human LPA1 ( pIC50 = 0.98 μM ); mouse LPA1 ( pIC50 = 0.73 μM )

AM095 是一种有效的 LPA1 受体拮抗剂，因为它抑制 GTPγS 与过表达重组人或小鼠 LPA1 的中国仓鼠卵巢 (CHO) 细胞膜结合，IC50 值分别为 0.98 和 0.73 μM。 AM095 抑制过表达小鼠 LPA1 的 CHO 细胞 (IC50=778 nM) 和人 A2058 黑色素瘤细胞 (IC50=233 nM) 的 LPA 驱动的趋化性。 AM095 在人 LPA1 GTPγS 结合测定中的 IC50 与我们之前发布的化合物 AM966 (IC50=0.98±0.17 μM) 和 Debio-0719 化合物 (IC50=0.60±0.04 μM) 相当[1]。 AM095 抑制稳定转染人或小鼠 LPA1 的 CHO 细胞中 LPA 诱导的钙流。 AM095 拮抗人或小鼠 LPA1 转染的 CHO 细胞中 LPA 诱导的钙流的 IC50 分别为 0.025 和 0.023 μM[2]。

AM095 具有较高的口服生物利用度和中等的半衰期，并且在口服和静脉给药后在大鼠和犬中测试的剂量下具有良好的耐受性。大鼠口服（10 mg/kg）给药后，AM095 血浆浓度在 2 小时达到峰值，Cmax 为 41 μM，此后到 24 小时降至 10 nM。静脉注射 (2 mg/kg) 给药后，15 分钟内观察到 Cmax 为 12 μM，到 24 小时也降至约 10 nM，t1/2 为 1.79 小时。在狗中，单次口服剂量 5 mg/kg 在给药 15 分钟内产生 21 μM 的峰值血浆浓度，然后在 24 小时内降至 10 nM。相比之下，2 mg/kg 的静脉注射剂量在 15 分钟内导致 Cmax 为 11 μM，并在 8 小时内降至 15 nM，产生 1.5 小时的 t1/2[1]。

在测定中，使用 hLPA1/CHO 和 mLPA1/CHO 细胞。将蛋白酶抑制剂、10 mM HEPES、pH 7.4、1 mM 二硫苏糖醇和约 20 mL 冰冷的膜缓冲液添加到 hLPA1/CHO 或 mLPA1/CHO 细胞的细胞沉淀中。对细胞进行超声处理，并将细胞裂解物在 4°C 下以 2000 rpm 离心 10 分钟。 4°C、25,000 rpm 进一步离心上清液 70 分钟。使用 Potter-Elvehjem 组织研磨机，将膜沉淀重悬于 5 mL 冰冷的膜缓冲液中并均质化。使用 Bradford 蛋白质检测试剂盒可计算最终蛋白质浓度。加入 25 至 40 μg hLPA1/CHO 或 mLPA1/CHO 膜和 0.1 nM [35S]-GTPηS 缓冲液（50 mM HEPES、0.1 mM NaCl、10 mM MgCl22 的冷缓冲液洗涤 3 次。 30°C 下孵育 30 分钟。板干燥后，使用 Packard TopCount NXT 微孔板闪烁计数器测量 cpm。

在体外，AM095是一种强效的LPA受体拮抗剂，因为它抑制了GTPγS与过表达重组人或小鼠LPA的中国仓鼠卵巢（CHO）细胞膜的结合，IC值分别为0.98和0.73μM，并且没有表现出LPA激动作用。在功能测定中，AM095抑制了过表达小鼠LPA 8321（IC 832=778 nM）和人A2058黑色素瘤细胞（IC 833=233 nM）的CHO细胞的LPA驱动的趋化性[3]。

Mice had their left kidney operated on either by UUO or sham surgery. To put it briefly, the left kidney is exposed by a longitudinal, upper left incision. A 6/0 silk thread is inserted between the renal artery and the ureter after the artery has been identified. To ensure complete ureter ligation, the thread is wound around the ureter and knotted three times. The skin is sutured shut, the kidney is returned to the abdomen, and staples are used to close the incision. The healthy control kidney was the contralateral (right) kidney. Oral gavage of AM095 (30 mg/kg) or the vehicle (water) is administered 1 to 4 hours prior to UUO and on an as-needed basis after that. The kidneys are removed and cut in half for histopathological and biochemical examination of the fibrosis after the mice are put to sleep for eight days using CO2 inhalation. A kidney sample is fixed in 10% neutral buffered formalin and stained with Masson's trichrome in order to measure the amount of fibrosis. To analyze the collagen content biochemically, the other half of the kidney is frozen at -80°C.

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Wild type (WT), and LPA₁-knockout (KO) and LPA₂-KO mice were injected subcutaneously with bleomycin or phosphate buffered saline (PBS) once daily for 28 days. Dermal thickness, collagen content, and numbers of cells positive for α-smooth muscle actin (α-SMA) or phospho-Smad2 were determined in bleomycin-injected and PBS-injected skin. In separate experiments, a novel selective LPA₁ antagonist AM095 or vehicle alone was administered by oral gavage to C57BL/6 mice that were challenged with 28 daily injections of bleomycin or PBS. AM095 or vehicle treatments were initiated concurrently with, or 7 or 14 days after, the initiation of bleomycin and PBS injections and continued to the end of the experiments. Dermal thickness and collagen content were determined in injected skin.[1]

In vivo, we demonstrated that AM095: 1) had high oral bioavailability and a moderate half-life and was well tolerated at the doses tested in rats and dogs after oral and intravenous dosing, 2) dose-dependently reduced LPA-stimulated histamine release, 3) attenuated bleomycin-induced increases in collagen, protein, and inflammatory cell infiltration in bronchalveolar lavage fluid, and 4) decreased kidney fibrosis in a mouse unilateral ureteral obstruction model. Despite its antifibrotic activity, AM095 had no effect on normal wound healing after incisional and excisional wounding in rats. These data demonstrate that AM095 is an LPA₁ receptor antagonist with good oral exposure and antifibrotic activity in rodent models. [3]

[1]. Amelioration of dermal fibrosis by genetic deletion or pharmacologic antagonism of lysophosphatidic acid receptor 1 in a mouse model of scleroderma. Arthritis Rheum. 2011 May;63(5):1405-15.

[2]. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. FASEB J. 2014 Feb;28(2):880-90.

[3]. Pharmacokinetic and pharmacodynamic characterization of an oral lysophosphatidic acid type 1 receptor-selective antagonist. Journal of Pharmacology and Experimental Therapeutics (2011), 336(3), 693-700.

Objective: Scleroderma (systemic sclerosis [SSc]), is characterized by progressive multiorgan fibrosis. We recently implicated lysophosphatidic acid (LPA) in the pathogenesis of pulmonary fibrosis. The purpose of the present study was to investigate the roles of LPA and two of its receptors, LPA₁ and LPA₂, in dermal fibrosis in a mouse model of SSc. Methods: Wild type (WT), and LPA₁-knockout (KO) and LPA₂-KO mice were injected subcutaneously with bleomycin or phosphate buffered saline (PBS) once daily for 28 days. Dermal thickness, collagen content, and numbers of cells positive for α-smooth muscle actin (α-SMA) or phospho-Smad2 were determined in bleomycin-injected and PBS-injected skin. In separate experiments, a novel selective LPA₁ antagonist AM095 or vehicle alone was administered by oral gavage to C57BL/6 mice that were challenged with 28 daily injections of bleomycin or PBS. AM095 or vehicle treatments were initiated concurrently with, or 7 or 14 days after, the initiation of bleomycin and PBS injections and continued to the end of the experiments. Dermal thickness and collagen content were determined in injected skin. Results: The LPA₁ -KO mice were markedly resistant to bleomycin-induced increases in dermal thickness and collagen content, whereas the LPA₂-KO mice were as susceptible as the WT mice. Bleomycin-induced increases in dermal α-SMA+ and phospho-Smad2+ cells were abrogated in LPA₁-KO mice. Pharmacologic antagonism of LPA₁ with AM095 significantly attenuated bleomycin-induced dermal fibrosis when administered according to either a preventive regimen or two therapeutic regimens. Conclusion: These results suggest that LPA/LPA₁ pathway inhibition has the potential to be an effective new therapeutic strategy for SSc, and that LPA₁ is an attractive pharmacologic target in dermal fibrosis.[1]

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Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1-3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase-protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.[2]

C27H23N2NAO5

478.471698045731

478.15

C, 67.78; H, 4.85; N, 5.85; Na, 4.80; O, 16.72

1345614-59-6

AM095 free acid; 1228690-36-5; 1345614-59-6 (sodium)

53303875

Light yellow to khaki solid powder

4.932

107.98

1

6

8

35

673

1

O=C([O-])CC1=CC=C(C2=CC=C(C3=C(NC(O[C@@H](C4=CC=CC=C4)C)=O)C(C)=NO3)C=C2)C=C1.[Na+]

BDKDADFSIDCQGB-GMUIIQOCSA-M

InChI=1S/C27H24N2O5.Na/c1-17-25(28-27(32)33-18(2)20-6-4-3-5-7-20)26(34-29-17)23-14-12-22(13-15-23)21-10-8-19(9-11-21)16-24(30)31;/h3-15,18H,16H2,1-2H3,(H,28,32)(H,30,31);/q;+1/p-1/t18-;/m1./s1

sodium;2-[4-[4-[3-methyl-4-[[(1R)-1-phenylethoxy]carbonylamino]-1,2-oxazol-5-yl]phenyl]phenyl]acetate

AM095 sodium; AM095; AM-095; 1345614-59-6; AM095; AM-095 Sodium; AM095 sodium; AM-095; sodium;2-[4-[4-[3-methyl-4-[[(1R)-1-phenylethoxy]carbonylamino]-1,2-oxazol-5-yl]phenyl]phenyl]acetate; AM 095

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

DMSO : ~83.33 mg/mL (~174.16 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.35 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 (4.35 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.35 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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配方 4 中的溶解度: 5 mg/mL (10.45 mM) in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。