Sub-study I Pharmacokinetics (PK) and Pharmacodynamics (PD).
Sub-study Ia:
On day 0, seven-weeks old male SHR will be randomized into three groups. Rats in groups A (n=12) and B (n=12) will receive a bolus administration of a maximum (630 μg/kg, 0,5 mL) or half of the maximum (315 μg/kg, 0,5 mL) doses of our investigational drug, respectively, into the tail vein. Rats in group C (n=12) will receive bolus i.v. (tail vein) injection of vehicle (PBS, 0,5 mL) (Table 1). The doses proposed for Sub-study Ia will be a maximum 2 μM based on the minimum effective concentration from cytotoxicity studies (16 μM). All three groups (A, B, and C) will be submitted to in vivo PK investigations in a time-dependent fashion, consisting of a collection of blood samples from the tail vein of the same animals at baseline (300μl of blood collection), and 5 (300ul of blood collection), 30 minutes (terminate and blood collection) (6 SHR per group), 10 (300ul of blood collection), 60 (300ul of blood collection) and 120 minutes (terminate and blood collection) (6 SHR per group) after the i.v. administration of our drug or vehicle. With the purpose of the PD and PK investigations, the concentration of our investigational drug in the bloodstream will be determined (using HPLC method), and cGMP will be measured (via ELISA). Sub-study Ia will also determine the optimal dose of our investigational drug that should be intravenously administrated in Sub-study Ib and its half-life.

Sub-study 1b:
On day 0, 15-week-old male SHR will be randomized into two groups. Rats in group D (n=12) will receive a bolus administration of the optimal dose of our investigational drug based on the results of sub-study Ia via tail vein injection. Rats in group E (n=12) will receive bolus i.v. (tail vein) injection of vehicle (PBS, 0,5 mL) (Table 2). This study is designed to define the acute effect of our investigational drug in a time-dependent fashion. A collection of blood samples from the tail vein of the same animals will be performed at baseline (300ul of blood), at half-life assessed in sub-study Ia (300ul of blood), and at 120 minutes (terminate and blood collection) after the i.v. administration of our investigational drug or vehicle. Blood pressure assessment will be performed as described in procedure 3 (at baseline, 5, 10, 30, 60, and 120 minutes after drug injection). On week 14 a subset of animals will go under a minimally invasive surgical process for telemetry system implantation (procedure 4) for continuous blood pressure measurement. Sub-study Ib will also determine the frequency (once or twice a day) of oral administration for Sub-study II.

Sub-study IIb Oral administration
On day 0, 15-weeks-old male SHR will be randomized into five groups. Rats in group F (n = 23) will be treated with Vehicle via oral gavage for one week. Rats in group G (n = 23) will be treated with our investigational Drug via oral gavage (once or twice a day, depending on the results obtained in Sub-study Ib) for one week. Rats in group H (n = 23) will be treated with MCUF-651 Drug via oral gavage once per day for one week (doi:10.1073/pnas.2109386118). In group I (n = 23), on day 15, animals will be implanted with one 7-day osmotic pump containing 0.1 nmol/kg BNP1-45 (Table3). Rats in group J (n = 23) will be treated with our investigational Drug via oral gavage for one week, plus s.c. delivery of recombinant rat BNP1-45 (0.1 nmol/kg, via osmotic minipump) (Table3). Both BNP and the investigational drug proposed to be tested in the current pilot study can selectively stimulate a natriuretic peptide receptor and generate cGMP, a second messenger that effectively regulates vascular tone in vivo. Therefore, we hypothesized that we could induce sustained blood pressure reduction by enhancing cGMP generation. Of note, the rat brain natriuretic peptide 1-45 (or rat BNP1-45) has been isolated from rat hearts and shown to be a circulating form of rat BNP1-45. Hence, BNP administration will be used as an internal control for the current protocol. Every other day, rats will undergo conscious blood pressure measurements using the tail-cuff method (Procedure 3). A subset of rats (n=6) will undergo a minimally invasive surgical procedure (day -7) to implant a telemetry device (Procedure 4) for acquiring direct blood pressure data from conscious rats. For blood pressure acquisition, each cage is placed over the receiver panel that is connected to the computer for data acquisition. The rats are unrestrained and free to move within their cages. Telemetry data is recorded every 10 minutes for 2 minutes for 24 hours/day (12-hr light-dark cycle). Despite the telemetry devices will be implanted only in 50% of animals, we will recover meaningful information on the primary parameter of observation (i.e. blood pressure) due to the reduced inter-animal variability (each animal will be his/her own control) using direct measurement of blood pressure level. Drug treatment will start seven days after surgery (Procedure 4) by performing oral gavage of the experimental Drug (Procedure 2)(Group D) or by implanting an osmotic minipump (Procedure 5) to deliver BNP1-45 (Group E), after a minimally invasive surgical procedure. Telemetry data will be collected for the following two weeks during treatment (days 0 to 14). In the first week (days 0 to 7) the animals will be randomized to receive either Vehicle or the Peptide via oral gavage. At the beginning of the second week of intervention (day 8) the rats that received Vehicle in the first week will have the peptide administrated via gavage or vice versa. By doing that each animal will serve as a control to itself. At the end of week two, all rats will be euthanized and tissue and blood collected for ex vivo examinations (molecular biological and biochemical assays).

We are proposing the adjustment for experiments pharmacokinetics and pharmacodynamics study, oral administration (described in detail below and in the FOTS protocol) to be performed in above mentioned groups. Therefore, we reduced the number of SHR rats to 175 (updated below) to complete the experiments.
1. Purpose. We have recently discovered a new small molecule that selectively stimulates an endogenous hormonal system known to be impaired in patients with hypertension. Activation of this endogenous system, may be beneficial in patients who are not responsive to current anti-hypertensive medications and, therefore, remain at high risk of cardiovascular events and death. Here, we purpose an in vivo preclinical study to identify the most effective dose of our small molecule in reducing high blood pressure in Spontaneously Hypertensive Rats (SHR).
2. Distress. Based on regulations, limited blood sampling with less 10% sampling of circulating blood is classified as mild distress.
3. Expected benefit. We expect that our investigational drug will induce sustained and adequate blood pressure reduction and lead to the development of new medication for the treatment of drug-resistant hypertension.
4. Number of animals, and what kind. A total of 175 SHR will be used to determine the most effective dose of our investigational drug in reducing high blood pressure.
5. How to adhere to 3R. This study investigates the most effective dose of a new compound that modulates arterial blood pressure. Replacement: The use of animals is necessary at this stage where a new compound has been discovered in silico and successfully tested in vitro. An effective preclinical dose that elicits sustained blood pressure reduction must be tested in vivo. Refinement: The procedures used for this study has been employed by the applicant in several other studies and are minimally invasive (i.e. intravenous bolus injection, oral gavage, tail-cuff blood pressure measurement, and telemetry device implantation for blood pressure monitoring), causing mild distress to the animals. Reduction: Our group has expertise with the animal model, the procedures proposed in the current protocol, and the pathway targeted by the investigational drug. The number of rats per group was calculated based on power analysis to have the minimum number to detect statistical differences among groups for the blood pressure measurements. The proposed protocol is based on a previously approved study, FOTS protocol (#12582).

We request an extension of this protocol until 3/6/2025

Request for modification June 16.2023
Addition of a new stain (Wistar Rats). See attached file.

Request for modification October 21.2023
Forty male SHRs will be included in this new set of studies. See the attached file for a complete description of the changes.

Susceptibility of Atlantic salmon families against CMS