BMS-986224

Kd: 0.3 nM (APJ receptor)[1]

BMS-986224 对人 APJ 的 EC50 为 0.02 nM，并完全阻断毛喉素介导的 cAMP 合成。中国仓鼠卵巢-K1 或 HEK293 ZF 细胞在 β-arrestin 募集、ERK 磷酸化和 APJ 内化方面对 BMS-986224 (0-100 nM) 完全响应[1]。强效选择性 APJ 受体激动剂 BMS-986224 显示出类似于 (Pyr1) apelin-13[1] 的信号传导谱。

在 RHR 模型中，BMS-986224（0.192 mg/kg 或 3 mg/kg；SC 输注；每日）通过将每搏输出量和心输出量增加至健康动物中观察到的水平，产生与依那普利不同的效果，但无法预防心脏肥大和纤维化[1]。

GPCR选择性分析[1]
进行GPCR选择性测定以评估BBMS-986224与其他GPCR（腺苷A2A；肾上腺素能α1B、1D、2A和2C，肾上腺素能β1和β2；大麻素CB1；多巴胺D1和D2；组胺H1和H2；毒蕈碱M2；阿片样物质μ和κ；血清素5HT1B、5HT2B和5HT4）相互作用的可能性。如Alt等人所述，进行了这些测定。12使用膜过滤法进行的竞争性放射性配体结合测定用于评估BBMS-986224在来源于细胞过表达单个人类GPCR的膜中竞争放射性配体结合的能力。

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放射性配体结合分析[1]
将一系列浓度的BBMS-986224添加到含有[3H]apelin-13的细胞膜提取物中（约2倍的平衡解离常数[Kd]）。使用过量未标记的（Pyr1）apelin-13（80 nM）来确定0至120分钟的特异性结合。使用竞争性结合动力学的非线性回归对值进行全局拟合，该回归应用了结合实验中确定的[3H]apelin-13的Kon和Koff约束。使用竞争性放射性配体结合分析来评估BBMS-986224与过表达单个人GPCR的细胞膜中相应放射性配体的结合。

ERK磷酸化[1]
表达人APJ的HEK293 ZF细胞在50μL完全生长培养基中以30000个细胞/孔的速度在384孔聚-D-赖氨酸涂层板上生长和铺板3天。将完整的生长培养基替换为80μL/孔的无血清培养基，以进一步培养过夜。试验化合物[BBMS-986224]在DMSO中连续稀释，并以10-50nL/孔的浓度分配到384孔REMP板中。对于该测定，化合物用50μL HBSS/HEPES/0.1%BSA测定缓冲液进一步稀释。从细胞板中取出培养基，直接向细胞中加入25μL稀释的化合物。在37°C下孵育7分钟后，立即用检测试剂盒中提供的裂解缓冲液裂解细胞并搅拌15分钟。然后将细胞裂解液（4μL/孔）转移到384孔ProxiPlates中，并加入7μL/孔免疫球蛋白G（IgG）检测试剂。在黑暗中孵育2小时后，在EnVision平板阅读器上测量信号。按照cAMP测定所述测定化合物效力。

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基于BRET的生物传感器检测1]
用Tyrode缓冲液（137 mM NaCl、1 mM CaCl2、0.9 mM KCl、1 mM MgCl2、3.6 mM NaH2PO4、5.5 mM葡萄糖、12 mM NaHCO3和25 mM HEPES，pH 7.4）洗涤细胞，并向每个孔中加入90μL Tyrode的缓冲液。细胞在室温下在新缓冲液中平衡≥30分钟；然后将10×腔肠素底物（10μL）添加到每个孔中（最终浓度为2μM的紫色腔肠素）。然后将不同浓度的测试化合物（BBMS-986224和（Pyr1）apelin-13）添加到每个孔（HP D300数字分配器；Tecan）中，并在室温下孵育细胞5-15分钟。然后使用BioTek的Synergy Neo多模式阅读器和BRET2BBMS-986224过滤器410/80和515/30收集BRET读数。通过计算GFP（515/30nm）发出的光与Rluc（410/80nm）发射的光的比率来确定BRET信号。使用非刺激对照将BRET信号值转换为激活百分比为0%，（Pyr1）apelin-13最大反应为100%。使用4参数逻辑斯谛方程，利用这些归一化值生成了Sigmoid浓度-响应曲线，以确定不同化合物的EC50。

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

129

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.17 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.17 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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