SOW_GemPharmatech follow up_2024 FINAL.pdf

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

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

Using humanized mouse models of NNMT and SULT1A1. This is a modification of PO 75N95023P00824 P00001- GemPharmatech, LLC in follow up efficacy studies

Statement of Need and Purpose:

NCATS needs to evaluate humanized NNMT and SULT1A1 in follow up studies. NCATS has developed small molecule inhibitors of NNMT and SULT1A1 that have differential activities against the human and mouse forms of the enzyme. Therefore, it is imperative that we develop mouse models that contain the human form of the enzymes to test our molecules in various models. An additional 50♂ B6-hNNMT homozygous mice at 4-6 weeks of age needs to be produced for evaluation in diet induced obesity models; this will be done in 8X5 groups with lead inhibitor and controls. The hSULT1A1 will be used to conduct a MTD and PK study with YC1 And followed up with liver pathology.

Background Information and Objective:

Biochemical testing of inhibitors from the NNMT and SUL1A1 projects have shown lower potency towards the human version of the enzyme vs the mouse version in biochemical and cellular assays. This has forced the use of large amounts of compound in animal studies without a true readout of the therapeutic potential in human. Thus, it is imperative that humanized mouse models are developed for dosing to determine the maximum tolerated dose and subsequent doses for PK/PD and efficacy experiments. Using 75N95023P00824 P00001 such mice are being generated and this modification will allow their in vivo evaluation.

Period of Performance:

12 months ARO

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

Specific Requirements:

There are two parts to this project:

PART 1 – MTD study and PK study

PART 2 – Efficacy study on hNNMT mice fed with HFD

PART 1 – MTD study and PK study

STUDY DESIGN - MTD study Mouse strain: hNNMT mice (6-8 weeks old), male, total 10 mice.

In vivo part:

(1) Mice will enter the animal facility for one week of acclimatization. The body weight will be recorded one week before test article administration, and fasted for at least 4 h on the day of administration.

(2) The first mouse will be orally administered the test article with a single dose of 175 mg/kg and the injection volume is 10μL/g. Then the mouse will be observe 30 min, 2 h, 4 h, 8 h, and 24 h, 48 h after injection and daily observation will continue until day 14 after injection.

(3) If the first mouse is live at 48 h after administration, then inject the second mouse, the second mouse will be orally administered the test article with a single dose of 550 mg/kg. If the second mouse is dead at 48h after administration, another two mice will be orally administered the test article with a single dose of 175 mg/kg to observe their living for two weeks. So the dose of 175 mg/kg can be determined as the maximum tolerated dose.

(4) If the mouse administered a single dose of 550mg/kg is live at 48 h after administration, then the third mouse will be orally administered the test article with a single dose of 2000 mg/kg . If the third mouse is dead at 48h after administration, another two mice will be orally administered the test article with a single dose of 550 mg/kg to observe their living for two weeks. So the dose of 550 mg/kg can be determined as the maximum tolerated dose.

(5) If the mouse administered a single dose of 2000mg/kg is live at 48 h after administration, another two mice will be orally administered the test article with a single dose of 2000 mg/kg to observe their living for two weeks. So the dose of 2000 mg/kg can be determined as the maximum tolerated dose.

(6) 2000 mg/kg is considered the upper dose limit for testing.

In vitro part:

(1) Serum from mice that survive at 48 h after administration will be collected to analysis liver function

(ALT,AST).

(2) The heart, liver, spleen, lung, kidney, brain, stomach, small intestine (jejunum), colon, femur, gastrocnemius, uterus, and ovary will be collected 14 days after administration to be grossly observed whether there are obvious abnormal. Then these tissues will be fixed and embedded in paraffin.

STUDY DESIGN - PK study Mouse strain: hNNMT mice (6-8 weeks old), male, total 27 mice.

Group Number of mice

Treatment Dosage (mg/kg)

Route Frequency Timepoint of plasma collection

G1

3 TA

TBD-low does p.o.

single Pre-dose, 30min,4h,24h

3 TA single 5min, 1h, 6h, 48h 3 TA single 15min, 2h, 8h, 72h

G2

3 TA

TBD-middle does p.o.

single Pre-dose, 30min,4h,24h

3 TA single 5min, 1h, 6h, 48h 3 TA single 15min, 2h, 8h, 72h

G3

3 TA

TBD-high does p.o.

single Pre-dose, 30min,4h,24h

3 TA single 5min, 1h, 6h, 48h 3 TA single 15min, 2h, 8h, 72h

~ TBD – to be determined PK analysis:

This part includes Method Transfer and Sample Analysis for quantification of a small molecule drug in mouse plasma.

(1) Method Transfer:

The client should provide the LC-MS method (internal standard, ion transition, ion source parameters, ionization mode, etc.) and sample pretreatment method.

(2) Sample Analysis:

Sample analysis of small molecule drug in mouse plasma using the above qualified LC-MS method.

PART 2 – Efficacy study on hNNMT mice fed with HFD

STUDY DESIGN

Mouse strain: C57BL/6 mice and hNNMT mice, male, total 40 mice.

Group Number of mice

Model Treatment Dosage (mpk)

Route Schedule Total doses

G1 8 B6-CD Vehicle - p.o. QD x 4W 28 dose G2 8 hNNMT-HFD Vehicle - p.o. QD x 4W 28 dose G3 8 hNNMT-HFD TA TBD p.o. QD x 4W 28 dose G4 8 hNNMT-HFD TA TBD p.o. QD x 4W 28 dose G5 8 hNNMT-HFD TA TBD p.o. QD x 4W 28 dose

~ TBD – to be determined

Study details:

(1) Note: The 27 hNNMT mice will be washed for at least two weeks after the PK experiment and continued to be enrolled for efficacy experiments.

(2) DIO modeling: After all mice entered the animal facility, they will be acclimated for 3 days or more.

hNNMT mice will be fed with 60% HFD for at least 12 weeks (Body weight will be monitored weekly) to establish a DIO model. Besides, C57BL/6J mice of similar age fed with chow diet at the same time will be enrolled as healthy controls.

(3) Grouping: Before grouping, all mice will be fasted (5~6h) and their blood will be collected for insulin, leptin, blood glucose and blood lipid (TG,TC,LDL-C, HDL-C) measurement. Fat and lean mass will be measured with QNMR. Then mice will be randomly divided into 8 groups with 8 mice in each group according to the body weight, fasting blood glucose, fat and lean mass.

(4) Administration: The date of the first agent administration will be denoted as day 0. The administration frequency is QW, and the administration route and dosage is TBD. All mice will be treated for 4 weeks.

(5) Data will be collected in efficacy period:

a. Body weight will be measured daily.

b. Food intake will be measured twice a week. Food intake will be measured as the average amount of 4 mice per cage.

c. Fasting blood glucose will be measured once a week.

d. Using QNMR before dosing, after dosing for 2 weeks and 4 weeks to measure lean mass and fat mass changes.

e. OGTT will be performed once before study endpoint.

Study endpoint:

Study endpoint will be 4 weeks from day 0.

(1) Mice will be weighted and fasted (5~6h) , then their blood will be collected for insulin, leptin, blood glucose, blood lipid (TG, TC, LDL-C, HDL-C) and liver function (ALT, AST). Remaining serum/plasma will be frozen and stored at -80℃.

(2) The liver will be collected and weighted at study endpoint. Part of liver will be homogenized for detection of liver TC and TG content. The middle part of the left, middle and right lobes of the liver will be embedded and stained with H&E for NAS score. Remaining liver tissue will be frozen and stored at - 80℃.

Estimated timeline: 8 months from initiation

List of agents to be tested

Agent name Note 1 TA Provided by Client or GPT *Final cost of agents provided by GemPharmatech will be calculated upon study completion and incorporated into the final invoice unless otherwise agreed upon.

See protocol for detailed study design.

Timeline of the Project MTD study and PK study - 3 months

Efficacy study - 5 months A timeline entered herein is the best estimate from GemPharmatech and will be valid only if the SOW is agreed by the Parties and fully executed. The Project will commence only after the payment terms defined in this SOW have been satisfied.

Study will initiate within 2 weeks of receipt of all agents/reagents at GemPharmatech Co. Ltd.

LEVEL OF EFFORT:

N/A

GOVERNMENT RESPONSIBILITIES

NCATS will supply compounds, earlier PK methods, and formulation methods to CRO

DELIVERY OR DELIVERABLES

Upon study endpoint, a final report will be provided to the Client within three (3) weeks through electronic delivery, unless otherwise specified and agreed. The client shall review the final report from GemPharmatech and provide comments within two (2) weeks. If no comments are received within the two (2) week time period, it shall be deemed that it is accepted by the Client and the Project defined in the SOW is completed.

REPORTING REQUIREMENTS

Progress reports of NNMT mice production and progress in efficacy and tolerability studies due upon request from NCATS.

OTHER CONSIDERATIONS

Travel:

N/A

Key Personnel:

Patnaik, Samarjit (NIH/NCATS) [E] <patnaiks@mail.nih.gov>

Information System Security Plan:

Data Rights:

Data belongs to NCATS

Section 508—Electronic and Information Technology Standards:

Publications and Publicity:

Contractor will not publish any information without informing NCATS. All information is confidential and NCATS will solely determine date for publication or publicity or patenting.

Confidentiality of Information:

All information shared between NCATS and contractor is confidential. The time or method for disclosure will be determined by NCATS.