CYP enzymes by a Test Article 1 Test Article (TA); non-GLP

2.5.2.2.1 Task 1 Protocol / Work Plan: Standard format document, to delineate objectives, key personnel, test article/test system information, experimental methods, data analysis, project timetable

2.5.2.2.1.1 Report shall be in PDF format and contain introduction, summary, materials and methods, data tables and figures, results and conclusions, and supporting information

2.5.2.2.1.2 Report is due within 30 days of task completion. Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.2.2.2 Task 2: Test article solubility testing

2.5.2.2.2.1 Simple dissolution: Solubility testing in stock solvent and one incubation medium

2.5.2.2.3 Task 3: Human hepatocyte culture (CYP1A2,2B6,3A4 mRNA experiment)

2.5.2.2.3.1 3 separate lots of cryopreserved sandwich cultured human hepatocytes Thawing, seeding, maintenance and treatment of cells for up to 72 h with 24 h media replacement

2.5.2.2.4 Task 4: mRNA analysis by qRT-PCR

2.5.2.2.4.1 Measure mRNA for CYP1A2, CYP2B6 and CYP3A4 at 7 TA treatment concentrations, negative control (flumazenil) and positive controls (CYP1A2: omeprazole, CYP2B6: phenobarbital, CYP3A4: rifampin), n=3 Fold-induction change endpoint; EC50/Emax determination where possible]

2.5.2.2.5 Task 5: Cytotoxicity testing concurrent with main assay

2.5.2.2.5.1 Additional cell culture of 3 separate lots of cryopreserved hepatocytes Lactate dehydrogenase release assay at 7 TA concentrations, positive and negative controls, n=3

2.5.2.2.6 Task 6: LC-MS/MS method development

2.5.2.2.6.1 MS infusion, tuning, transition selection, parameter optimization for instrument sensitivity and selectivity for Test Article LC parameter development in three incubation matrices, to include column retention, peak shape, sensitivity, and carryover minimization optimization at relevant concentration range

2.5.2.2.7 Task 7: Spent media analysis (TA stability in test system)

2.5.2.2.7.1 LC-MS/MS method development, absolute quantitation with calibration curve. Collection and analysis of spent media at 0, 0.25, 4 and 24 h time points, 7 TA concentrations, n=3, TA quantitation

2.5.2.3 Item 3 In vitro human CYP reaction phenotyping of a Test Article 1 Test Article (TA); non-GLP

2.5.2.3.1 Task 1 Protocol / Work Plan: Standard format document, to delineate objectives, key personnel, test article/test system information, experimental methods, data analysis, project timetable

2.5.2.3.1.1 Report shall be submitted in PDF and contain materials and methods, data and results tables and figures, summary and conclusions, and supporting information

2.5.2.3.1.2 Report is due within 30 days of task completion. Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.2.3.2 Task 2: Test article solubility testing

2.5.2.3.2.1 Simple dissolution: Solubility testing in stock solvent and one incubation media

2.5.2.3.3 Task 3: LC-MS/MS method development

2.5.2.3.3.1 MS infusion, tuning, transition selection, parameter optimization for instrument sensitivity and selectivity for Test Article LC parameter development in one incubation matrix, to include column retention, peak shape, sensitivity, and carryover minimization optimization at relevant concentration range

2.5.2.3.4 Task 4: LC-MS/MS method qualification

2.5.2.3.4.1 LC-MS/MS Method qualification for one Test Article (Substrate loss) in human liver microsomes and rCYP media One precision & accuracy batch with duplicate calibration curves and six replicates of three QC levels Dilution linearity, carryover, interference testing (analyte to IS) and reinjection stability evaluation in each media

2.5.2.3.5 Task 5: Preliminary human liver microsome assay

2.5.2.3.5.1 Incubation of Test Article in human liver microsomes to establish conditions 3 TA concentrations, 0 and 4 time points at 1 protein concentration, n=2 3 TA concentrations, 0 and 1 time point at 3 protein concentration, n=2 1 Positive control substrate analyzed for relative substrate loss Zero cofactor, Zero time and zero protein negative controls LC-MS/MS analysis for 1 analyte (Test Article substrate) with duplicate calibration curves; determination of initial rate conditions where possible

2.5.2.3.6 Task 6: Recombinant CYP enzyme assay

2.5.2.3.6.1 Incubation of Test Article in recombinant CYP enzymes 2 TA concentrations, 0 and 1 time point, n=3 rCYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 and negative control (empty vector) Positive control substrate for each CYP, 0 and 1 time point, n=2 LC-MS/MS analysis for 1 analyte (Test Article substrate) with duplicate calibration curves; determination of extent of metabolism where possible

2.5.2.3.7 Task 7: Chemical inhibition assay in human liver microsomes

2.5.2.3.7.1 Incubation of Test Article in human liver microsomes ± chemical inhibitors for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4. 1 TA concentrations, 0 and 1 time point, n=3 Solvent control incubations serve as negative control 1 Positive control substrate analyzed for relative substrate loss LC-MS/MS analysis for 1 analyte (Test Article substrate) with duplicate calibration curves; percent inhibition determination where possible

2.5.2.4 Item 4 Plasma protein binding assessment of Test Article using Rapid Equilibrium Dialysis 1 Test Article (TA); non-GLP

2.5.2.4.1 Task 1 Protocol / Work Plan: Standard format document, to delineate objectives, key personnel, test article/test system information, experimental methods, data analysis, project timetable

2.5.2.4.1.1 Report shall be submitted in PDF and contain table of contents, introduction, summary, materials and methods, results and conclusions, deviations, data tables and figures, and supporting information.

2.5.2.4.1.1.1 PDF meets eCTD specifications for electronic submission for regulatory submission and will be performed in a standardized fashion that is appropriate for FDA filings

2.5.2.4.1.2 Report is due within 30 days of task completion. Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.2.4.2 Task 2 Test article solubility testing

2.5.2.4.2.1 Simple dissolution: Solubility testing in stock solvent and one incubation medium

2.5.2.4.3 Task 3: LC-MS/MS method development for TA

2.5.2.4.3.1 MS infusion, tuning, transition selection, parameter optimization for instrument sensitivity and selectivity for Test Article LC parameter development in one incubation matrix, to include column retention, peak shape, sensitivity, and carryover minimization optimization at relevant concentration range Calibration curve range suitability check

2.5.2.4.4 Task 4: Preliminary experiments for plasma protein binding

2.5.2.4.4.1 4species (mouse, rat, dog, and human) Stability test: 1 TA concentration, 0 and 3 time points (0, 4, 6, 24 h), n=2 Time to equilibrium assessment: 1 TA concentration, 0 and 3 time points (0, 4, 6, 24 h), n=2 Nonspecific binding and recovery assessment: 1 TA concentration, 0 and 3 time points (0, 4, 6, 24 h), n=2 LC-MS/MS absolute quantitation; fu, % stability, % nonspecific binding and % recovery determinations where possible

2.5.2.4.5 Task 5: Plasma protein binding determination

2.5.2.4.5.1 4 species (mouse, rat, dog, and human) 3 TA concentrations; 0 and 1 time point; n=3 Positive control (warfarin) incubation and analysis LC-MS/MS absolute quantitation; fu, % bound and % recovery determinations where possible

2.5.2.5 Item 5 In vitro evaluation of a Test Article as an inhibitor and substrate of human transporters 1 Test Article (TA); non-GLP

2.5.2.5.1 Task 1 Protocol / Work Plan: Standard format document, to delineate objectives, key personnel, test article

2.5.2.5.1.1 Report shall be submitted in pdf and contain introduction, summary, materials and methods, data tables and figures, results and conclusions, and supporting information

2.5.2.5.1.2 Report is due within 30 days of task completion. Government POC/team will get all the reports via electronic delivery methods (via emails).

2.5.2.5.2 Task 2: Test article solubility testing

2.5.2.5.2.1 Simple dissolution: Solubility testing in stock solvent and up to three incubation media types

2.5.2.5.3 Task 3: LC-MS/MS method development

2.5.2.5.3.1 MS infusion, tuning, transition selection, parameter optimization for instrument sensitivity and selectivity for Test Article LC parameter development in three incubation matrices, to include column retention, peak shape, sensitivity, and carryover minimization optimization at relevant concentration range

2.5.2.5.4 Task 4: Preliminary experiments

2.5.2.5.4.1 TA Stability testing (low & high concentration), up to 4 incubation media types TA Cytotoxicity testing (3 concentrations) in Caco-2 cells, MDCKII control cells and HEK293 control cells TA Nonspecific binding and recovery (4 concentrations) in incubation plates containing Caco-2, MDCKII control and HEK293 control cells and incubation media LC-MS/MS absolute quantitation; % stability, % cytotoxicity and % nonspecific binding determinations where possible

2.5.2.5.5 Task 5: Transwell ABC Transporter IC50 determinations (FDA Guidance Panel)

2.5.2.5.5.1 Bidirectional permeability of probe substrate in the presence of the TA 7TA concentrations, 1time pint, 1 probe substrate, n+3, 1 positive control substrate ± 2 control inhibitors P-gp Caco-2 cells (digoxin ± valspodar ± verapamil) BCRP expressing MDCKII cells (prazosin ± Ko143 ± lopinavir) LC-MS/MS sample analysis; IC50 determinations where possible

2.5.2.5.6 Task 6: SLC Transporter IC50 determinations (FDA Guidance Panel)

2.5.2.5.6.1 Uptake of the probe substrate in HEK293 cells in the presence of the TA

2.5.2.5.6.1.1 7 TA concentrations, 1 time point, 1 probe substrate, n=3 1 positive control substrate ± 2 control inhibitors Transporter expressing and control HEK293 cells OATP1B1 (estradiol glucuronide ± rifampin ± cyclosporine) OATP1B3 (estradiol glucuronide ± rifampin ± cyclosporine) OAT1 (p-aminohippurate ± probenecid ± novobiocin) OAT3 (estrone sulfate ± probenecid ± ibuprofen) OCT1 (tetraethylammonium bromide ± quinidine ± verapamil) OCT2 (metformin ± quinidine ± cimetidine) MATE1 (metformin ± cimetidine ± pyrimethamine) MATE2-K (metformin ± cimetidine ± pyrimethamine) Radiometric sample analysis; Percent inhibition endpoint

2.5.2.5.7 Task 7 ABC Transporter substrate determinations (transwell assay)

2.5.2.5.7.1 Bidirectional permeability of the TA in MDCKII cells

2.5.2.5.7.1.1 2 TA concentrations, 1 TA concentration ± 2 inhibitors, 2 time points, n=3

2.5.2.5.7.1.2 2 TA concentrations, 2 time points, n=3 in control MDCKII cells 1 Positive control substrate, 1 time point ± 2 control inhibitors P-gp expressing MDCKII cells (digoxin ± valspodar ± verapamil) BCRP expressing MDCKII cells (prazosin ± Ko143 ± lopinavir) LC-MS/MS Sample analysis; efflux ratio determinations

2.5.2.5.8 Task 8: Preliminary experiments for BSEP vesicles

2.5.2.5.8.1 Simple dissolution: Solubility testing in BSEP buffer TA Stability testing (low & high concentration) in BSEP buffer TA Nonspecific binding and recovery (2 concentrations) in control vesicles

2.5.2.5.9 Task 9: Vesicle ABC percent inhibition determinations: BSEP Accumulation of the probe substrate into transporter expressing vesicles in the presence of TA 2 TA concentrations, 1 timepoint, 1 probe substrate ± ATP, n=2

2.5.2.5.9.1 1 positive control substrate ± 2 control inhibitors ± ATP BSEP expressing vesicles (taurocholate ± cyclosporine ± glyburide) Radiometric sample analysis; IC50 determinations where possible

3.0 Contractor Qualifications

3.1 Vendor shall have the ability to provide Phased testing, that allows for testing to cease if test article fails an initial round of testing

3.2 Vendor must have at least 10 years of experience providing ADME-Tox services to most top pharma companies, determined as the top 20 global pharma companies by revenue in 2024

3.3 Vendor must have at least 10 years of of experience dealing with regulatory affairs and perform studies in line with FDA requirements. Reports must be submitted in specific electronic filings compatible with FDA submission, eCTD.

3.4 Vendor must be able to provide the following services for FDA filing

3.4.1 IC50 design to evaluate drug candidates as direct, time-dependent and metabolism-dependent inhibitors of CYP enzymes in human liver microsomes in a single experiment. Direct and time-dependent CYP inhibition generally represent inhibition caused by the drug candidate itself.

3.4.2 Metabolism-dependent inhibition represents inhibition caused by metabolites of drug candidates formed after preincubation with a co-factor. The design features low protein concentrations and short incubation times to minimize artifacts caused by protein binding, metabolic instability of the test article and excessive metabolism of the marker substrate.

3.4.3 Options for follow-up studies to support the further characterization of those drug candidates that are identified as potential direct or metabolism-dependent inhibitors.

3.4.4 CYP enzyme inhibition studies and analysis include the following features:

3.4.4.1 Robotic incubations

3.4.4.2 LC-MS/MS analysis

3.4.4.3 Efficient 3-curve IC50 study designs calculated from several concentrations of the drug candidate

3.4.4.4 Direct and metabolism-dependent positive controls included specific for each enzyme

3.4.4.5 Optional follow-up mechanistic studies

3.4.4.6 Automated data retrieval and data processing

3.4.4.7 CFR 58, Part 11 compliant systems

3.5 Vendor must have transporter inhibition and substrate services for multiple transporters both included and not included in ICH M12 guidelines.

3.6 Vendor must have at least one scientist referenced in the ICH M12 drug-drug interaction guidance (referenced by the FDA here)

3.7 Vendor must sell primary human hepatocytes.

3.8 Vendor must provide biospecimens used by top developers of cell/gene therapies, determined as top companies by revenue in 2024.

3.9 Vendor must have over 10 years of experience providing and using hepatocytes.

3.10 Vendor must have relevant scientific staff with 10+ years of experience with the Vendor.

4.0 Other Contract Requirements

4.1 Period of Performance

4.1.1 This requirement is estimated to be a two-year period of performance (no option years) with the initial period being 9/20/2025-9/19/2026

4.2 Type of contract

4.2.1 Firm-fixed price/Indefinite Delivery/Definite Quantity (IDIQ) contract

4.3 Task Orders

4.3.1 Separate task orders will be ordered against contract at time of individual study need.

4.3.2 Funding will be obligated on individual task orders.

4.3.3 Each task order shall contain a SOW addendum addressing the specific tasks associated with the task order and any milestones required.

4.4 Invoicing

4.4.1 Vendor shall submit invoices within 45 days of each service.

4.4.2 Each order will be paid via certified invoice submitted through Tungsten.

5.0 Estimated tasks

Performance of each task and successful generation reports that meet regulatory standards will indicate completion of task and the invoice will be for the indicated price of each task.