BSEP ( IC50 = 4.8 μM ); MRP4 ( IC50 = 6.2 μM ); MDR3 ( IC50 = 7.5 μM ); LPA1

在 LPA1+ 细胞和肺切片中观察到 [18F]BMT-083133 结合的 BMS-986020（0.1-10 nM；预孵育）浓度依赖性位移。在 0.1 nM 浓度下，健康小鼠、博莱霉素小鼠和 IPF 肺的位移百分比分别为 18%、24% 和 31%。在 10 nM 时，位移百分比分别为 73%、76% 和 64%。 [18F]BMT-083133 是一种靶向 LPA1 的放射性配体，被开发为一种转化研究工具，用于使用体外放射自显影 (ARG) 评估 BMS-986020 的肺 LPA1 参与情况[4]。

中风是导致死亡的主要原因。中风幸存者往往患有长期功能性残疾。本研究通过使用短暂性大脑中动脉闭塞（tMCAO）的小鼠模型，证明了选择性溶血磷脂酸受体1（LPA1）拮抗剂BMS-986020（BMS）在肺纤维化和银屑病临床试验中对缺血性卒中后急性和亚急性损伤的神经保护作用BMS-986020再灌注后立即给药可显著减轻急性脑损伤，包括脑梗塞、神经功能缺损和tMCAO后第1天的细胞凋亡。即使在再灌注后3小时给药，BMS-986020的神经保护作用也得以保留。BMS-986020对急性损伤的神经保护作用与缺血后脑内小胶质细胞活化和脂质过氧化的减弱有关。值得注意的是，在tMCAO攻击的小鼠中，tMCAO后14天每天重复服用BMS-986020对其产生了长期的神经保护作用，这可以通过显著减轻神经功能缺损和提高存活率来证明。它还减轻了缺血后脑组织的损失和细胞凋亡。从机制上讲，它显著增强了受损大脑的神经发生和血管生成。除了存活率外，单次服用BMS-986020也能提供类似的长期神经保护。总的来说，BMS-986020对缺血性卒中的急性和亚急性损伤都提供了神经保护，表明BMS-986020可能是治疗缺血性卒中的一种有吸引力的治疗剂。[5]

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BMS-986020改变了体内胆汁稳态，导致大鼠和人类全身胆汁酸升高。相比之下，在预期的临床相关浓度下，结构上不同的LPA1拮抗剂BMS-986020在体外没有抑制BSEP（IC50=19.6µM）、MRP4（>50µM）或MDR3（>50μM），也没有抑制人肝细胞中的胆汁酸流出（≤50µM。此外，BMS-986020不会增加大鼠或猴子的胆汁酸。总之，用BMS-986020观察到的肝胆作用可能是该分子特有的脱靶作用，而不是通过LPA1的拮抗作用介导的。这些结果表明，LPA1拮抗剂的结构变化可能导致IPF和其他纤维化疾病患者的安全性不同。[2]

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在143名随机分配的患者中，108名完成了26周的给药阶段。35名患者提前停药。治疗组之间的患者基线特征相似（安慰剂：n=47；600mg qd：n=48；600mg bid：n=48）。与安慰剂相比，接受BMS-986020bid治疗的患者FVC下降速度明显较慢（分别为-0.042 L；95%CI，-0.106至-0.022 vs-0.134 L；95%CI，-0.201至-0.068；P=0.049）。在两个BMS-986020治疗组中都观察到肝酶的剂量相关升高。由于三例胆囊炎病例在揭盲后被确定与BMS-986020有关，该研究提前终止。 结论：与安慰剂相比，BMS-986020600mg bid治疗26周显著减缓了FVC的下降速度。BMS-986020的两种方案都与肝酶升高有关[2]。

TUNEL检测[5]
为了确定BMS-986020对细胞凋亡的影响，根据制造商的方案，在tMCAO后1天和15天使用原位细胞死亡检测试剂盒进行TUNEL免疫测定。将低温恒温器脑切片在4%PFA中后固定10分钟，并在冰上用0.1%Triton X-100中的0.1%柠檬酸钠渗透2分钟。然后用TUNEL检测试剂盒标记脑切片1小时，用PBS洗涤，并用VECTASHIELD安装介质安装。使用荧光显微镜用DP72相机拍摄图像。

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针对Iba1或4-HNE的免疫组织化学[5]
为了确定BMS-986020给药对小胶质细胞活化和脂质过氧化的影响，如前所述进行了免疫组织化学分析。简而言之，低温恒温器脑切片用1%过氧化氢氧化15分钟，并用0.3%Triton X-100中的1%胎牛血清（FBS）封闭。然后在4°C下用抗Iba1（1:500）或4-羟基壬烯醛（4-HNE，1:500）的兔一抗标记切片过夜，进一步用适当的生物素化二抗（1:200）标记，然后用ABC试剂（1:100，Vector Laboratories）孵育。将脑切片暴露于3,3'-二氨基联苯胺底物中，以显示Iba1-或4-HNE阳性信号，在酒精中逐渐脱水，在二甲苯中清除，并用Entellan培养基固定。

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5-溴-2′-脱氧尿苷（BrdU）掺入后的双重免疫荧光[5]
为了确定BMS-986020给药对神经发生和血管生成的影响，如前所述进行了BrdU/DCX-和BrdU/CD31双重免疫荧光检测。简而言之，在tMCAO攻击后13天和14天，以12小时的间隔对小鼠施用BrdU（PBS中50mg/kg，i.p.）四次。对于双重免疫荧光，脑切片用2N HCl孵育以使DNA变性，然后用0.1 M硼酸盐缓冲液中和。然后用0.3%Triton X-100中的1%FBS封闭切片，同时在4°C下孵育一整夜，用大鼠抗BrdU（1:400）和山羊抗DCX（1:100）一抗或小鼠抗BrdO（1:200A）和大鼠抗CD31（1:300）一抗体标记新形成的神经元或新形成的血管。然后将切片与与Cy3或AF488偶联的相应二抗（1:1000）一起孵育，并用VECTASHIELD安装培养基安装。使用共聚焦显微镜获得图像。

使用补充有 0.4% 胎牛血清、37.5 mg/mL Ficoll 70、25 mg/mL Ficoll 400 和 1% 抗坏血酸的 Dulbecco's Modified Eagle Medium (DMEM) + GlutaMax 在 48 孔板中培养人肺成纤维细胞。使用稀释后的 1 ng/mL 转化生长因子 β 1 (TGF-β1) 或 20 µM LPA（含或不含 BMS-986020（0.01、0.05、0.1、0.5、1 或 5 µM））刺激细胞，一式四份。二甲基亚砜 (DMSO) 或载体 (0.05% DMSO) 中。细胞在 37°C、95% O2 和 5% CO2 的环境中生长十二天。第四天和第八天，更换培养基。在生物标志物测量之前，上清液保存在-20°C下。在第 0 天（开始药物治疗之前）和第 12 天，使用 alamarBlue 测量细胞代谢。在第 4、8 和 12 天测量乳酸脱氢酶 (LDH) 释放。

After MCA occlusion, mice were randomly assigned into a BMS-986020 or a vehicle (1% DMSO in 10% Tween-80)-administered group. To determine whether BMS-986020 could exert neuroprotective effects against acute brain injuries in tMCAO-challenged mice, BMS-986020 was administered via oral gavage at different dosages (0.5, 2, 5, and 10 mg/kg) immediately after reperfusion. For the time window experiment, BMS-986020 was orally administered at 3 h after reperfusion. To determine long-term neuroprotective effects of BMS-986020 against sub-acute brain injuries, BMS-986020 was orally administered once immediately after reperfusion for the single administration group or daily for the repeated administration group (administration for fourteen consecutive days).[5]

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IM136003 was a phase 2, parallel-arm, multicenter, randomized, double-blind, placebo-controlled trial. Adults with IPF (FVC, 45%-90%; diffusing capacity for carbon monoxide, 30%-80%) were randomized to receive placebo or 600 mg BMS-986020 (once daily [qd] or bid) for 26 weeks. The primary end point was rate of change in FVC from baseline to week 26.[3]

[1]. Lysophospholipid receptors in drug discovery. Exp Cell Res. 2015 May 1;333(2):171-7.

[2]. LPA1 antagonists BMS-986020 and BMS-986234 for idiopathic pulmonary fibrosis: Preclinical evaluation of hepatobiliary homeostasis. European Respiratory Journal.

[3]. Randomized, Double-Blind, Placebo-Controlled, Phase 2 Trial of BMS-986020, a Lysophosphatidic Acid Receptor Antagonist for the Treatment of Idiopathic Pulmonary Fibrosis. Chest. 2018 Nov;154(5):1061-1069.

[4]. Autoradiographic evaluation of [18F]BMT-083133, a lysophosphatidic acid receptor 1 (LPA1) radioligand. The jornal of nuclear medicine.

[5]. BMS-986020, a Specific LPA1 Antagonist, Provides Neuroprotection against Ischemic Stroke in Mice. Antioxidants logo Antioxidants (Basel). 2020 Nov 8;9(11):1097.

BMS-986020 is under investigation in clinical trial NCT02017730 (To Evaluate The Relationship Between Plasma Drug Levels And Receptor Binding in Lung Using PET (Positron Emission Tomography) In Healthy Volunteers).

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BMS-986020 is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 2 investigational indications.

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Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing lung disease with limited effective treatment options. The LPA1 pathway has been implicated in the etiology and pathogenesis of IPF and is a promising therapeutic target for fibrotic diseases. LPA1 antagonists, including BMS‑986020 and BMS-986234, are being investigated for IPF. Differences in structure and pharmacology of LPA1 antagonists could impact their efficacy and safety profile. In a Phase 2 trial, BMS-986020 compared with placebo significantly slowed lung function decline but, in some patients, showed hepatobiliary effects; the mechanisms underlying these effects were investigated in vitro and in vivo. In vitro, BMS-986020 inhibits bile acid and phospholipid transporters, BSEP (IC50=4.8 µM), MRP4 (6.2 µM), and MDR3 (7.5 µM), which may reduce bile acid and phospholipid efflux, and alter bile composition and flow. [2]

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Lysophospholipids (LPs), including lysophosphatidic acid (LPA), sphingosine 1-phospate (S1P), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LysoPS), are bioactive lipids that transduce signals through their specific cell-surface G protein-coupled receptors, LPA1-6, S1P1-5, LPI1, and LysoPS1-3, respectively. These LPs and their receptors have been implicated in both physiological and pathophysiological processes such as autoimmune diseases, neurodegenerative diseases, fibrosis, pain, cancer, inflammation, metabolic syndrome, bone formation, fertility, organismal development, and other effects on most organ systems. Advances in the LP receptor field have enabled the development of novel small molecules targeting LP receptors for several diseases. Most notably, fingolimod (FTY720, Gilenya, Novartis), an S1P receptor modulator, became the first FDA-approved medicine as an orally bioavailable drug for treating relapsing forms of multiple sclerosis. This success is currently being followed by multiple, mechanistically related compounds targeting S1P receptor subtypes, which are in various stages of clinical development. In addition, an LPA1 antagonist, BMS-986020 (Bristol-Myers Squibb), is in Phase 2 clinical development for treating idiopathic pulmonary fibrosis, as a distinct compound, SAR100842 (Sanofi) for the treatment of systemic sclerosis and related fibrotic diseases. This review summarizes the current state of drug discovery in the LP receptor field.[1[

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diopathic pulmonary fibrosis (IPF) causes irreversible loss of lung function. The lysophosphatidic acid receptor 1 (LPA1) pathway is implicated in IPF etiology. Safety and efficacy of BMS-986020, a high-affinity LPA1 antagonist, was assessed vs placebo in a phase 2 study in patients with IPF.[3]

C29H26N2O5

482.5271

482.184

C, 72.19; H, 5.43; N, 5.81; O, 16.58

1257213-50-5

BMS-986020 sodium; 1380650-53-2

49792850

White to yellow solid powder

1.3±0.1 g/cm3

664.8±55.0 °C at 760 mmHg

355.9±31.5 °C

0.0±2.1 mmHg at 25°C

1.647

4.99

102

2

6

8

36

764

1

O([H])C(C1(C2C([H])=C([H])C(C3C([H])=C([H])C(C4=C(C(C([H])([H])[H])=NO4)N([H])C(=O)O[C@]([H])(C([H])([H])[H])C4C([H])=C([H])C([H])=C([H])C=4[H])=C([H])C=3[H])=C([H])C=2[H])C([H])([H])C1([H])[H])=O

GQBRZBHEPUQRPL-LJQANCHMSA-N

InChI=1S/C29H26N2O5/c1-18-25(30-28(34)35-19(2)20-6-4-3-5-7-20)26(36-31-18)23-10-8-21(9-11-23)22-12-14-24(15-13-22)29(16-17-29)27(32)33/h3-15,19H,16-17H2,1-2H3,(H,30,34)(H,32,33)/t19-/m1/s1

1-[4-[4-[3-methyl-4-[[(1R)-1-phenylethoxy]carbonylamino]-1,2-oxazol-5-yl]phenyl]phenyl]cyclopropane-1-carboxylic acid

AM152; AM 152; AM-152; AP-3152 free acid; BMS-986020; 1257213-50-5; AP-3152 free acid; 38CTP01B4L; (R)-1-(4'-(3-Methyl-4-(((1-phenylethoxy)carbonyl)amino)isoxazol-5-yl)-[1,1'-biphenyl]-4-yl)cyclopropane-1-carboxylic acid; Cyclopropanecarboxylic acid, 1-[4'-[3-methyl-4-[[[(1R)-1-phenylethoxy]carbonyl]amino]-5-isoxazolyl][1,1'-biphenyl]-4-yl]-; UNII-38CTP01B4L; 1-[4-[4-[3-methyl-4-[[(1R)-1-phenylethoxy]carbonylamino]-1,2-oxazol-5-yl]phenyl]phenyl]cyclopropane-1-carboxylic acid; BMS-986020; BMS986020; BMS 986020

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: 97~125 mg/mL (201.0~259.1 mM)
Ethanol: ~97 mg/mL

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.31 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 (4.31 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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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。