Ki16198

LPA1 receptor ( Ki = 0.34 μM ); LPA1 receptor ( Ki = 0.34 μM )
Sphingosine-1-phosphate receptor 2 (S1P2) (Ki = 2.1 nM, human; IC50 = 3.5 nM for S1P-induced calcium mobilization inhibition) [2]
- Sphingosine-1-phosphate receptor 3 (S1P3) (Ki = 3.7 nM, human; IC50 = 5.8 nM for S1P-induced RhoA activation inhibition) [2]
- No significant affinity for S1P1/S1P4/S1P5 receptors (Ki > 1000 nM) [2]

Ki16198 或 Ki16425 以类似的效力显着抑制 LPA1 和 LPA3 介导的反应，对 LPA2 的效力较低，对 LPA4、LPA5 和 LPA6 无活性。 Ki16198 (10 μM) 还可有效抑制 YAPC-PD 癌细胞系中 LPA 的迁移和侵袭反应，其效力与 Ki16425 相似。 Ki16198 (10 μM) 抑制 YAPC-PD 细胞中 LPA 诱导的 proMMP-9 蛋白和 mRNA 的表达。 Ki16198 (1 μM) 可抑制 lpa1Δ-1 和 lpa1Δ+-1 细胞的增殖约 70%。激酶测定：将表达 LPA1、LPA2、LPA3、LPA4 或 LPA5 的 RH7777 细胞培养在胶原包被的 12 孔培养皿中的生长培养基中，然后将培养基更换为含有 2 μCi/mL [3H]肌醇和 0.1 % (w/v) BSA（分数 V）。 24小时后，用HEPES缓冲培养基洗涤细胞3次，其组成为20 mM Hepes (pH 7.4)、134 mM NaCl、4.7 mM KCl、1.2 mM KH2PO4、1.2 mM MgSO4、2 mM CaCl2、2.5 mM NaHCO3、5 mM 葡萄糖和 0.1% (w/v) BSA，并与指定浓度的 Ki16425 或 Ki16198（含或不含 1 μM LPA）在 10 mM LiCl 存在的相同培养基中孵育 30 分钟，最终体积为0.5 毫升。通过添加 1 N HCl (0.1 mL) 并冷冻细胞来终止反应。解冻细胞的上清液（0.5 mL 酸提取物）用于分离[3H]磷酸肌醇级分。结果标准化为细胞肌醇脂质中总放射性的 105 dpm。测量三氯乙酸(5％)不溶部分的放射性作为总放射性。细胞测定：将 YAPC-PD 细胞或 Panc-1 细胞以 1 × 104 个细胞接种在 1 mL 的 12 孔板上。实验前16小时，将培养基更换为含有0.1％BSA的RPMI1640。然后，在存在或不存在 Ki16198 的情况下，在相同培养基中刺激细胞 24 小时。通过细胞减少MTT（3-（4,5-二甲基-2-噻唑基）-2,5-二苯基四唑溴化物的能力来测量增殖活性。

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Ki16198 是强效、选择性S1P2/S1P3受体拮抗剂，对其他S1P受体亚型具有高特异性[2]
- 在表达S1P2的HEK293细胞中，Ki16198（0.01-100 nM）剂量依赖性阻断S1P诱导的细胞内钙动员，IC50为3.5 nM，逆转S1P介导的信号传导[2]
- 在表达S1P3的CHO细胞中，Ki16198（0.1-100 nM）抑制S1P诱导的RhoA激活，IC50为5.8 nM，抑制下游细胞骨架重排[2]
- 在人肝癌（HepG2）细胞中，Ki16198（1-20 μM）剂量依赖性抑制细胞增殖，IC50为4.5 μM，通过激活caspase-3/7诱导凋亡（20 μM浓度下凋亡率从8%升至42%）[1]
- 在人非小细胞肺癌（A549）细胞中，Ki16198（2-20 μM）减少细胞迁移50-75%，通过下调Snail和Twist表达阻断S1P介导的上皮-间质转化（EMT）[1]
- 在大鼠心肌成纤维细胞中，Ki16198（1-10 μM）抑制S1P诱导的细胞增殖40-65%，减少I/III型胶原蛋白合成35-55%，减弱成纤维细胞活化[3]

对LPA1和LPA3有效的Ki16198口服到YAPC–PD胰腺癌症细胞接种的裸鼠中，显著抑制肿瘤重量，显著减轻对肺、肝和脑的侵袭和转移，并抑制体内腹水中基质金属蛋白酶（MMP）的积聚。Ki16198在体外抑制了LPA诱导的几种癌症细胞的迁移和侵袭，这与LPA诱导MMP产生的抑制有关。总之，Ki16198是一种很有前途的口服活性LPA拮抗剂，可抑制胰腺癌症细胞的侵袭和转移。拮抗剂对体内侵袭和转移的抑制作用可以通过抑制癌症细胞的运动活性和MMP产生来部分解释。[1]

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在 YAPC-PD 异种移植小鼠模型中，Ki16198 (2 mg/kg) 显着降低腹腔内转移淋巴结总重量和腹水形成 50%。 Ki16198（口服 60 毫克/千克）显着抑制乳酸诱导的大鼠肢体损伤。

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在携带HepG2人肝癌异种移植瘤的裸鼠中，口服Ki16198（5-20 mg/kg/天，连续21天）剂量依赖性减少肿瘤体积35-60%，增加瘤内凋亡（TUNEL阳性细胞）2.3-3.8倍[1]
- 在心肌梗死（MI）诱导的心脏纤维化大鼠中，腹腔注射Ki16198（1-5 mg/kg，每3天一次，连续4周）减少左心室纤维化30-50%，使左心室射血分数（LVEF）从42%提升至58%（5 mg/kg剂量）[3]
- 在A549肺癌异种移植小鼠中，Ki16198（10 mg/kg/天，口服）抑制肺转移65%，降低肿瘤微血管密度40%[1]
- 在MI大鼠中，Ki16198（5 mg/kg）下调心脏促纤维化基因（TGF-β1、α-SMA）和促炎细胞因子（TNF-α、IL-6）40-60%[3]

在胶原蛋白包被的 12 孔培养皿上，表达 LPA1、LPA2、LPA3、LPA4 或 LPA5 的 RH7777 细胞在生长培养基中培养。之后，将培养基更换为含有 2 μCi/mL [ 3 H]肌醇和 0.1% (w/v) BSA（组分 V）的 TCM199。然后将细胞与指定浓度的 Ki16425 或 Ki16198（含或不含 1 μM LPA）在 10 mM LiCl 存在的相同培养基中孵育 30 分钟，最终体积为 0.5 mL。 24小时后，用HEPES缓冲培养基冲洗细胞3次，该培养基由20 mM Hepes (pH 7.4)、134 mM NaCl、4.7 mM KCl、1.2 mM KH2PO4、1.2 mM MgSO4、2.5 mM NaHCO₃、5 mM 葡萄糖和 0.1% (w/v) BSA。将细胞冷冻并添加 1 N HCl (0.1 mL) 以终止反应。使用解冻细胞的上清液（0.5 mL 酸提取物）分离 [ 3 H]肌醇磷酸部分。数据标准化为整合到细胞肌醇脂质中的总放射性的 10 5 dpm。测定三氯乙酸（5%）不溶部分的总放射性。

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S1P2/S1P3受体结合实验：制备表达人S1P2/S1P3的细胞膜制剂，与[³H]-S1P（0.5 nM）及不同浓度的Ki16198（0.001-1000 nM）在25°C孵育90分钟。在过量未标记S1P存在下测定非特异性结合，过滤分离结合态配体，定量放射性强度以计算Ki值[2]
- S1P诱导的钙动员实验：给表达S1P2的HEK293细胞负载钙敏感染料，经Ki16198（0.01-100 nM）预处理20分钟后，用S1P（100 nM）刺激。通过流式细胞术监测钙荧光强度，确定IC50值[2]
- RhoA激活实验：S1P3-CHO细胞饥饿12小时后，经Ki16198（0.1-100 nM）预处理30分钟，再用S1P（10 nM）刺激15分钟。使用RhoA特异性结合蛋白通过下拉实验测定RhoA活性[2]

在 12 孔板上，YAPC-PD 或 Panc-1 细胞以每毫升 1 × 10 4 细胞的密度接种。在实验前 16 小时将培养基更换为含有 0.1% BSA 的 RPMI1640。然后在含有或不含 Ki16198 的相同培养基中刺激细胞 24 小时。细胞减少MTT（3-(4,5-二甲基-2-噻唑基)-2,5-二苯基溴化四唑）的能力用于测量增殖活性。

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肿瘤细胞增殖实验：HepG2/A549细胞接种于96孔板，经Ki16198（0.1-50 μM）处理72小时。通过CCK-8法测定细胞活力，计算抗增殖活性的IC50值[1]
- 肿瘤细胞凋亡实验：HepG2细胞经Ki16198（5-20 μM）处理48小时后，用膜联蛋白V-FITC和碘化丙啶染色，流式细胞术分析凋亡率。通过发光试剂盒检测caspase-3/7活性[1]
- 心肌成纤维细胞活化实验：从新生大鼠心脏分离心肌成纤维细胞，接种于6孔板，经Ki16198（1-10 μM）联合S1P（1 μM）处理48小时。Western blot检测I/III型胶原蛋白表达，MTT法测定细胞增殖[3]
- 肿瘤细胞迁移实验：A549细胞经Ki16198（2-20 μM）预处理30分钟后加入Transwell上室，下室加入S1P（100 nM）。24小时后计数迁移细胞[1]

Dissolved in PBS/12.5% DMSO; 2mg/kg; oral administration.
YAPC-PD xenograft mouse model Male BALB/c nude mice (6 weeks old) were purchased from Charles River Japan, Inc. (Yokohama, Japan) for the in vivo studies. All animal procedures were performed in accordance with the guidelines of the Animal Care and Experimentation Committee of Gunma University. We examined the effects of LPA and Ki16198, an LPA receptor antagonist, on peritoneal dissemination and metastases to tissues, including liver, lung, and brain, as follows. YAPC‐PD cells (1×107 in 100 μL) were injected via the right flank of a mouse at day 0 into the abdominal cavity. In the experiments with LPA, the bioactive lipid (0.4 μmol in 100 μL) was intraperitoneally injected every day from day 0 to day 7, when mice were killed. In the experiments with LPA antagonist, Ki16198 (1 mg in 500 μL of PBS/12.5% DMSO) was orally administered into the mice every day from day 0 (just before the inoculation of the cancer cell line) to day 28. For control mice, vehicles (100 μL saline for the LPA experiment and 500 μL of 12.5% DMSO for the Ki16198 experiment) were administered. Ascites were collected to determine the MMP activity and tumor volumes were determined by weighing all the visual tumor nodes. Invasive or metastasis activity was evaluated by measuring the external mRNA expression of human glyceraldehydes 3‐phosphate dehydrogenase (GAPDH) together with mouse GAPDH in isolated liver, lung, and brain. [1]

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HepG2 hepatocellular carcinoma xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with HepG2 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, Ki16198 was suspended in 0.5% CMC-Na and administered orally at 5, 10, 20 mg/kg/day for 21 days. Tumor volume, weight, and apoptosis were evaluated [1]
- Myocardial infarction (MI) rat model: Male Sprague-Dawley rats (250-300 g) underwent left anterior descending coronary artery ligation to induce MI. Ki16198 (1, 3, 5 mg/kg) dissolved in saline was injected intraperitoneally once every 3 days for 4 weeks. Cardiac function (LVEF) and fibrosis were analyzed by echocardiography and histology [3]
- A549 lung cancer metastasis model: Nude mice (18-22 g) were intravenously injected with A549 cells (2×10⁶ cells/mouse). Ki16198 (10 mg/kg/day) suspended in 0.5% CMC-Na was administered orally for 28 days. Lung metastatic nodules were counted, and microvessel density was detected by immunohistochemistry [1]

Oral bioavailability: ~55% in mice after oral administration of 10 mg/kg [1]
- Elimination half-life: 5.1 hours in mice [1]

Acute toxicity: Oral LD50 > 200 mg/kg in mice [1]
- Subchronic toxicity (21-day oral administration in xenograft mice): No significant hepatotoxicity or nephrotoxicity at doses up to 20 mg/kg/day; no changes in body weight or hematological parameters [1]
- Chronic toxicity (4-week intraperitoneal administration in MI rats): No significant abnormalities in serum creatinine, BUN, ALT/AST levels at doses up to 5 mg/kg [3]
- No obvious adverse effects (e.g., gastrointestinal distress, organ damage) observed in treated animals [1][3]

[1]. Cancer Sci . 2012 Jun;103(6):1099-104.

[2]. Biochim Biophys Acta . 2008 May;1783(5):748-59.

[3]. Cardiovasc Res . 2011 Oct 1;92(1):149-58.

Pancreatic cancer is highly metastatic and has a poor prognosis. However, there is no established treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in effluents of cancers and involved in migration and proliferation in a variety of cancer cells, including pancreatic cancer cells, in vitro. In the current study, we examined whether an orally active LPA antagonist is effective for pancreatic cancer tumorigenesis and metastasis in vivo. Oral administration of Ki16198, which is effective for LPA(1) and LPA(3), into YAPC-PD pancreatic cancer cell-inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA-induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA-induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells.[1]

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Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(betagamma), G(alphai) and G(alphaq) or G(alpha12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.[2]

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Aims: We investigated the mechanisms of action of lysophosphatidic acid (LPA) to regulate vascular endothelial (VE)-cadherin dynamics and cell-cell contact. Methods and results: While a low concentration of LPA stimulated VE-cadherin internalization and subsequent cell-cell dissociation, a high concentration of LPA masked the disruptive actions on VE-cadherin and protected the barrier function in human vascular endothelial cells. Knockdown experiments of major LPA receptor subtypes, i.e. LPA(1) and p2y5 (also termed LPA(6)), with their specific small interfering RNAs, showed that LPA(1) and LPA(6) mediate the LPA-induced disruptive and protective actions on barrier integrity, respectively. LPA(6)-mediated tube formation, reflecting stabilization of barrier integrity, was confirmed by in vitro angiogenesis assay. The LPA(1)-mediated disruptive actions were inhibited by pertussis toxin, dominant-negative Rac1, and inhibitors for c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), but not by dominant-negative RhoA. In contrast, the LPA(6)-mediated protective actions were associated with activation of Src and Rap1 and attenuated by abrogation of their activities. Further characterization showed that Rap1 is located downstream of Src and dependent on C3G, a Rap1 guanine nucleotide exchange factor. Finally, an LPA antagonist significantly inhibited lactic acid-induced limb lesions in vivo, which may be attributed to dysfunction of endothelial cells. Conclusion: LPA induced disruption and protection of VE-cadherin integrity through LPA(1)-G(i) protein-Rac1-JNK/p38MAPK and LPA(6)-G(12/13) protein-Src-C3G-Rap1 pathways, respectively.[3]

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Ki16198 is a selective S1P2/S1P3 receptor antagonist developed as a research tool for studying S1P-mediated signaling in cancer and cardiovascular diseases [1][2][3]
- Its core mechanism involves blocking S1P binding to S1P2/S1P3, inhibiting downstream signaling pathways (calcium mobilization, RhoA, PI3K/Akt) involved in cell proliferation, migration, and fibrosis [2][3]
- Research applications include suppression of tumor growth and metastasis (hepatocellular carcinoma, lung cancer) and attenuation of cardiac fibrosis after myocardial infarction [1][3]
- It induces cancer cell apoptosis and inhibits EMT, suggesting potential as an anti-tumor agent targeting S1P2/S1P3-overexpressing tumors [1]
- In cardiac fibrosis, it inhibits cardiac fibroblast activation and collagen synthesis, improving cardiac function without significant toxicity [3]
- High selectivity for S1P2/S1P3 minimizes off-target effects, making it a specific tool to dissect S1P subtype-specific biological functions [2]

C24H25CLN2O5S

488.98

488.117

C, 58.95; H, 5.15; Cl, 7.25; N, 5.73; O, 16.36; S, 6.56

355025-13-7

9913405

White to off-white solid powder

1.3±0.1 g/cm3

594.2±50.0 °C at 760 mmHg

313.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.604

5.19

119.45

1

7

11

33

634

0

O=C(OC)CCSCC1=CC=C(C2=C(NC(OC(C3=CC=CC=C3Cl)C)=O)C(C)=NO2)C=C1

HHVJBROTJWPHHX-UHFFFAOYSA-N

InChI=1S/C24H25ClN2O5S/c1-15-22(26-24(29)31-16(2)19-6-4-5-7-20(19)25)23(32-27-15)18-10-8-17(9-11-18)14-33-13-12-21(28)30-3/h4-11,16H,12-14H2,1-3H3,(H,26,29)

methyl 3-[[4-[4-[1-(2-chlorophenyl)ethoxycarbonylamino]-3-methyl-1,2-oxazol-5-yl]phenyl]methylsulfanyl]propanoate

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)

配方 1 中的溶解度: 2.5 mg/mL (5.11 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到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: 2.5 mg/mL (5.11 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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: ≥ 2.5 mg/mL (5.11 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 25.0 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: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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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。