Sample_Task_Order__2_-_Protocol_REV_12-21-15.pdf

SAMPLE PROTOCOL

TASK ORDER #2

Pharmacokinetic-Pharmacodynamic-Efficacy Evaluation of [NSC(s) XXXXX] in

[Cell Line TBD] Tumor Xenografts in Nude Mice

SPONSOR: Toxicology and Pharmacology Branch

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

MONITOR: TBD

CONTRACT NUMBER: TBD

CONTRACTOR: TBD

PROJECT MANAGER: TBD

PROJECT DIRECTOR: TBD

KEY DATES:

hudakad Typewritten Text RFP Number: N02CM67000-11 hudakad Typewritten Text Attachment 19-2P

Institutional No. xxxxxx PAGE 2 OF 7

I. OBJECTIVE

The objectives of this evaluation are to determine the efficacy of the LDHA inhibitor(s), NSC(s) XXXXX against [cell line] xenografts in mice and to obtain plasma and tumor samples at various times to characterize the plasma and tumor pharmacokinetics and pharmacodynamics of the compound(s).

II. MATERIALS AND METHODS

A. Test Article and Vehicle:

1. Name of Test Article:

TBD

2. Vehicle:

TBD

3. Characterization and Documentation of Methods of Synthesis, Fabrication or Derivation or test article and vehicle:

a. Test Article:

Compound identity, strength, quality, stability and purity and documentation of methods of synthesis, fabrication or derivation are the responsibility of the NCI and will be communicated to the CRO.

b. Vehicle:

The vehicle (and its components) is stable throughout the date of expiration on the container labels when stored according to the manufacturer’s instructions.

B. Test System:

1. Species, Strain Supplier and Test System Justification:

Athymic nude (nu/nu NCr) mice obtained from an approved commercial supplier will be used in this evaluation.

2. Initial Age, Sex and Weight:

On the first day of dosing, the weight ranges of mice will be approximately 20-24 grams and the mice will be approximately 6 to 7 weeks of age. Only females will be used.

3. Care and Housing:

General procedures for animal care and housing will be in accordance with the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011 and the U.S.

Department of Agriculture through the Animal Welfare Act (Public Law 99-198). Appropriate caging and bedding or cage board (not

Institutional No. xxxxxx PAGE 3 OF 7 cedar or pine chips) will be used. No contaminants shall be present in the bedding that could interfere and affect the results of the evaluation. Environmental parameters will be set to maintain conditions specified in the facility SOPs. Environmental conditions will be within specified limits of at least 90 percent of scheduled observations.

4. Diet and Water Supply:

Diet is to be certified, commercial, dry rodent chow provided ad libitum. Water source will be the public supply given ad libitum. No contaminants will be present in the feed or water that could interfere and affect the results of the evaluation.

5. Acclimation:

All animals will be isolated upon arrival, and the Attending Veterinarian or designee will document that the animals are healthy and free from disease and parasites. The animals will be acclimated to the housing room for at least 5 days prior to dosing.

Only healthy animals will be placed on test.

6. Animal Identification:

All mice will be given a unique identification number by ear punch or other approved method.

7. Human Tumor Xenograft:

Tumor cells will be obtained from the American Type Culture Collection (ATCC) or other approved source. Cells shall be passaged in vitro according to the supplier’s recommendations. A suspension of 1x107 cells shall be implanted subcutaneously in sufficient host animals to conduct the evaluation described below.

Tumors will be staged when the median tumor size reaches approximately 200 mg (or a designated size as per NCI direction).

C. Experimental Design

1. Randomization:

In order to obtain groups that are comparable by tumor weight, all mice will be randomly assigned to each treatment group using a computer-based body weight stratification procedure, randomizing for tumor volumes calculated as follows:

Tumor volume = 1/2(length × width2)

2. Group Assignments:

Upon randomization, 59 female mice will be assigned to the vehicle control group and 51 mice will be assigned to each of four treatment groups. Eight mice will be used as untreated controls.

Institutional No. xxxxxx PAGE 4 OF 7

Group # Mice Cpd. Route Dose

(mg/kg)

Conc. in Formulation

(mg/mL)

Dose Volume (mL/kg)

Schedule

59 (16 for efficacy)

Vehicle IP 0 0 10 QD x 14

51 (8 for efficacy)

NSC 1 IP Level 1 TBD 10 QD x 14

51 (8 for efficacy)

NSC 1 IP Level 2 TBD 10 QD x 14

51 (8 for efficacy)

NSC 2 IP Level 1 TBD 10 QD x 14

51 (8 for efficacy)

NSC 2 IP Level 2 TBD 10 QD x 14

6 8 Untreated Controls

3. Route of Administration and Reason for Choice:

The test compound will be given as daily intraperitoneal injections for 14 days in order to characterize the effects on tumor growth and the pharmacokinetic-pharmacodynamic time course in plasma and tumor.

4. Formulation Preparation

[Prepared twice-weekly; detailed procedures to be specified]

5. Dosing Procedure:

All mice in each group will receive either vehicle (Group 1) or formulated test article (Groups 2-5) by intraperitoneal injection on days 1 to 14. The dose volume will be 10 mL/kg of body weight for all dose groups. The amount of drug administered to each mouse will be based on each animal’s most recently determined individual body weight.

6. Measurements:

a. Clinical signs:

The mice will be observed at least once daily during the dosing period and at least twice-weekly thereafter. Any adverse clinical signs recorded.

Institutional No. xxxxxx PAGE 5 OF 7

b. Body Weight:

Mice shall be weighed prior to dosing each day and the weight shall be used to adjust the dose volume for test article administration.

c. Tumor Volume:

Tumor volumes for all mice remaining on study will be measured by caliper at least twice weekly. Note that cohorts of 8 mice/group (16 in control group) will remain after the dosing/sampling period and shall have tumor volume measurements at the above interval until the end of the evaluation (~50-60 days post-implant).

d. Plasma and Tumor Sample Collection:

Plasma and tumor samples will be collected as follows:

Day 1 Collections

Harvest tumors from 5 mice in Groups 1-5 at 2, 4, 8, 16, and 24 hr after dose 1 (the latter just prior to dose 2), cut into halves and flash freeze.

Collect blood in EDTA tubes at times of sacrifice - spin, flash freeze plasma.

Harvest tumor and plasma samples from all Group 6 (untreated) mice at a convenient time on Day 1

Day 4 Collections

Harvest tumors from 3 mice in Groups 1-5 at 2 hr after dose 4 and 24 hours (just prior to dose 5), cut into halves and flash freeze.

Collect blood in EDTA tubes at times of sacrifice - spin, flash freeze plasma.

Day 9 Collections

Harvest tumors from 3 mice in Groups 1-5 at 2 hr after dose 9 and 24 hours (just prior to dose 10), cut into halves and flash freeze.

Collect blood in EDTA tubes at times of sacrifice - spin, flash freeze plasma.

Day 14 Collections

Harvest tumors from 3 mice in Groups 1-5 at 2 hr and 24 hours after dose 14, cut into halves and flash freeze.

Collect blood in EDTA tubes at times of sacrifice - spin, flash freeze plasma.

Actual collection times will be recorded.

Blood samples will be placed on wet ice until centrifuged to obtain plasma. Plasma samples will be transferred into appropriately labeled tubes and placed on dry ice until stored in a freezer set to maintain at least -70 ºC until all samples are ready for shipping.

Tumors will be excised from the implant site (according to facility SOP), cut into halves and flash frozen. Each tumor piece will be placed in a separate cryo vial and stored at least -70 ºC until all samples are ready for shipping..

d. Necropsy Procedure:

Necropsy is not required in this evaluation. Moribund

Institutional No. xxxxxx PAGE 6 OF 7 animals and animals with excessive or necrotic tumor growth will be euthanized according to facility SOP.

e. Bioanalytical and Pharmacodynamic Analysis:

Plasma and tumor levels of the test article, as well as pharmacodynamic measurements will be conducted at another NCI-designated facility.

All samples from the evaluation shall be shipped on dry ice in one batch to:

[To be specified in final protocol]

III. QUALITY ASSURANCE

A. Type of Evaluation This evaluation will not require compliance with the FDA Good Laboratory Practice Regulations.

B. Standard Operating Procedures

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

C. Protocol Amendments

All changes in or revisions of an approved protocol and the reasons therefore will be documented, signed, and dated by the Project Manager/Project Director and the NCI COR. Amendments will be maintained with the protocol. Verbal approval for changes in the protocol may be granted by the NCI COR, but a written amendment will follow.

IV. REPORTING AND DISCUSSION OF DATA

B. Report and Data Analysis

All animal related data (clinical observations, body weights or other) will be provided in an excel spreadsheet suitable for post hoc analysis.

Documentation of tumor and plasma collection times, sample handling and sample numbers shall be provided in a spreadsheet to be included with the sample shipment and in a duplicate file sent electronically to the

NCI COR.

Institutional No. xxxxxx PAGE 7 OF 7

V. REGULATORY REFERENCES

A. Facilities and Animal Husbandry:

Animal care will be in compliance with facility SOPs, the Guide for the Care and Use of Laboratory Animals, (Institute of Laboratory Animal Research, Division on Earth and Life Sciences, National Research Council; National Academy Press; Washington, DC;

2011), and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198).

B. Animal Welfare Act Compliance:

By signing this protocol, the Sponsor signifies that there are no generally accepted alternatives to the use of animals, and that the evaluation described by this protocol does not unnecessarily duplicate previously conducted or reported experiments.

Procedures used in this protocol are designed to conform to accepted practices and to minimize or avoid causing pain, distress, or discomfort in the animals. In those circumstances in which required test procedures are likely to cause more than momentary or slight pain or distress, the animals will receive appropriate analgesics or anesthetics unless the withholding of these agents has been justified in writing by the Project Director and approved by the facility’s Institutional Animal Care and Use Committee (IACUC).

C. Safety Compliance:

This evaluation will comply with all applicable federal and state regulations, including the CDC/NIH’s BMBL current edition, OSHA standard 1910.1450 “Occupational Exposure to Hazardous Chemical in Laboratories” and by applying recommended best practices or other recognized safe practices and codes. The evaluations will be conducted in a manner that ensures that the health, safety, and welfare of our employees, the public, and the environment are maintained.

Protocol Approvals:

Project Manager/Project Director: _____

TBD Date

NCI's Representative:

TBD Date