Kd: 0.3 nM (APJ receptor)[1]

BMS-986224 has an EC50 of 0.02 nM for human APJ and completely blocks forskolin-mediated cAMP synthesis. Chinese hamster ovary-K1 or HEK293 ZF cells fully respond to BMS-986224 (0-100 nM) in terms of β-arrestin recruitment, ERK phosphorylation, and APJ internalization[1]. Strong and selective APJ receptor agonist BMS-986224 displays a signaling profile resembling that of (Pyr1) apelin-13[1].

In the RHR model, BMS-986224 (0.192 mg/kg or 3 mg/kg; SC infusion; daily) produced effects distinct from enalapril by increasing stroke volume and cardiac output to levels observed in healthy animals, but without preventing cardiac hypertrophy and fibrosis[1].

GPCR Selectivity Assays [1]
GPCR selectivity assays were conducted to assess the potential for BBMS-986224 to interact with other GPCRs (adenosine A2A; adrenergic alpha 1B, 1D, 2A, and 2C, and adrenergic beta 1 and beta 2; cannabinoid CB1; dopamine D1 and D2; histamine H1 and H2; muscarinic M2; opioid mu and kappa; serotonin 5HT1B, 5HT2B, and 5HT4). The assays were performed as described in Alt et al.12 Competitive radioligand binding assays using membrane filtration methods were used to assess the ability of BBMS-986224 to compete the binding of the radioligands in membranes derived from cells overexpression the individual human GPCRs.

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Radioligand Binding Assays[1]
A range of BBMS-986224 concentrations was added to cell membrane extract containing [3H] apelin-13 (~2-fold equilibrium dissociation constant [Kd]). Excess unlabeled (Pyr1) apelin-13 (80 nM) was used to determine specific binding from 0 to 120 min. Values were fit globally using nonlinear regression for competitive binding kinetics, which applied the Kon and Koff constraints for [3H] apelin-13 determined in the association binding experiments. Competitive radioligand binding assays were used to assess the binding of BBMS-986224 versus respective radioligands in membranes derived from cells overexpressing individual human GPCRs.

ERK Phosphorylation[1]
HEK293 ZF cells expressing human APJ were grown and plated in 384-well poly-D-Lysine-coated plates at 30,000 cells/well in 50 μL complete growth medium for 3 days. The complete growth medium was replaced with 80 μL/well serum-free medium for further overnight incubation. Test compounds [BBMS-986224] were serially diluted in DMSO and dispensed into 384-well REMP plates at 10–50 nL/well. For the assay, the compounds were further diluted with 50 μL of HBSS/HEPES/0.1% BSA assay buffer. The medium was removed from the cell plate and 25 μL of diluted compound was added directly to the cells. After incubation for 7 min at 37°C, the cells were immediately lysed with the lysis buffer provided in the assay kits and agitated for 15 min. Cell lysate (4 μL/well) was then transferred to 384-well ProxiPlates and 7 μL/well of immunoglobulin G (IgG) detection reagent was added. After a 2-h incubation in the dark, the signal was measured on an EnVision plate reader. Compound potency was determined as described for the cAMP assay.

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BRET-based Biosensor Assays1]
Cells were washed with Tyrode’s buffer (137 mM NaCl, 1 mM CaCl2, 0.9 mM KCl, 1 mM MgCl2, 3.6 mM NaH2PO4, 5.5 mM glucose, 12 mM NaHCO3, and 25 mM HEPES, pH 7.4), and 90 μL of Tyrode’s buffer was added to each well. Cells were equilibrated in the new buffer at room temperature for ≥30 min; 10× coelenterazine substrate (10 μL) was thenadded to each well (prolume purple coelenterazine at a final concentration of 2 μM). The test compounds (BMS-986224 and (Pyr1) apelin-13) at different concentrations were then added to each well (HP D300 digital dispenser; Tecan) and the cells incubated at room temperature for 5–15 min. BRET readings were then collected using a Synergy Neo Multi-Mode reader from BioTek with BRET2BBMS-986224 filters 410/80 and 515/30. The BRET signal was determined by calculating the ratio of the light emitted by GFP (515/30 nm) over the light emitted by the Rluc (410/80 nm). BRET signal values were converted into percentage of activation using the non-stimulated control as 0% and (Pyr1) apelin-13 maximal response as 100%. Sigmoidal concentration–response curves were generated with those normalized values using a 4-parameter logistic equation to determine EC50 of the different compounds.

Animal/Disease Models: Male SD (Sprague-Dawley) rats (renal hypertensive rat model)[1]
Doses: 0.192 mg/kg or 3 mg/kg
Route of Administration: SC infusion; daily; Initiated 3 days before surgery and continued for 7 days after surgery
Experimental Results: The achieved steady-state plasma concentrations during 10-day infusion were 102 and 2686 nmol/L at low dose and HD, respectively. At the low dose, BMS-986224 increased SV and CO without affecting other measured parameters, including the measured diastolic parameters, cardiac fibrosis, and heart weight in RHR.

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(Pyr1) Apelin-13, BBMS-986224, and Dobutamine: Acute Hemodynamic Analyses in Normal Rats[1]
Following a 10-min equilibration period, vehicle (n=14) or BBMS-986224 (1, 10, or 100 μg/kg/min; n=8, 6, and 7, respectively) was infused intravenously over 15 min (Supplemental Figure I B(i)). We also included dobutamine, a positive inotropic agent, as a comparator. A dose‑escalation and a 15-min IV infusion of 1 μg/kg/min dose of dobutamine was conducted (see Methods and Supplemental Figure IC). Plasma exposures were measured in separate groups of anesthetized and cannulated animals (Supplemental Figure I A(ii) and I B(ii)). Following equilibration (Pyr1) apelin-13 (6 μg/kg/min), BBMS-986224 (1, 10, and 100 μg/kg/min), or vehicle were infused over 15 min (BMS-986224 and vehicle) or 20 min ((Pyr1) apelin-13). Blood samples were collected 5, 10, 20, 22, and 30 min after infusion start for (Pyr1) apelin-13 and 5, 15, 20, and 30 min after infusion start for BMS‑986224 and vehicle. In the (Pyr1) apelin-13 experiments, blood was collected using an optimized blood collection protocol and concentrations subsequently determined using liquid chromatography-tandem mass spectrometry as described previously.[1]

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BBMS-986224: Cardiovascular Effects in the Chronic Renal Hypertensive Rat Model[1]
Subcutaneous Drug Administration[1]
For the infusion study, animals were implanted subcutaneously with an osmotic minipump (ALZET® Osmotic Pump Model 2ML2; DURECT Corporation). For the SC infusion, the study arms were BMS-986224 (0.192 mg/kg/day or 3 mg/kg/day, n=15 per dose), vehicle (n=15), enalapril (40 mg/kg/day, n=15), and sham-operated controls (n=10). BBMS-986224, enalapril, or vehicle infusion was initiated 3 days before surgery and continued for 7 days after surgery (Supplemental Figure I D). Key measurements were systolic blood pressure (SBP) in conscious animals on Day 6 via tail cuff (CODA; Kent Scientific Products), and echocardiography (ECHO) of cardiac structure and function (stroke volume [SV], CO, heart rate [HR], LV mass, LV end-diastolic volume [LVEDV] and ejection fraction [EF], and isovolumic relaxation time [IVRT]) on Day 7. Plasma samples were collected at Day 7 for measuring BBMS-986224 and heart failure biomarkers. The experiment was repeated in a larger cohort (n=20 per group).

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Oral Drug Administration[1]
BBMS-986224 doses in the twice-daily (BID) study were selected to target a plasma concentration of 200 nM at peak (0.1 mg/kg/day BID) or at trough (1 mg/kg/day BID). In the low-dose once-daily (QD) study, 0.06 mg/kg QD dose was targeted to achieve a peak concentration of 100 nM and 0.2 mg/kg dose to cover an area under the plasma drug concentration–time curve (AUC) 0–24 of 3.4 μM*h. Vehicle was given PO and SC in separate animal groups, and enalapril maleate (40 mg/kg/day) was administered SC. Test compound preparation is described in full in Supplemental Table II. Sham-operated controls were also included. Treatment was initiated 3 days before surgery and continued for 8 days after surgery (Supplemental Figures I E, I F). ECHO was performed at BMS-986224 trough on Day 7 and peak on Day 8. Plasma samples were collected at trough and peak on Day 6 and repeated at peak on Day 8.

[1]. In Vitro and In Vivo Evaluation of a Small-Molecule APJ (Apelin Receptor) Agonist, BMS-986224, as a Potential Treatment for Heart Failure. Circ Heart Fail. 2021;14(3):e007351.

Background: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224.

Methods: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, β-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output.

Results: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril.

Conclusions: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.[1]

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Our studies have several limitations. Although BMS-986224 was designed to closely resemble the signaling profile and receptor interaction characteristics of (Pyr1) apelin-13, it is possible that unknown differences between these compounds could limit extrapolation of preclinical findings with BMS-986224 to those seen with apelin peptides in humans. It should also be noted that only male rats were used in our studies. While the sustained effect on CO seen in the RHR model is promising, the mechanism responsible for these changes is not clear, and more studies are needed to assess their relevance to human disease.

In conclusion, we identified a novel, potent, selective, and orally bioavailable small-molecule APJ receptor agonist that recapitulated APJ receptor signaling properties and in vivo cardiovascular effects of endogenous (Pyr1) apelin-13. We showed that increased cardiac function could be sustained in a disease model with prolonged oral administration, supporting further evaluation of BMS-986224 in the clinical setting. Whether the favorable effects of BMS-986224 observed in preclinical models can be translated to human HF remains to be determined.[1]

C24H23CLN4O6

498.915624856949

498.13

2055200-88-7

137106310

Off-white to yellow solid powder

1.8

2

9

9

35

812

0

C1(=O)NC(COCC)=C(C2=C(OC)C=CC=C2OC)C(O)=C1C1=NN=C(CC2=NC=C(Cl)C=C2)O1

AGZKELPIAJYRDT-UHFFFAOYSA-N

InChI=1S/C24H23ClN4O6/c1-4-34-12-15-19(20-16(32-2)6-5-7-17(20)33-3)22(30)21(23(31)27-15)24-29-28-18(35-24)10-14-9-8-13(25)11-26-14/h5-9,11H,4,10,12H2,1-3H3,(H2,27,30,31)

3-[5-[(5-chloropyridin-2-yl)methyl]-1,3,4-oxadiazol-2-yl]-5-(2,6-dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxy-1H-pyridin-2-one

BMS-986224; 2055200-88-7; N5O33BF03F; UNII-N5O33BF03F; CHEMBL4873876; BMS986224; 2(1H)-Pyridinone,3-(5-((5-chloro-2-pyridinyl)methyl)-1,3,4-oxadiazol-2-yl)- 5-(2,6-dimethoxyphenyl)-6-(ethoxymethyl)-4-hydroxy-; 3-(5-((5-Chloropyridin-2-yl)methyl)-1,3,4-oxadiazol-2-yl)-5-(2,6-dimethoxyphenyl)-6- (ethoxymethyl)pyridine-2,4-diol;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 20.83 mg/mL (41.75 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.17 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)