Services for the Characterization of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Properties of Small-molecule Drug Candidates Using St...

1.0 DESCRIPTION The Department of Health and Human Services (HHS), National Institutes of Health (NIH), National Cancer Institute (NCI) intends to procure services, on a sole source basis, from Cyprotex US, LLC, 313 Pleasant Street, Watertown, Massachusetts 02472-2418, United States.The publicizing and response time of the notice for this requirement is in accordance with Federal Acquisition Regulation (FAR) 5.203. This acquisition will be processed under FAR Part 12 - Acquisition for Commercial Items and will be made pursuant to the authority in FAR Part 13.106-1(b)(1); and is exempt from the requirements of FAR Part 6. The North American Industry Classification System code is 541380 and the business size standard is $19 million. 2.0 BACKGROUNDA central mission of the Developmental Therapeutics Program (DTP) of the Division of Cancer Treatment and Diagnosis (DCTD), within the National Cancer Institute (NCI), National Institutes of Health (NIH) is to facilitate the discovery and

development of new agents with demonstrable anticancer activity. To achieve this goal, DCTD supports peer-reviewed projects aligned in a consolidated pipeline, the NCI's Experimental Therapeutic (NExT) Program. NExT provides awardees with access to contracted resources to advance the drug development process from discovery, development, and clinical trials. DTP also supports meritorious NCI grantees through the Stepping Stones program by conducting smaller, focused studies designed to fill identified gaps in the data and potentially improve the investigator’s chances of receiving further support.Pharmacokinetic, metabolism and toxicity studies are an integral part of the drug discovery and development process. Pharmacokinetic studies in animals provide information on absorption, distribution, exposure, time course, excretion, and metabolism of new compounds. Toxicity studies in animals provide information on the adverse effects of new compounds. These data can sometimes be used to predict the behavior of a compound in humans. DTP has the ability to acquire such data in animals. However, it has been shown that carefully designed in vitro studies (e.g., with cells, tissues, enzymes) can be used to predict, to a reasonable degree, the behavior of a compound that may be seen in in vivo. This can be useful for selecting compounds with optimized physical, chemical, and biological properties when large numbers of analogs are being synthesized and screened during the discovery phase. Subsequently, more detailed in vitro assays can help inform the nomination of a lead or clinical candidate. Collectively, in vitro assays for metabolism, transporters, permeability, solubility, etc. have come to be termed, “in vitro ADMET.” 3.0 SCOPEThis acquisition includes services to carry out a list of defined in vitro ADMET assays. Compounds to be tested in each assay shall be selected by NCI based on priorities within the NExT and Stepping Stones (or other) programs. No personally identifiable information (PII) shall be included. The contractor shall run compounds in the specified assays and return the data to the NCI Technical Point of Contact (TPOC) electronically, in accordance with section 9.0 below. The Contractor shall only generate screening data using already developed standard methods. This purchase order does not include interpreting the data, prioritizing compounds, or participating in project meetings. 4.0 CONTRACTOR REQUIREMENTS / TASKS 4.1 COMPOUND ONEThe Contractor shall characterize the specified in vitro ADMET properties of a small, drug-like molecule provided by the NCI. Within 30 business days of award, the test article shall be supplied by NCI to the Contractor as a dry powder (~5 mg) or as a stock solution in DMSO as specified in the assay protocols. The test article shall be a small, drug-like molecule with MW 600. Residual test articles shall be destroyed. The contractor shall evaluate one (1) compound in the assays specified below, as follows:4.1.1 CYP450 Inhibition (IC50 Determination)The test article shall be incubated at increasing concentrations with pooled human liver microsomes in the presence of NADPH and a probe substrate for each major CYP enzyme. Selective CYP inhibitors of each CYP shall be screened alongside the test articles as positive controls. After optimal incubation at 37 °C, the reactions shall be terminated. The supernatant shall be removed, and the probe substrate metabolite concentration shall be analyzed by LC-MS/MS. A decrease in the formation of the metabolite compared to vehicle control shall be used to calculate an IC50 value for the test article.4.1.2 CYP450 Reaction Phenotyping (7 Isoforms)The test article shall be incubated at 37 oC with human CYP enzymes obtained from a commercial source. A control shall be run for each test article in mock microsomes (no CYP) to detect non-CYP degradation. At the specified times, an aliquot shall be removed from each experimental and control reaction and stopped with ice-cold methanol. Samples shall be centrifuged to remove precipitated protein. The supernatants shall be analyzed by LC-MS/MS to quantitate the percent parent remaining. Data shall be fit to a first-order decay model to determine half-life elimination rate constant, and CYP-dependent intrinsic clearance. 4.1.3 UGT Reaction Phenotyping (7 Isoforms)cDNA expressed human UGT enzyme preparations and test article shall be pre-incubated at 37 °C prior to the addition of UDGPA to initiate the reaction. Incubations shall also be performed using control microsomes (no UGT enzymes present) to reveal any non-enzymatic degradation. Compounds known to be metabolized specifically by each UGT isoform shall be used as controls. Test article shall be incubated in duplicate for up to 60 minutes with each isoform. The reactions shall be stopped by the addition of methanol and incubated on ice for at least ten minutes. The samples shall be centrifuged to remove precipitated protein and the supernatants analyzed by LC-MS/MS to quantitate the parent remaining. Data shall be fit to a first-order decay model to determine half-life, elimination rate constant, and UGT-dependent intrinsic clearance.4.1.4 Microsomal Metabolic Stability (mouse, rat, dog, nonhuman primate, human)The test article shall be incubated with mouse, rat, dog, nonhuman primate, and human liver microsomes at 37 oC. The reaction contains a microsomal protein buffer with standard co-factors. A control shall be run for each test article omitting NADPH to detect NADPH-free degradation. At predetermined time points, an aliquot shall be removed from each experimental and control reaction and mixed with an equal volume of ice-cold acetonitrile containing internal standard to stop the reaction and precipitate proteins. The samples shall be centrifuged to remove precipitated protein, and the supernatants shall be analyzed by LC-MS/MS to quantify % parent remaining over time. Data shall be fit to a first-order decay model to determine the rate constant, half-life, and intrinsic clearance.4.1.5 Caco-2 Permeability (Bi-directional) Caco-2 cells grown in tissue culture flasks shall be trypsinized, suspended in medium, and the suspensions shall be applied to wells of 96 well plates. The cells shall be allowed to grow and differentiate for three weeks, feeding at 2-day intervals.For Apical to Basolateral (A→B) permeability, the test article or reference compound shall be added to the apical (A) side and amount of permeation shall be determined on the basolateral (B) side; for Basolateral to Apical (B→A) permeability, the test article or reference compound shall be added to the B side and the amount of permeation shall be determined on the A-side. The A-side shall contain 100 μM lucifer yellow dye in a standard transport buffer and the B-side buffer shall be transport buffer only. Caco-2 cells shall be incubated with test articles in these buffers for at least 2 h; at the end of the assay, donor and receiver side solution samples shall be collected, quenched with 100% methanol containing an internal standard and centrifuged to obtain supernatant for donor and receiver side samples which shall be analyzed by LC-MS/MS to determine peak area ratios. Permeability and efflux ratio shall be calculated using standard equations.4.1.6 MDCK-MDR1 Permeability (Bi-directional)MDCK-MDR1 cells grown in tissue culture flasks shall be trypsinized, suspended in medium, and the suspensions shall be applied to wells of 96 well plates. The cells shall be allowed to grow and differentiate for five days, feeding at 2-day intervals.For Apical to Basolateral (A→B) permeability, the test article shall be added to the apical (A) side and amount of permeation shall be determined on the basolateral (B) side; for Basolateral to Apical (B→A) permeability, the test article shall be added to the B-side and the amount of permeation shall be determined on the A-side. The A-side shall contain 100 μM lucifer yellow dye in a standard transport buffer and the B-side buffer shall be transport buffer only. Caco-2 cells shall be incubated with test articles in these buffers for at least 2 h; at the end of the assay, donor and receiver side solution samples shall be collected, quenched with 100% methanol containing an internal standard and centrifuged to obtain supernatant for donor and receiver side samples which shall be analyzed by LC-MS/MS to determine peak area ratios. Permeability and efflux ratio shall be calculated using standard equations.4.1.7 hERG Channel Inhibition (IC50)The effect of test compounds on the hERG cardiac ion channel, expressed in mammalian cells (HEK-293), shall be assessed using the electrophysiology platform QPatch HTX (or proposed alternate approved by NCI). The QPatch HTX platform is an automated electrophysiology system that follows the general principles of conventional whole-cell patch-clamping. Test compounds shall be tested at 3 concentrations (cumulative concentration-response). The percent change in hERG tail-current shall be calculated and used, where appropriate, to calculate an IC50 value (test compound concentration that produces 50 % inhibition). Percent inhibition and, where appropriate, an IC50 shall be reported for the test compound.4.1.8 Plasma protein binding (M, R, D, H)Test article and reference compound shall be added in duplicate to plasma (pH 7.4) from the indicated species. This mixture shall be dialyzed in a Rapid Equilibrium Dialysis device per the manufacturers’ instructions against PBS and incubated on an orbital shaker. At the end of the incubation, aliquots from both plasma and PBS sides shall be collected and acetonitrile containing an analytical internal standard (IS) shall be added to precipitate the proteins and release the test article. After centrifugation, the supernatant shall be analyzed by LC-MS/MS to obtain peak area ratios (analyte/IS) for determining the fraction unbound of the test article. 4.1.9 Blood/Plasma Partitioning (M, R, D, H)The test article and reference compound shall be incubated separately with species-specific whole blood for 60 min at 37 °C. A separate aliquot of plasma is spiked with each compound and incubated alongside the whole blood. Samples shall be analyzed by LC-MS/MS. The mean blood-to-plasma ratio value and standard deviation shall be reported for each test compound.4.1.10 Turbidimetric Aqueous SolubilitySerial dilutions of test article shall be prepared in DMSO at 100x the desired final concentration. Test article solutions shall be diluted 100-fold into buffers of specified pH in a 96-well plate and mixed. After time, the presence of precipitate shall be detected by turbidity (absorbance at 540 nm). An absorbance value of greater than mean + 3x standard deviation of the blank (after subtracting the background) shall be considered an indication of turbidity. For brightly colored compounds, a visual inspection of the plate shall be performed to verify the solubility limit determined by UV absorbance. The solubility limit shall be reported as the highest experimental concentration with no evidence of turbidity.4.1.11 Metabolite ID 1 Time Point with Microsomes (Human, Rat, Mouse, Dog, Nonhuman Primate) The metabolite profile of the test article (structure provided) shall be generated in samples from microsomal stability assays (Human, Rat, Mouse, Dog, Nonhuman Primate). The data provided shall include one or two time points analysis and UNIFI Report. Structure Elucidation, and Top 5 Metabolites shall also be reported.4.2 COMPOUND TWOThe Contractor shall characterize the specified in vitro ADMET properties of one additional small, drug-like molecule provided by the NCI. The test article shall be supplied by NCI to the Contractor as a dry powder (~5 mg) or as a stock solution in DMSO as specified in the assay protocols. The test article shall be small, drug-like molecules with MW 600. Residual test articles shall be destroyed. The contractor shall evaluate one (1) additional compound in the assays specified below, as follows:4.2.1 Microsomal Metabolic Stability (dog, nonhuman primate)The test article shall be incubated with dog and non-human primate liver microsomes at 37 oC. The reaction contains microsomal protein buffer with standard co-factors. A control shall be run for each test article omitting NADPH to detect NADPH-free degradation. At predetermined time points, an aliquot shall be removed from each experimental and control reaction and mixed with an equal volume of ice-cold acetonitrile containing internal standard to stop the reaction and precipitate proteins. The samples shall be centrifuged to remove precipitated protein, and the supernatants shall be analyzed by LC-MS/MS to quantify % parent remaining over time. Data shall be fit to a first-order decay model to determine the rate constant, half-life, and intrinsic clearance.4.2.2 Hepatocyte Stability (dog, nonhuman primate)The test articles shall be incubated in singlicate with pooled cryopreserved dog and nonhuman primate hepatocytes at 37 °C. The cells shall be thawed; viable cells counted and equilibrated according to the supplier’s directions. After 30 min equilibration at 37 °C with gentle agitation, the test compound shall be added into the cells to give the desired final concentration. The cell suspension shall be incubated at 37 °C and samples removed at various times and mixed with an equal volume of ice-cold methanol containing an analytical internal standard. Stopped reactions shall be kept on ice for at least ten minutes. The samples shall be centrifuged to remove precipitated protein, and the supernatants shall be analyzed by LC-MS/MS to quantitate the % parent remaining over time. Data shall be fit to a first-order decay model to determine the rate constant, half-life, and intrinsic clearance.5.0 TYPE OF ORDERThis is a firm fixed price purchase order. 6.0 NON-SEVERABLE SERVICESThe services specified in each contract line item (CLIN) have been determined to be non-severable services – a specific undertaking or entire job with a defined end product of value to the Government.7.0 PERIOD OF PERFORMANCEThe anticipated period of performance shall be from September 16, 2023 to September 15, 2024. 8.0 PLACE OF PERFORMANCEAll work under this purchase order shall be performed in the Contractor’s facility(ies).9.0 REPORT(S)/DELIVERABLES AND DELIVERY SCHEDULEMethods used and data generated shall be reported to the TPOC as each assay is completed for each compound. All written deliverables shall be sent electronically to the NCI Technical Point of Contact (TPOC), in a Microsoft-compatible format, such as Microsoft Excel or Microsoft Word, unless approved by the TPOC. The TPOC shall review the contents of all draft deliverables. If no comments or requests for revisions are provided to the Contractor within fourteen (14) business days, the deliverables shall be considered acceptable. Final copies of approved drafts shall be delivered to the NCI TPOC within five (5) business days after receipt of the Government’s comments. The Contractor shall provide final written deliverables to the TPOC no later than 12 months after the date of the award. TPOC: TBD at awardAll deliverables shall be sent per the following deliverable schedule:DELIVERABLE / DELIVERABLE DESCRIPTION / FORMAT REQUIREMENTS / DUE DATEFinal Study Reports - Individual reports (14 total) for each compound and assay performed sent electronically in Microsoft Word, Excel, or PDF, unless an alternate format is approved by the TPOC; due upon completion of each assay over the anticipated 12-month period of performance.Section 508 of the Rehabilitation Act, as amended by the Workforce Investment Act of 1998 (P.L. 105-220) requires that when Federal agencies develop, procure, maintain, or use information and communication technology (ICT), it shall be accessible to people with disabilities. Federal employees and members of the public who have disabilities must have access to, and use of, information and data that is comparable to people without disabilities. Remediation of any materials that do not comply with the applicable Section 508 requirements as set forth below, shall be the responsibility of the Contractor.Products, platforms and services delivered as part of this work statement that are ICT, or contain ICT, must conform to the Revised 508 Standards, which are located at 36 C.F.R. § 1194.1 & Apps. A, C & D, and available at https://www.access-board.gov/guidelines-and- standards/communications-and-it/about-the-ict-refresh/final-rule/text-of-the-standards- and-guidelinesPer Section 508 and as mandated under HHS Policy for Section 508 Compliance and Accessibility of Information and Communications Technology (ICT) (07/2020) all documents or electronic files provided to the NIH NCI under contract must be conformant with Section 508 standards and accessible to persons with disabilities. Conformance shall be confirmed by use of material provided at HHS OS Factsheets & Reference Guides and verified through the use of the HHS Checklist Documents (WCAG 2.0 Refresh); in addition, contractors and vendors are encouraged to make use of the instructional materials and checklists at GSA Section 508.gov’s Create Accessible Digital Products.10.0 UNIQUE QUALIFICATIONS OF THE CONTRACTORIn FY22, a requirement for similar services was solicited competitively and awarded to Cyprotex US, LLC under Purchase Order #75N91022P00919, which expires on 9/15/2023. Under the previous award, NCI/DTP acquired services for the “Characterization of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Properties of Small-molecule Drug Candidates Using Standardized In Vitro Methods.” Four compounds of interest to DTP were evaluated in this package of assays.The current acquisition includes a follow-on request to the previous purchase order award. Under the current acquisition, the NCI/DTP requires comparative analysis of metabolism in microsomes from additional assays. These additional data will allow full comparison across species and the determination of which non-clinical species best reflects human metabolism. This will be critical for the selection of species for in vivo toxicology and pharmacokinetic studies.The data acquired under the proposed acquisition will collectively be used for comparisons across species. The same techniques must be used on the current assays, as all studies be performed in a consistent, standardized, and scientifically comparable way. Therefore, it is critically important to use the same laboratory for the current requirement to ensure scientific comparability of the new results with previous results from the studies previously conducted. Procuring the services through another source could result in substantial inter-laboratory variation, impeding the interpretability of the data. 11.0 RESPONSE INSTRUCTIONSThis notice is not a request for competitive quotations. However, if any interested party believes it can meet the above requirements, it may submit a proposal or quote for the Government to consider. The response and any other information furnished must be in writing and must contain material in sufficient detail to allow NCI to determine if the party can perform the requirement. All responses must be sent via email to Contracting Officer, Megan Kisamore, at megan.kisamore@nih.gov by no later than 3:00 PM EST, on Monday, July 10, 2023 (7/10/23). A determination by the Government not to compete this proposed requirement based upon responses to this notice is solely within the discretion of the Government. Information received will be considered solely for the purpose of determining whether to conduct a competitive procurement. In order to receive an award, Contractors must be registered and have valid certification through SAM.gov. Reference: 75N91023Q00116 on all correspondence.