75N91021R00003-Attachments.pdf

PACKAGING AND DELIVERY OF PROPOSAL ATTACHMENT 1

Attachment 1 - Packaging and Delivery of Proposals for Use with the NIH electronic Contract Proposal Submission (eCPS) Website

I. PROPOSAL SUBMISSION

A. eCPS

1. Proposals must be submitted via the electronic Contract Proposal Submission (eCPS) website at https://ecps.nih.gov.

2. Proposals submitted by facsimile or e-mail will not be accepted.

3. Follow the “How to Submit an Electronic Proposal” instructions provided on the eCPS website at: https://ecps.nih.gov/home/howto. Please note that creating an account to submit may take up to three (3) business days. Please apply for a new account early to allow enough time for the registration process.

4. Offerors are solely responsible for submitting proposals and any modifications or revisions so as to reach the Government office designated above by the date and time specified in the solicitation. If your proposal is not received by the date and time specified in the solicitation, it will be considered a “late proposal,” in accordance with FAR Clause 52.215-1 Instructions to Offerors – Competitive Acquisition and HHSAR 352.215-70, Late Proposals and Revisions (December 18, 2015)].

B. Creating and Naming Files:

1. Create one PDF file of your Technical Proposal, including all attachments. The

Technical Proposal should be created in a PDF format that enables word searches to the maximum extent practicable. Forms and/or documents requiring signature(s) may be scanned, but must be merged into the Technical Proposal PDF file.

2. Create one PDF file of your Business Proposal, including all attachments:

The Business Proposal should be created in a PDF format that enables word searches to the maximum extent practicable. Forms and/or documents requiring signature(s) may be scanned, but must be merged into the Business Proposal PDF file.

Additionally, the “Breakdown of Proposed Estimated Costs (plus Fee) with Excel Spreadsheet” (http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx) must be included in the Business Proposal.

3. Create your Business Document Excel. The Excel file should be the “Breakdown of Proposed Estimated Costs (plus Fee) with Excel Spreadsheet” (http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx) included in the Business Proposal in its original Excel format, not PDF. Multiple Excel files may be included, as necessary.

4. Each of the proposals, Technical and Business, must be separate and complete in itself.

Do not reference one proposal in another.

https://ecps.nih.gov/ https://ecps.nih.gov/home/howto http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx http://oamp.od.nih.gov/sites/default/files/DGS/contracting-forms/spshexcl_dec2012.xlsx

PACKAGING AND DELIVERY OF PROPOSAL ATTACHMENT 1

5. File naming convention: It is requested that the filenames for your Technical Proposal, Business Proposal, and Excel Workbook include the name of the offeror, the solicitation number and the type of proposal (i.e., Technical, Business, or Excel Workbook).

Examples:

Technical Proposal: XYZ Company_NIHAI2012001_Technical.pdf Business Proposal: XYZ Company_NIHAI2012001_Business.pdf Excel Workbook: XYZ Company_NIHAI2012001_Business.xlsx

II. FORMATTING AND PAGE LIMITATIONS:

A. Formatting for proposals

1. Proposal page layout shall be letter size 8.5” x 11" for all pages.

2. Proposals shall not include links to internet web site addresses (URLs) or otherwise direct readers to alternate sources of information.

3. Proposals shall not include audio or video files of any type.

4. Font size must be 10 to 12 points.

5. Spacing should be no more than 15 characters per inch. Within a vertical inch, there must be no more than six lines of text.

6. Margins must be at least one-inch on all sides.

7. Failure to adhere to the formatting requirements above may impact whether your proposal is reviewed in its entirety.

B. Page limitations:

1. The total page count of the Technical Proposal shall not exceed 150 pages. The page limits for Technical Proposal excludes any response to the Sample Protocols. The total page count for the Technical Proposals for Sample Protocols shall not exceed 15 pages (per Sample Protocol). Pages in excess of this limitation will be removed from the proposal and will not be provided to the reviewers to read or evaluate.

Proposal pages shall be numbered “Page 1 of 120, “Page 2 of 120” etc. The page limits for the Technical Proposals excludes any sample protocol proposals.

2. Total page count does not include: Title and Back Page; Table of Contents; Section Dividers that do not contain information other than title of Section.

3. Each Curriculum Vitae (CV) shall not exceed 3 pages.

4. Pages exceeding limitations will be removed from the proposal and will not be considered.

PROPOSAL INTENT RESPONSE FORM

RFP No:

RFP Title:

Please review the Request for Proposal (RFP). Furnish the information requested below and return this page to the Contracting Officer/Contract Specialist identified on Section A-Solicitation/Contract Form by

Your expression of intent is not binding but will greatly assist us in planning for proposal evaluation.

Choose one of the following Options:

Do intend to submit a proposal Do Not intend to submit a proposal

If you are not responding to this RFP, please provide your reason(s):

Please provide the following contact information:

Name (First, Middle Initial, Last):

Title:

Organization:

E-mail:

Attachment 3, Dated 11/20/2020 75N91021R00003

STATEMENT OF WORK

PRECLINICAL TOXICOLOGY OF SMALL MOLECULE DRUGS

DEVELOPED FOR CANCER AND OTHER INDICATIONS

1. SCOPE

Independently and not as an agent of the Government, the Contractor shall furnish all necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government, as needed to support the Developmental Therapeutics Program (DTP) with conducting toxicology and pharmacology studies related to small molecule drugs. The timeline or schedule for support activities will be specified by the NCI Contracting Officer’s Representative (COR) based on the study. Projects could be stopped, restarted, and/or reprioritized at any given time at the discretion of the Government based on need and immediate findings. Written instructions will be provided by the Government if a project needs to be changed.

TASKS

Toxicology and Pharmacology work conducted under this small molecule contract will involve the following types of tasks. For each task across any and all studies, the Contractor shall expend hours in planning, ordering, initiating work and documenting findings including, but not limited to:

a. Validating the analytical methodology to quantify drug levels in dosing solutions, biological fluids, and tissues as required. Measuring drug plasma and tissue levels in rodents, beagle dogs and/or non-human primates treated with the agent under study.

Calculating and reporting all important pharmacokinetic parameters from the derived data.

b. Determining bioavailability of drug after oral and/or intraperitoneal, subcutaneous or intramuscular administration. Calculating and reporting all relevant pharmacokinetic parameters from the derived data.

c. Determining acute toxicity, (including, but not limited to, clinical observations, body weights, clinical pathology, histopathology) and plasma and tissue drug levels in rodents, beagle dogs and/or non-human primates over specified time periods.

d. Assessing subacute toxicity (including, but not limited to, clinical pathology, hematology, histopathologic evaluation of tissues, clinical observations) in rodents, beagle dogs and/or non-human primates.

e. Assessing cardiotoxicity, neurotoxicity, and immunotoxicity in rodents, beagle dogs and/or non-human primates as specified by the COR.

f. Assessing mutagenicity in vitro and in vivo (e.g., Bacterial Reversal Mutation Assay, Mouse Lymphoma Assay, Chromosomal Aberration Assessment).

g. Assessing the metabolic profiles of drugs using hepatocytes in vitro.

2. TYPES OF TOXICOLOGY AND PHARMACOLOGY STUDIES

The types of studies that the Contractor shall perform according to the requirements of the protocol(s) developed for each test article by the Contracting Officer’s Representative (COR) include, but are not limited to, the following:

a. Analytical: Drug identity analysis (e.g., using IR, NMR, MS, or other assay methodology specified by the COR), validation of dose concentration analysis procedures that are supplied by the NCI, and/or validation of the requisite methodology for the assay of drug in biological fluids. The Contractor shall initiate work immediately upon drug receipt.

b. Pharmacokinetic: Plasma elimination kinetics shall be determined after single intravenous doses of drug in one or more of the following species: rodents, dogs, and non-human primates. Other routes of administration such as oral, intraperitoneal, subcutaneous and intramuscular may be necessary to evaluate as well. Bioavailability of non-parenteral routes and plasma clearance rates shall be determined in order to establish the dose required to produce effective drug concentrations in plasma for future toxicity studies.

c. Exploratory or Dose Range-Finding Toxicology: For each drug, establish a maximum tolerated dose (MTD) and dose limiting toxicities (DLT) in rodents and/or beagle dogs. The use of an alternative species such as non-human primates, rabbits, or mini-pigs may be required for certain agents under evaluation. The types of studies required in this phase may include exploratory toxicity studies and single or multiple dose range-finding studies, as specified in the protocols.

d. IND-Directed Toxicology: For each drug, establish the MTD, DLTs, and reversibility of toxicity in rodents and/or dogs. The use of an alternative species such as non-human primates, rabbits, and mini-pigs may be required for certain agents under evaluation.

The types of studies required in this phase may include single or multiple-dose studies. Studies shall be conducted under Good Laboratory Practice regulations.

Protocols for the following types of studies may be issued:

i. Single or multiple daily dose schedules such as Dx1, q3hr x 3, Dx5, Dx28, etc.

ii. Continuous administration to mice via Alzet osmotic pumps and to beagle dogs, rats and non-human primates via infusion pumps for periods of from one hour up to 30 days.

iii. Twenty-eight days or more of repeated administration of drug to rodents, beagle dogs and/or non-human primates.

iv. Special studies such as cardiotoxicity, reproductive, neurotoxicity, and immunotoxicity may be requested as part of an existing study or in a separate study.

e. In vitro and in vivo mutagenicity assays/assessments, such as the Bacterial Reversal

Mutation Assay, the Mouse Lymphoma Assay, and the Chromosomal Aberration Assessment).

f. In vitro metabolic profile analyses of drugs using hepatocytes.

3. GENERAL REQUIREMENTS

The Contractor shall engage in the following project management tasks:

a. Participate in planning and progress review meetings:

i. The Contractor shall plan for and participate in meetings to review requirements and timelines, discuss technical plans and approaches, and identify any anticipated problems/obstacles to completing the requirements. Meetings shall be attended by Contractor key personnel (including subcontractor personnel as appropriate), the COR, and other representatives at the discretion of the COR (e.g., CO, Subject Matter Experts, investigator representatives).

ii. Key personnel or other senior staff coordinator(s) shall be available for ad hoc meetings/teleconferences with the COR immediately upon a meeting request.

b. As needed and upon permission of the CO and COR, travel may be required to disseminate technical and scientific information and/or present results at a national meeting and/or conference.

4. GOVERNMENT FURNISHED PROPERTY/SUPPLIES

a. Dogs, rats, rabbits, mini-pigs, non-human primates and specialty animals such as cathetered rats shall be purchased by the contractor under this contract as needed to perform the projects listed above.

b. The Government will furnish the test articles and some of the control articles used in these studies.

c. Available analytical chemistry information including chemical identity data and methodology for dose concentration analysis, plasma drug analysis procedures, formulation information, available literature, etc. will be supplied by the Government.

d. Specialized equipment for infusion studies (pumps, jackets, collars, etc.) shall be furnished by the contractor.

5. HUMAN MATERIALS

Human refuse materials such as blood, serum, plasma, bone marrow, liver, kidney, other tissues, etc. may be necessary for use on this contract. If necessary, samples will be coded and anonymized such that the donors cannot be identified. The Contractor shall be responsible for any local IRB approvals required for work with human samples as described herein and for implementation of, and adherence to, all federal, local, and institutional policies regarding the occupational health and safety of personnel working with animal and/or human plasma, urine, or other tissues.

6. SHIPPING

The Contractor shall be responsible for shipping samples generated in-house or received externally to other laboratories at the request of the COR.

7. HEALTH AND SAFETY

Each agent will be considered potentially hazardous. Therefore, all necessary precautions must be taken to protect personnel and the environment against possible exposure to the agents being tested. The Contractor shall perform all work associated with this contract in accordance with all applicable Federal, State and local regulations including transportation and disposal of hazardous waste.

8. ACCESS TO DATA

The Government requires that all data accumulated under the projected contract be immediately available for its review and that provisions are made to maintain confidentiality of all data. Authority to release data may be granted only by the Contracting Officer together with the COR and must be in writing.

All individual animal data shall be provided to the COR in a format that allows integration into DTP databases. Excel or other tab delimited file formats are acceptable.

Other formats will be considered but compatibility shall be confirmed. In addition, data shall be supplied in SEND-compliant format for those studies specified by the COR.

The Contractor shall also prepare study reports with study data generated in the electronic Common Technical Document (eCTD) format.

Date: 20 November 2020

Attachment 4, Dated 11/20/2020 1

PROTOCOL

FIVE-DAILY DOSE RANGE-FINDING TOXICITY STUDY of [compound name] in RATS GLP ☐ Non-GLP ☒

SPONSOR: Toxicology & Pharmacology Branch (TPB)

Developmental Therapeutics Program Division of Cancer Treatment and Diagnosis National Cancer Institute National Institutes of Health Bethesda, MD 20892

TPB REPRESENTATIVE:

TBD

Attachment 4, Dated 11/20/2020 2

STUDY OBJECTIVE

Study Objective The objective of this study is to determine target organ toxicities in rats that receive [compound name] when administered once daily for 5 days as a one-hr IV infusion.

STUDY DESIGN

Species Sprague Dawley rat Initial Age Range 8-12 weeks Initial Body Weight Range

~150-200 g (males); ~125-175 g (females)

Justification for Species

The rat is an accepted species that is used in preclinical safety evaluations of drugs that are intended for human use.

Animal Housing General procedures for animal care, housing and environmental conditions shall be in accordance with the current Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations, current requirements stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and current requirements as stated by the U.S. Department of Agriculture through the Animal Welfare Act and Animal Welfare Regulations (November 2013). The testing facility’s standard operating procedures for animal identification, quarantine, housing, feeding, handling, and care of animals shall be followed.

Route of Administration

1-hr iv infusion

Schedule of Administration

Once daily for 5 days

Group Assignments

GROUP Number of Males*

Number of Females*

DOSE

(mg/kg)

DOSE

(mg/m2)

TOTAL DOSE

(mg/kg)

TOTAL DOSE

(mg/m2)

1 VCTL1 13 13 0 0 0 0

2 13 13 TBD

3 13 13 TBD

4 13 13 TBD

*3 rats/sex/dose group will be assigned to the PK subgroup for plasma determination only. The remaining rats will be in the main study group.

Test Article (TA) Identification, Formulation and Analysis Test Article Compound identity, strength, quality, stability and purity, and documentation of methods of synthesis, fabrication or derivation will be provided by the NCI.

Confirmation of identity will be done immediately upon receipt of each shipment of the compound. Yes ☐ No ☒ Sufficient quantity of drug shall be reserved for archiving from each lot and shipment used. Yes ☒ No ☐

1 Vehicle control

Attachment 4, Dated 11/20/2020 3

Vehicle and Storage Conditions

Sterile water for injection. Store at room temperature.

Storage of TA At 2 to 8°C, protected from light.

TA Formulation

Dissolve in sterile water and dilute to achieve final concentrations for the respective mg/kg doses.

Formulations will be prepared daily by weighing a known quantity of test article and combining it with the appropriate volume of vehicle. The formulations will be administered within one hour of preparation. Formulations will be kept on ice until the dose is administered.

Dose Formulation Analysis

Yes ☐ No ☒

Dose concentration analysis shall be performed on the dose formulation(s).

Dose formulations must be within ±10% of the theoretical concentration.

Homogeneity analysis will not be conducted as part of this study.

Test Article Exposure Assessment – Bioanalysis, Pharmacokinetics (PK) Plasma Drug Levels

Blood collection shall be performed according to the laboratory's SOP, but in no case will animals be bled from the treatment site. Blood samples shall be obtained from each animal at the time points indicated below.

Whole blood will be centrifuged and the plasma collected will be frozen at ≤20 °C until analysis. The time between blood collection and the freezing of plasma samples should be less than 1 hr.

For animals in a moribund condition, a blood sample shall be drawn and processed to plasma for plasma drug analysis prior to euthanasia.

Plasma samples will be analyzed for drug concentration using an LC/MS-MS method.

PK Time Points Day 1 immediately prior to the end of infusion, 5, 15, 30, 60, 120 min, and 4, 8, and 24 hrs post-dose (prior to Day 2 dose); and on Day 5 immediately prior to the end of infusion.

PK Sample Volume

0.7 mL

PK Data Analysis (PK Parameters) Non-compartmental analysis (Tmax, Cmax, AUC, CL, etc.) on all dose groups.

Attachment 4, Dated 11/20/2020 4

MEASUREMENTS

Clinical Observations

Pre-dose – Observe animals daily and record all baseline clinical signs beginning on study Day -3 (after quarantine period).

Dosing Period – All adverse clinical signs shall be recorded at least twice daily during the dosing period and at least once daily thereafter or more often if clinical signs are noted.

Body Weights Predose – On approximately Day -1 (after quarantine period), baseline body weights shall be recorded and used for randomization of animals into treatment groups.

Dosing Period – Body weights shall be recorded prior to test article/vehicle administration on Days 1-5, and on Days 6, 8, 12, 15, and prior to euthanasia at approximately the same time each day prior to feeding or necropsy.

Hematology*

Erythrocyte count Hemoglobin Leukocyte count, differential (absolute) Mean corpuscular hemoglobin Mean corpuscular volume Platelet count

Hematocrit Leukocyte count, total Leukocyte count, differential (relative) Mean corpuscular hemoglobin concentration Nucleated red blood cell count Reticulocyte count, absolute and relative

Clinical Chemistry*

Alanine aminotransferase Albumin/Globulin ratio Aspartate aminotransferase Cholesterol Creatinine Gamma glutamyl transferase Glucose Protein, total Urea nitrogen

Albumin Alkaline phosphatase Bilirubin, total Creatine kinase Globulin Lactate dehydrogenase Triglycerides BUN/Creatinine ratio

Electrolytes* All ☐ None ☒

Calcium ☐ Chloride ☐ Inorganic phosphorus ☐

Potassium ☐ Sodium ☐ Sodium/Potassium ratio ☐

Coagulation* Yes ☐ No ☒

Activated partial thromboplastin time ☐ Fibrinogen ☐ Prothrombin time ☐

Urinalysis Yes ☐ No ☒

The following parameters will be measured (the time of collection may be modified by the Study Director with agreement by the COR):

Albumin ☐ Appearance ☐ Bilirubin ☐ Chloride ☐ Color ☐ Creatinine ☐ Glucose ☐ Ketones ☐ Leukocytes ☐ Nitrite ☐ Occult blood ☐ pH ☐ Potassium ☐ Sedimentation ☐ Sodium ☐ Specific gravity ☐ Total protein ☐ Urea nitrogen ☐ Urobilinogen ☐ Volume ☐

*see Blood Collections for Clinical Pathology table for collection time points

Attachment 4, Dated 11/20/2020 5

Blood Collections for Clinical Pathology GROUP Day -2 a Days 3 and 6 * Days 12 and 19**

Number of

Males

Number of

Females

Number of Males/timept

Number of Females/timept

Number of Males/timept

Number of Females/timept

1 VCTL 10 10 5 5 5 5

2 10 10 5 5 5 5 3 10 10 5 5 5 5 4 10 10 5 5 5 5 aBlood drawn for baseline measurements. *Five rats/sex/dose group that will be euthanized on Day

6. **5 rats/sex/dose group that will be euthanized on Day 19.

The tubes used for hematology will contain K3EDTA as an anticoagulant. The tubes used for serum chemistry will not contain anticoagulant but may contain serum separator gel. The tubes used for coagulation parameters will contain sodium citrate as the anticoagulant. Separation of cellular components from serum shall be performed within 60 minutes of blood collection.

NECROPSY PROCEDURES

Unscheduled Necropsy Moribund animals shall be weighed, blood collected for clinical pathology determinations, and necropsied with histopathology evaluation as for scheduled necropsies. The authorization to euthanize moribund animals will be made by the Study Director or other qualified individual after examination of the animals. If an animal is found dead outside of normal working hours, it shall be necropsied as soon as possible with the carcass refrigerated (not frozen) in the interim period (not to exceed 24 hr). Body weight shall be taken and tissues will not be discarded because of postmortem autolysis.

Scheduled Necropsy

GROUP Day 6 Day 19 Day Number of

Males Number of

Females Number of

Males Number of

Females Number of

Males Number of

Females

1 VCTL 5 5 5 5

2 5 5 5 5 3 5 5 5 5 4 5 5 5 5

Animals shall be terminated following the most recent AVMA Guidelines for the Euthanasia of Animals (2013) and the testing facility’s IACUC recommendations and facility SOPs.

Body weights and clinical pathology determinations shall be taken on the day of necropsy. The necropsy shall include examination of the external surface of the body; all orifices; the cranial, thoracic, abdominal and pelvic cavities, and their contents; and collection of the tissues listed below. A complete necropsy and all antemortem observations shall be recorded for each animal and commented on or confirmed at necropsy. Animals which are clinically normal will also be indicated. A pathologist will be available to examine any unusual findings.

Organ Weights Yes ☐ No ☒ Adrenals, brain, epididymides, heart, kidney, liver, spleen, testes, thyroid, parathyroid, thymus, ovary, uterus

Attachment 4, Dated 11/20/2020 6

Tissue Collection Complete ☐ Partial ☒ All tissues listed below shall be examined, trimmed, and fixed in 10% cold, neutral-buffered formalin.

☐Adrenal glands (2) ☐ Aorta (thoracic) ☒ Bone, femoral head with articular surface ☐ Bone marrow, sternum ☐ Bone marrow-rib, costochondral junction ☒ Brain ☒ Cecum ☒ Colon ☒ Duodenum ☒ Epididymides (2) ☐ Esophagus ☐ Eyes (2), including optic nerves ☐ Gall bladder ☒ Gross lesions ☒ Heart ☒ Ileum ☒ Jejunum ☒ Kidneys (2) ☒ Liver ☒ Lungs ☒ Lymph nodes (bronchial, mandibular, mesenteric)

☐ Mammary gland (when present in regular abdominal skin section)

☒ Ovaries ☐ Pancreas ☐ Parathyroid gland (when present in regular thyroid gland section)

☐ Pituitary gland

☐ Prostate gland ☐ Salivary gland (mandibular) ☒ Sciatic nerve ☐ Skeletal muscle ☐ Skin (ventral abdomen, injection site) ☒ Spinal cord, thoracolumbar (cervical and posterior lumbar spinal cord examined if nervous system signs present)

☒ Spleen ☐ Stomach (cardiac, fundic, and pyloric) ☒ Testes (2) ☒ Thymus ☒ Thyroid gland ☐ Tonsils (2) ☐ Trachea ☒ Urinary bladder (fundus) ☒ Uterus (with cervix) ☒Dorsal root ganglion ☒ The identification mark from each animal shall be preserved in fixative.

Histopathology A sample of all fixed tissues shall be embedded in paraffin blocks. Sections of approximately 4 to 6 micrometers shall be cut, mounted on glass slides and stained with hematoxylin and eosin according to the testing facility’s SOPs.

All fixed tissues will be examined microscopically by a board certified veterinary pathologist. All lesions will be categorized either as drug-related or non-drug-related. All morphologies shall follow, to the best extent possible, the published criteria of the International Harmonization of Nomenclature and Diagnostic criteria (http:/www.goreni.org/). Records of gross findings and clinical pathology results will be available to the pathologist when examining the microscopic slides.

Special Collections Bone marrow smears Collected: Yes ☐ No ☒ Stained and evaluated: Yes ☐ No ☒

Attachment 4, Dated 11/20/2020 7

QUALITY ASSURANCE

Regulatory Compliance This nonclinical laboratory study will ☐ / will not ☒ require compliance with the FDA Good Laboratory Practice Regulations and monitoring by the testing facility’s Quality Assurance Unit.

Standard Operating Procedures

All operations pertaining to this study, unless specifically defined in the protocol, will be performed according to the standard operating procedures of the testing facility and any deviations will be documented.

Protocol Amendments All changes in or revisions of an approved protocol and the reasons therefore will be documented, signed, and dated by the Principal Investigator, Study Director and the TPB Representative. Amendments will be maintained with the protocol. Verbal approval for a protocol change may be granted by the TPB Representative, but a written amendment will follow.

Disposition of Records and Pathology Materials ☐ Will require archiving Specimens (wet tissue, paraffin blocks, tissue slides), documentation records and raw data will be archived in accordance with FDA Good Laboratory Practice Regulations. The study protocol including all amendments and deviations, the final study report including all appendices, all raw data, and other documentation relevant to the study shall be stored for a period of two years. In addition, all tissue slides and paraffin blocks from the study will be stored for a period of two years.

At the end of the retention period, the Sponsor shall be contacted for instructions regarding further storage or transfer of the records, tissue slides and paraffin blocks. The formalin-fixed wet tissues shall be stored for two years following the in-life phase of a study. At the end of the retention period, the Sponsor shall be contacted prior to disposal of the wet tissues. Following confirmation from the Sponsor, the wet tissues shall be discarded in accordance with state and federal regulations.

☒ Will not require archiving

Specimens (wet tissue, paraffin blocks, tissue slides), documentation records and raw data will be retained during the duration of the study.

Upon completion of the study and issuance of the final study report, the Sponsor shall be contacted for instructions regarding further storage, transfer or disposal of the records, formalin-fixed wet tissues, tissue slides and paraffin blocks.

REPORTING AND DISCUSSION OF DATA

Statistical Analysis Group means and standard deviations will be calculated when appropriate for body weights and clinical pathology parameters.

Group mean and standard deviations will also be calculated, as appropriate, for the PK data.

Status Updates Status updates summarizing the progress of the study (unaudited) will be provided when requested by the TPB Representative. These updates will detail the status of the study on the reporting date, any problems encountered and proposed means of resolution. Real time in-life data will be made available in draft form.

Attachment 4, Dated 11/20/2020 8

Draft and Final Reports A draft report shall be due 45 working days after the necropsy date. The audited final report will be due 15 working days after review by the TPB Representative. In addition, the data and results of this study will be submitted as datasets in Excel and in SEND format. These shall be due when the audited final report is due. The report will be formatted as per FDA guidance in the electronic Common Technical Document (eCTD) format for IND filing.

PROTOCOL APPROVAL:

Study Director: Date:

Principal Investigator: Date:

TPB Representative: Date:

Attachment 4, Dated 11/20/2020 9

TWENTY-EIGHT DAY IND-DIRECTED TOXICITY STUDY of [compound name] in RATS GLP ☒ Non-GLP ☐

Division of Cancer Treatment and Diagnosis National Cancer Institute National Institutes of Health

Attachment 4, Dated 11/20/2020 10

STUDY OBJECTIVE

Study Objective The objective of this study is to determine target organ toxicities and their reversibility in rats that receive [compound name] when administered via oral gavage. The administration schedule consists of a single daily administration for 28 days with a 2-week recovery period.

Species Sprague Dawley rat Initial Age Range 8-12 weeks Initial Body Weight Range

~150-200 g (males); ~125-175 g (females)

Justification for Species

The rat is an accepted species that is used in preclinical safety evaluations of drugs that are intended for human use.

Animal Housing General procedures for animal care, housing and environmental conditions shall be in accordance with the current Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations, current requirements stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and current requirements as stated by the U.S. Department of Agriculture through the Animal Welfare Act and Animal Welfare Regulations (November 2013). The testing facility’s standard operating procedures for animal identification, quarantine, housing, feeding, handling, and care of animals shall be followed.

Route of Administration

Oral gavage

Schedule of

Once daily for 28 days

Males*

Number of Females*

DOSE

(mg/kg)

DOSE

(mg/m2)

TOTAL DOSE

(mg/kg)

TOTAL DOSE

(mg/m2)

1 VCTL2 13 13 0 0 0 0

2 13 13 TBD

3 13 13 TBD

4 13 13 TBD

*3 rats/sex/dose group will be assigned to the PK subgroup for plasma determination only. The remaining 10 rats/sex/dose group will be in the toxicology group.

of methods of synthesis, fabrication or derivation will be provided by the NCI.

Confirmation of identity will be done immediately upon receipt of each shipment of the compound. Yes ☒ No ☐

2 Vehicle control

Attachment 4, Dated 11/20/2020 11

Vehicle and Storage Conditions

Sterile water for injection. Store at room temperature.

Storage of TA At -30 to -15°C, protected from light and stored over inert gas (i.e. nitrogen).

TA Formulation

Dissolve in sterile water and dilute to achieve final concentrations for the respective mg/kg doses.

Formulations will be prepared daily by weighing a known quantity of test article and combining it with the appropriate volume of vehicle. The formulations will be administered within one hour of preparation. Formulations will be kept on ice until the dose is administered.

Dose Formulation Analysis

Yes ☒ No ☐

Dose concentration analysis shall be performed on the dose formulation(s).

Dose formulations must be within ±10% of the theoretical concentration.

Homogeneity analysis will not be conducted as part of this study.

The analytical method to determine the concentration of the dose formulation shall be HPLC/UV. The method will be supplied by the NCI. The analytical method shall be validated prior to initiation of this study.

Levels

Blood collection shall be performed according to the laboratory's SOP, but in no case will animals be bled from the treatment site. Blood samples shall be obtained from each animal at the time points indicated below.

Whole blood will be centrifuged and the plasma collected will be frozen at ≤20 °C until analysis. The time between blood collection and the freezing of plasma samples should be less than 1 hr.

For animals in a moribund condition, a blood sample shall be drawn and processed to plasma for plasma drug analysis prior to euthanasia.

PK Time Points Day 1 at 30 min, and 1, 2, 4, 8, and 24 hrs post-dose (prior to Day 2 dose); Days 28 prior to dosing and at 2 hr post-dose.

PK Sample Volume

0.7 mL

Attachment 4, Dated 11/20/2020 12

MEASUREMENTS

Clinical Observations

Pre-dose – Observe animals daily and record all baseline clinical signs beginning on study Day -3 (after quarantine period). Food consumption will be qualitatively measured on Day -3.

Dosing Period – All adverse clinical signs shall be recorded at least twice daily during the dosing period and at least once daily thereafter or more often if clinical signs are noted. Food consumption will be qualitatively measured daily during the dosing period and recovery period.

Body Weights Predose – On approximately Day -1 (after quarantine period), baseline body weights shall be recorded and used for randomization of animals into treatment groups.

Dosing Period – Body weights shall be recorded prior to test article/vehicle administration on Days 1, 5, 8, 12, 15, 19, 22, 26, 28; Days 36, 43, 50, and prior to necropsy on Days 29 and 57 at approximately the same time each day prior to feeding or necropsy.

Leukocyte count, differential (absolute) Mean corpuscular hemoglobin Mean corpuscular volume Platelet count

Hematocrit Leukocyte count, total Leukocyte count, differential (relative) Mean corpuscular hemoglobin concentration Nucleated red blood cell count Reticulocyte count, absolute and relative

Aspartate aminotransferase Cholesterol Creatinine Gamma glutamyl transferase Glucose Protein, total Urea nitrogen

Albumin Alkaline phosphatase Bilirubin, total Creatine kinase Globulin Lactate dehydrogenase

Calcium ☐ Chloride ☐ Inorganic phosphorus ☐

Potassium ☐ Sodium ☐ Sodium/Potassium ratio ☐

Coagulation* Yes ☒ No ☐

Activated partial thromboplastin time ☒ Fibrinogen ☒ Prothrombin time ☒

Urinalysis Yes ☐ No ☒

The following parameters will be measured (the time of collection may be modified by the Study Director with agreement by the COR):

Albumin ☐ Appearance ☐ Bilirubin ☐ Chloride ☐ Color ☐ Creatinine ☐ Glucose ☐ Ketones ☐ Leukocytes ☐ Nitrite ☐ Occult blood ☐ pH ☐ Potassium ☐ Sedimentation ☐ Sodium ☐ Specific gravity ☐

Attachment 4, Dated 11/20/2020 13

Blood Collections for Clinical Pathology GROUP Day -2 a Days 8, 15, 29 Days 43 and 57

Number of

Males

Number of

Females

Number of Males/timept

Number of Females/timept

Number of Males/timept

Number of Females/timept

1 VCTL 10 10 10 10 5 5

2 10 10 10 10 5 5 3 10 10 10 10 5 5 4 10 10 10 10 5 5 aBlood drawn for baseline measurements.

The tubes used for hematology will contain K3EDTA as an anticoagulant. The tubes used for serum chemistry will not contain anticoagulant but may contain serum separator gel. The tubes used for pathology determinations, and necropsied with histopathology evaluation as for scheduled necropsies. The authorization to euthanize moribund animals will be made by the Study Director or other qualified individual after examination of the animals. If an animal is found dead outside of normal working hours, it shall be necropsied as soon as possible with the carcass refrigerated (not frozen) in the interim period

GROUP Day 29 Day 57 Day Number of

Males Number of

Females Number of

Males Number of

Females Number of

Males Number of

Females

1 VCTL 5 5 5 5

2 5 5 5 5 3 5 5 5 5 4 5 5 5 5

Animals shall be terminated following the most recent AVMA Guidelines for the Euthanasia of Animals (2013) and the testing facility’s IACUC recommendations and facility SOPs.

Body weights and clinical pathology determinations shall be taken on the day of necropsy. The necropsy shall include examination of the external surface of the body; all orifices; the cranial, thoracic, abdominal and pelvic cavities, and their contents; and collection of the tissues listed below. A complete necropsy and all antemortem observations shall be recorded for each animal and commented on or confirmed at necropsy. Animals which are clinically normal will also be indicated. A pathologist will be available to examine any unusual findings.

Attachment 4, Dated 11/20/2020 14

Organ Weights Yes ☒ No ☐ Adrenals, brain, epididymides, heart, kidney, liver, spleen, testes, thyroid, parathyroid, thymus, ovary, uterus

Tissue Collection Complete ☒ Partial ☐ formalin.

☒Adrenal glands (2) ☒ Aorta (thoracic) ☒ Bone, femoral head with articular surface ☒ Bone marrow, sternum ☒ Bone marrow-rib, costochondral junction ☒ Brain ☒ Cecum ☒ Colon ☒ Duodenum ☒ Epididymides (2) ☒ Esophagus ☒ Eyes (2), including optic nerves ☐ Gall bladder ☒ Gross lesions ☒ Heart ☒ Ileum ☒ Jejunum ☒ Kidneys (2) ☒ Liver ☒ Lungs ☒ Lymph nodes (bronchial, mandibular, mesenteric)

☒ Mammary gland (when present in regular abdominal skin section)

☒ Ovaries ☒ Pancreas ☒ Parathyroid gland (when present in regular thyroid gland section)

☒ Pituitary gland

☒ Prostate gland ☒ Salivary gland (mandibular) ☒ Sciatic nerve ☒ Skeletal muscle ☐ Skin (ventral abdomen, injection site) ☒ Spinal cord, thoracolumbar (cervical and posterior lumbar spinal cord examined if nervous system signs present)

☒ Spleen ☒ Stomach (cardiac, fundic, and pyloric) ☒ Testes (2) ☒ Thymus ☒ Thyroid gland ☒ Tonsils (2) ☒ Trachea ☒ Urinary bladder (fundus) ☒ Uterus (with cervix) ☒Dorsal root ganglion

A sample of all fixed tissues shall be embedded in paraffin blocks. Sections of approximately 4 to 6 micrometers shall be cut, mounted on glass slides and stained with hematoxylin and eosin according to the testing facility’s SOPs.

All fixed tissues will be examined microscopically by a board certified veterinary pathologist. All lesions will be categorized either as drug-related or non-drug-related. All morphologies shall follow, to the best extent possible, the published criteria of the International Harmonization of Nomenclature and Diagnostic criteria (http:/www.goreni.org/). Records of gross findings and clinical

Attachment 4, Dated 11/20/2020 15

Bone marrow smears Collected: Yes ☒ No ☐ Stained and evaluated: Yes ☐ No ☒

Regulatory Compliance This nonclinical laboratory study will ☒ / will not ☐ require compliance with the FDA Good Laboratory Practice Regulations and monitoring by the testing facility’s Quality Assurance Unit.

Standard Operating Procedures

All operations pertaining to this study, unless specifically defined in the protocol, will be performed according to the standard operating procedures of the testing facility and any deviations will be documented.

Protocol Amendments All changes in or revisions of an approved protocol and the reasons therefore will be documented, signed, and dated by the Principal Investigator, Study Director and the TPB Representative. Amendments will be maintained with the protocol. Verbal approval for a protocol

☒ Will require archiving Specimens (wet tissue, paraffin blocks, tissue slides), documentation records and raw data will be archived in accordance with FDA Good Laboratory Practice Regulations. The study protocol including all amendments and deviations, the final study report including all appendices, all raw data, and other documentation relevant to the study shall be stored for a period of two years. In addition, all tissue slides and paraffin blocks from the study will be stored for a period of two years.

At the end of the retention period, the Sponsor shall be contacted for instructions regarding further storage or transfer of the records, tissue slides and paraffin blocks. The formalin-fixed wet tissues shall be stored for two years following the in-life phase of a study. At the end of the retention period, the Sponsor shall be contacted prior to disposal of the wet tissues. Following confirmation from the Sponsor, the wet tissues shall be discarded in accordance with state and federal regulations.

☐ Will not require archiving

Specimens (wet tissue, paraffin blocks, tissue slides), documentation records and raw data will be retained during the duration of the study.

Upon completion of the study and issuance of the final study report, the Sponsor shall be contacted for instructions regarding further storage, appropriate for body weights and clinical pathology parameters.

Group mean and standard deviations will also be calculated, as appropriate, for the PK data.

Status Updates Status updates summarizing the progress of the study (unaudited) will be provided when requested by the TPB Representative. These updates will

Attachment 4, Dated 11/20/2020 16 detail the status of the study on the reporting date, any problems encountered and proposed means of resolution. Real time in-life data will be made available in draft form.

Draft and Final Reports A draft report shall be due 60 working days after the necropsy date. The audited final report will be due 15 working days after review by the TPB Representative. In addition, the data and results of this study will be submitted as datasets in Excel and in SEND format. These shall be due when the audited final report is due. The report will be formatted as per

Attachment 4, Dated 11/20/2020 17

FIVE DAILY DOSE PHARMACOKINETICS AND DOSE RANGE-FINDING TOXICITY STUDY of [compound name] in DOGS

GLP ☐ Non-GLP ☒

Division of Cancer Treatment and Diagnosis National Cancer Institute National Institutes of Health

Attachment 4, Dated 11/20/2020 18

STUDY OBJECTIVE

Study Objective The objective of this study is to determine target organ toxicities and plasma elimination kinetics in Beagle dogs that receive [compound name] once daily for 4 days via IV bolus and oral gavage.

Species Beagle dog Initial Age Range >8 months Initial Body Weight Range

>8 kg

Justification for Species

The dog is an accepted species that is used in preclinical safety evaluations of drugs that are intended for human use.

Animal Housing General procedures for animal care, housing and environmental conditions shall be in accordance with the current Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations, current requirements stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and current requirements as stated by the U.S. Department of Agriculture through the Animal Welfare Act and Animal Welfare Regulations (November 2013). The testing facility’s standard operating procedures for animal identification, quarantine, housing, feeding, handling, and care of animals shall be followed.

Route of Administration

IV and PO (gavage)

Schedule of

Once daily for 5 days

Males

Number of Females

DOSE

(mg/kg)

DOSE

(mg/m2)

TOTAL DOSE

(mg/kg)

TOTAL DOSE

(mg/m2)

1 VCTL3 2 2 0 0 0 0

2 2 2 TBD (PO)

3 2 2 TBD (PO)

4 2 2 TBD (IV)

of methods of synthesis, fabrication or derivation will be provided by the NCI.

Confirmation of identity will be done immediately upon receipt of each shipment of the compound. Yes ☐ No ☒

3 Vehicle control

Attachment 4, Dated 11/20/2020 19

Vehicle and Storage Conditions

Sterile water for injection (PO); 0.95% sterile saline (IV). Store at room temperature.

Storage of TA At 2-8°C, protected from light.

TA Formulation

Dissolve in sterile water and dilute to achieve final concentrations for the respective mg/kg doses.

Formulations will be prepared daily by weighing a known quantity of test article and combining it with the appropriate volume of vehicle. The formulations will be administered within one hour of preparation. Formulations will be kept on ice until the dose is administered.

Dose Formulation Analysis

Yes ☐ No ☒

Dose concentration analysis shall be performed on the dose formulation(s).

Dose formulations must be within ±10% of the theoretical concentration.

Homogeneity analysis will not be conducted as part of this study.

Levels

Blood collection shall be performed according to the laboratory's SOP, but in no case will animals be bled from the treatment site. Blood samples shall be obtained from each animal at the time points indicated below.

Whole blood will be centrifuged and the plasma collected will be frozen at ≤20 °C until analysis. The time between blood collection and the freezing of plasma samples should be less than 1 hr.

For animals in a moribund condition, a blood sample shall be drawn and processed to plasma for plasma drug analysis prior to euthanasia.

PK Time Points Pre-dose, 2 (iv only), 5 (iv only), 10, 20, 30 min, and 1, 2, 4, 8, 12, 24 hrs after dosing on both Days 1 and 4.

PK Sample Volume

2 mL

PK Data Analysis (PK Parameters) Non-compartmental analysis (Tmax, Cmax, AUC, CL, bioavailability, etc.) on all dose groups.

Attachment 4, Dated 11/20/2020 20

MEASUREMENTS

Clinical Observations

Pre-dose – Observe animals daily and record all baseline clinical signs beginning on study Day -3 (after quarantine period).

Dosing Period – All adverse clinical signs shall be recorded at least twice daily during the dosing period and at least once daily thereafter or more often if clinical signs are noted.

Body Weights Predose – On approximately Day -1 (after quarantine period), baseline body weights shall be recorded and used for randomization of animals into treatment groups.

Dosing Period – Body weights shall be recorded prior to test article/vehicle administration on Day 1 and on Days 8 and 15 at approximately the same time each day prior to feeding.

Leukocyte count, differential (absolute) Mean corpuscular hemoglobin Mean corpuscular volume Platelet count

Hematocrit Leukocyte count, total Leukocyte count, differential (relative) Mean corpuscular hemoglobin concentration Nucleated red blood cell count Reticulocyte count, absolute and relative

Aspartate aminotransferase Cholesterol Creatinine Gamma glutamyl transferase Glucose Protein, total Urea nitrogen

Albumin Alkaline phosphatase Bilirubin, total Creatine kinase Globulin Lactate dehydrogenase

Calcium ☐ Chloride ☐ Inorganic phosphorus ☐

Potassium ☐ Sodium ☐ Sodium/Potassium ratio ☐

Coagulation* Yes ☒ No ☐

Activated partial thromboplastin time ☒ Fibrinogen ☒ Prothrombin time ☒

Urinalysis Yes ☐ No ☒

The following parameters will be measured (the time of collection may be modified by the Study Director with agreement by the COR):

Albumin ☐ Appearance ☐ Bilirubin ☐ Chloride ☐ Color ☐ Creatinine ☐ Glucose ☐ Ketones ☐ Leukocytes ☐ Nitrite ☐ Occult blood ☐ pH ☐ Potassium ☐ Sedimentation ☐ Sodium ☐ Specific gravity ☐

Attachment 4, Dated 11/20/2020 21

Blood Collections for Clinical Pathology GROUP Day -2 a Days 2 (prior to dosing), 8, and

Days

Number of

Males

Number of

Females

Number of Males/timept

Number of Females/timept

Number of Males/timept

Number of Females/timept

1 VCTL 2 2 2 2 0 0

2 2 2 2 2 0 0 3 2 2 2 2 0 0 4 2 2 2 2 0 0 aBlood drawn for baseline measurements.

The tubes used for hematology will contain K3EDTA as an anticoagulant. The tubes used for serum chemistry will not contain anticoagulant but may contain serum separator gel. The tubes used for pathology determinations, and necropsied with histopathology evaluation as for scheduled necropsies. The authorization to euthanize moribund animals will be made by the Study Director or other qualified individual after examination of the animals. If an animal is found dead outside of normal working hours, it shall be necropsied as soon as possible with the carcass refrigerated (not frozen) in the interim period

Scheduled Necropsy (NONE)

GROUP Day Day Day Number of

Males Number of

Females Number of

Males Number of

Females Number of

Males Number of

Females

1 VCTL

Animals shall be terminated following the most recent AVMA Guidelines for the Euthanasia of Animals (2013) and the testing facility’s IACUC recommendations and facility SOPs.

Body weights and clinical pathology determinations shall be taken on the day of necropsy. The necropsy shall include examination of the external surface of the body; all orifices; the cranial, thoracic, abdominal and pelvic cavities, and their contents; and collection of the tissues listed below. A complete necropsy and all antemortem observations shall be recorded for each animal and commented on or confirmed at necropsy. Animals which are clinically normal will also be indicated. A pathologist will be available to examine any unusual findings.

Organ Weights Yes ☐ No ☐ Adrenals, brain, epididymides, heart, kidney, liver, spleen, testes, thyroid/parathyroid, thymus, ovary, uterus

Attachment 4, Dated 11/20/2020 22

Tissue Collection Complete ☐ Partial ☒ (UNSCHEDULED NECROPSY) formalin.

☐Adrenal glands (2) ☐ Aorta (thoracic) ☒ Bone, femoral head with articular surface ☐ Bone marrow, sternum ☐ Bone marrow-rib, costochondral junction ☒ Brain ☒ Cecum ☒ Colon ☒ Duodenum ☐ Epididymides (2) ☐ Esophagus ☐ Eyes (2), including optic nerves ☐ Gall bladder ☒ Gross lesions ☒ Heart ☒ Ileum ☒ Jejunum ☒ Kidneys (2) ☒ Liver ☒ Lungs ☒ Lymph nodes (bronchial, mandibular, mesenteric)

☐ Mammary gland (when present in regular abdominal skin section)

☒ Ovaries ☐ Pancreas ☐ Parathyroid gland (when present in regular thyroid gland section)

☐ Pituitary gland

☐ Prostate gland ☐ Salivary gland (mandibular) ☒ Sciatic nerve ☐ Skeletal muscle ☐ Skin (ventral abdomen, injection site) ☒ Spinal cord, thoracolumbar (cervical and posterior lumbar spinal cord examined if nervous system signs present)

☒ Spleen ☒ Stomach (cardiac, fundic, and pyloric) ☒ Testes (2) ☒ Thymus ☒ Thyroid gland ☐ Tonsils (2) ☐ Trachea ☒ Urinary bladder (fundus) ☒ Uterus (with cervix)

A sample of all fixed tissues shall be embedded in paraffin blocks. Sections of approximately 4 to 6 micrometers shall be cut, mounted on glass slides and stained with…