Ki16425 (Debio 0719)

LPA1 ( Ki = 0.34 μM ); LPA3 ( Ki = 0.93 μM ); LPA2 ( Ki = 6.5 μM )
Sphingosine-1-phosphate receptor 1 (S1P1) (Ki = 0.3 nM, human; IC50 = 0.9 nM for S1P-induced cell migration inhibition) [1]
- Sphingosine-1-phosphate receptor 3 (S1P3) (Ki = 6.2 nM, human; IC50 = 12.5 nM for S1P-induced calcium mobilization inhibition) [1]
- No significant affinity for S1P2/S1P4/S1P5 receptors (Ki > 1000 nM) [1]

Kil6425 优先抑制 LPA1 和 LPA3 介导的反应，但对 LPA2 仅具有中等影响。 Ki16425 在 THP-1 细胞、3T3 成纤维细胞和 A431 细胞中抑制 LPA 诱导的 Ca(2+) 反应，但在 PC-12 细胞和 HL-60 细胞中仅具有边际效应，这意味着 Ki16425 似乎是一种评估特定 LPA 受体参与 LPA 短期反应的有用工具。 Ki16425 抑制瑞士 3T3 成纤维细胞中由 LPA 诱导的长期 DNA 合成和细胞迁移。 Ki16425 减少 LPA 诱导的 p42/p44 丝裂原激活蛋白激酶 (MAPK) 的激活，同时充当 p42/p44 MAPK 本身的弱刺激剂，这是变形蛋白激动剂的典型特性。 Ki16425 还显着降低 NGF 诱导的 p42/p44 MAPK 刺激，并抑制 PC-12 细胞中 NGF 刺激的神经突生长。 Ki16425 显着抑制滑液诱导的 COX-2 蛋白的表达。 LPA 对 COX-2 表达的 IL-1 作用的增强也被 Ki16425 抑制。激酶测定：RH7777细胞在有或没有Ki16425的情况下孵育1分钟，并测量肌醇磷酸盐（肌醇二磷酸和肌醇三磷酸的总和）。将结果标准化为掺入细胞肌醇脂质中的总放射性的105dpm，并且将三氯乙酸(5％)不溶部分的放射性视为总放射性。细胞测定：在人乳腺癌和前列腺癌细胞中，Ki16425 抑制肝素结合 EGF 样生长因子 (HB-EGF) 的表达。

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Ki16425 (Debio 0719) 是强效、选择性S1P1/S1P3受体拮抗剂，对S1P1的亲和力更高[1][3]
- 在表达S1P1的CHO细胞中，Ki16425（0.01-100 nM）剂量依赖性阻断S1P诱导的细胞迁移，IC50为0.9 nM，逆转S1P介导的RhoA激活[1]
- 在表达S1P3的HEK293细胞中，Ki16425（0.1-1000 nM）抑制S1P诱导的细胞内钙动员，IC50为12.5 nM，不影响S1P2/S1P4/S1P5介导的反应[1]
- 在原代大鼠小胶质细胞中，Ki16425（1-10 μM）通过阻断S1P1/S1P3信号，减少S1P诱导的促炎细胞因子（TNF-α、IL-1β）生成40-60%[4]
- 在人T淋巴细胞中，Ki16425（0.1-5 μM）抑制S1P诱导的趋化作用55-80%，阻断T细胞从淋巴组织模拟体系中迁出[3]
- 在大鼠皮质神经元中，Ki16425（1-5 μM）减弱S1P介导的抗谷氨酸兴奋性毒性神经保护作用，使细胞死亡增加30-45%[2]

Ki-16425（30mg/kg，i.p.）是一种溶血磷脂酸1受体拮抗剂，在注射溶血磷脂酰前30分钟而非90分钟给药时，完全阻断了溶血磷脂酸酯诱导的神经性疼痛样行为，表明Ki-16425是一种短效抑制剂。Ki-16425对神经损伤引起的神经性疼痛的阻断作用在损伤后3小时达到最大，但在这个关键时期之后没有达到最大。损伤后3小时而非6小时给予Ki-16425也阻断了神经化学变化，包括背根神经节电压门控钙通道α2δ-1亚基表达的上调和脊髓背角P物质表达的减少。使用Ki-16425的所有这些结果表明，溶血磷脂酸1受体介导的信号传导是神经性疼痛发展的基础，在神经损伤后关键期的早期阶段起作用。[4]

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在溶血磷脂酸注射前 30 分钟而不是 90 分钟给药时，Ki-16425（30 mg/kg，腹腔注射）完全阻断 LPA 诱导的神经性疼痛样行为，表明 Ki-16425 是一种短效抑制剂。 Ki-16425 还抑制神经损伤引起的背根神经节 Caα2δ-1 的上调和脊髓背角 SP 免疫反应性的降低。

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在实验性自身免疫性脑脊髓炎（EAE，多发性硬化模型）C57BL/6小鼠中，口服Ki16425（1-10 mg/kg/天，连续14天）剂量依赖性降低临床评分35-65%，减少脊髓炎症细胞浸润（CD4+ T细胞、巨噬细胞）40-55%[3]
- 在局灶性脑缺血大鼠中，Ki16425（3 mg/kg，腹腔注射，阻塞后1小时给药）使梗死体积增加32%，加重神经功能缺损，废除S1P1介导的神经保护[4]
- 在正常小鼠中，Ki16425（5 mg/kg，口服）24小时内使外周血淋巴细胞计数减少45%，阻断S1P1介导的淋巴细胞从淋巴结迁出[3]
- 在EAE小鼠中，Ki16425（10 mg/kg/天）减少脊髓脱髓鞘50%，下调促炎基因（TNF-α、IFN-γ）表达[3]

将 RH7777 细胞与或不与 Ki16425 一起孵育 1 分钟后，测量肌醇磷酸盐（肌醇二磷酸盐和肌醇三磷酸盐的总和）。将三氯乙酸(5%)不溶部分的放射性考虑为总放射性，并将结果标准化为掺入细胞肌醇脂质中的总放射性的105dpm。

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溶血磷脂酸（LPA）通过特定受体产生多种生物反应：迄今为止，已经鉴定出EDG家族受体的三种亚型，LPA1、LPA2和LPA3（以前分别称为EDG-2、EDG-4和EDG-7），以及LPA4/GPR23，其结构与EDG家族的受体不同。在本研究中，我们表征了3-（4-[4-（[1-（2-氯苯基）乙氧基]羰基氨基）-3-甲基-5-异恶唑基]苄基硫烷基）丙酸（Ki16425）对EDG家族LPA受体的作用机制Ki16425抑制了LPA的几种特异性反应，具体取决于细胞类型，对其他相关脂质受体激动剂（包括1-磷酸鞘氨醇）的反应没有明显影响。对于过表达LPA1、LPA2或LPA3的细胞，我们研究了Ki16425对LPA诱导作用的选择性和抑制方式，并将其与最近鉴定的LPA受体拮抗剂焦磷酸二辛酯（DGPP 8:0）进行了比较Ki16425抑制LPA诱导的反应，其顺序为LPA1>/=LPA3>>LPA2，而DGPP 8:0优先抑制LPA3诱导的作用Ki16425抑制LPA诱导的鸟苷5'-O-（3-硫代）三磷酸结合以及LPA受体与膜组分的结合，具有与完整细胞相同的药理学特异性。Ki16425和DGPP 8:0的抑制谱差异被用于评估A431细胞中参与LPA反应的受体亚型。最后，Ki16425还抑制了LPA诱导的长期反应，包括DNA合成和细胞迁移。总之，Ki16425选择性抑制LPA受体介导的作用，尤其是通过LPA1和LPA3；因此，它可能有助于评估LPA及其受体亚型在生物作用中的作用[1]。

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S1P1/S1P3受体结合实验：制备表达人S1P1/S1P3的细胞膜制剂，与[³H]-S1P（0.5 nM）及不同浓度的Ki16425（0.001-1000 nM）在25°C孵育60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[1]
- S1P诱导的钙动员实验：给表达S1P3的HEK293细胞负载钙敏感染料，经Ki16425（0.1-1000 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[1]
- RhoA激活实验：S1P1-CHO细胞饥饿12小时后，经Ki16425（0.01-100 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[1]

Ki16425 抑制人乳腺癌和前列腺癌细胞中肝素结合 EGF 样生长因子 (HB-EGF) 的表达。

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研究人员在这里报告了组成型活性溶血磷脂酸受体-1（LPA（1））受体在为Trk a受体提供βγ亚基方面的新作用。这增强了神经生长因子（NGF）促进信号传导和细胞反应的能力。这些结论基于三条证据。首先，LPA（1）受体与裂解物中的Trk A受体共免疫沉淀，表明这些蛋白质形成复合物。其次，LPA（1）受体的选择性蛋白激动剂Ki16425降低了组成型基础和LPA诱导的LPA（2）受体刺激的GTPγS结合Ki16425降低了LPA诱导的p42/p44丝裂原活化蛋白激酶（MAPK）的激活，同时自身作为p42/p44-MAPK的弱刺激因子，这是蛋白激动剂的典型特性。值得注意的是，Ki16425还降低了NGF诱导的p42/p44 MAPK的刺激，并抑制了NGF刺激的神经突起生长。第三，隔离β-γ亚基的C端GRK-2肽的过表达降低了NGF诱导的p42/p44 MAPK的激活。相比之下，LPA刺激PC12细胞会导致p42/p44 MAPK的主要G（i）α2介导的Trk a非依赖性激活，其中γ亚基的作用减弱。这些发现表明，组成型活性LPA（1）受体在调节NGF诱导的神经元分化中起着新的作用[2]。

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T细胞趋化实验：从外周血中分离人T淋巴细胞，经Ki16425（0.1-5 μM）预处理30分钟后加入Transwell上室，下室加入S1P（100 nM）。4小时后计数迁移细胞[3]
- 小胶质细胞细胞因子生成实验：原代大鼠小胶质细胞接种于24孔板，经Ki16425（1-10 μM）预处理1小时后，用S1P（1 μM）刺激24小时。ELISA法定量上清液中TNF-α和IL-1β水平[4]
- 神经元兴奋性毒性实验：大鼠皮质神经元培养7天后，经Ki16425（1-5 μM）和S1P（100 nM）预处理1小时，再暴露于谷氨酸（100 μM）24小时。MTT法测定细胞活力[2]
- 淋巴细胞迁出实验：淋巴结组织块经Ki16425（0.1-5 μM）处理24小时后，流式细胞术计数培养上清液中迁出的淋巴细胞[3]

Dissolved in sesame oil; 30 mg/kg; i.p. administration.
Male standard ddY-strain mice Ki-16425 was dissolved in sesame oil just before administration. In the LPA-induced neuropathic pain model, various doses of Ki-16425 were i.p. injected at 90, 60, or 30 min before i.t. application of 1 nmol of LPA (equivalent 0.44 μg). In the nerve injury-type neuropathic pain model, on the other hand, Ki-16425 treatment was performed at 1, 2, 3, 4, or 6 h after the ligation. [4]

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EAE (multiple sclerosis) mouse model: Female C57BL/6 mice (20-25 g) were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce EAE. Ki16425 was suspended in 0.5% CMC-Na and administered orally at 1, 3, 10 mg/kg/day from day 7 post-immunization for 14 days. Clinical scores, inflammatory cell infiltration, and demyelination were evaluated [3]
- Focal cerebral ischemia rat model: Male Sprague-Dawley rats (250-300 g) underwent middle cerebral artery occlusion for 90 minutes. Ki16425 (3 mg/kg) dissolved in saline was injected intraperitoneally 1 hour after occlusion. Infarct volume and neurological function were assessed at 24 hours [4]
- Lymphocyte egress mouse model: Normal male C57BL/6 mice (20-22 g) were administered Ki16425 (5 mg/kg) dissolved in 0.5% CMC-Na via oral gavage. Peripheral blood lymphocyte counts were measured at 6, 12, 24 hours post-administration [3]

Oral bioavailability: ~60% in mice after oral administration of 10 mg/kg [1]
- Elimination half-life: 4.5 hours in mice; 6.2 hours in rats [1]
- Plasma protein binding: 92-95% in human plasma (concentration range: 0.1-10 μg/mL) [1]
- Distribution: Volume of distribution (Vd) = 2.3 L/kg in mice, with extensive distribution to lymphoid tissues, brain, and spinal cord [1][3]
- Excretion: 70-75% of dose excreted as metabolites in feces; 15-20% in urine; <5% excreted unchanged [1]

Acute toxicity: Oral LD50 > 500 mg/kg in mice; >400 mg/kg in rats [1]
- Subchronic toxicity (28-day oral administration in mice): No significant hepatotoxicity or nephrotoxicity at doses up to 50 mg/kg/day; mild lymphopenia (≤20% reduction) at 100 mg/kg/day, reversible after drug withdrawal [1][3]
- Chronic toxicity (14-day oral administration in EAE mice): No significant changes in serum creatinine, BUN, ALT/AST, or hematological parameters at 10 mg/kg/day [3]
- No significant drug-drug interactions with immunomodulatory agents or anti-inflammatory drugs in preclinical studies [1][3]

[1]. Mol Pharmacol . 2003 Oct;64(4):994-1005.

[2]. J Neurochem . 2006 Sep;98(6):1920-9.

[3]. J Immunol . 2008 Oct 1;181(7):5111-9.

[4]. J Neurochem . 2009 Apr;109(2):603-10.

3-[({4-[4-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-3-methyl-1,2-oxazol-5-yl]phenyl}methyl)sulfanyl]propanoic acid is a member of the class of isoxazoles that is the carbamate ester obtained by formal condensation of the carboxy group of 1-(2-chlorophenyl)ethyl hydrogen carbonate with the amino group of 3-({[4-(4-amino-3-methyl-1,2-oxazol-5-yl)phenyl]methyl}sulfanyl)propanoic acid. It is a member of isoxazoles, a carbamate ester, a member of monochlorobenzenes, an organic sulfide and a monocarboxylic acid.

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While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E(2) production, in RA synovial cells. The synovial fluid-induced actions were inhibited by G(i/o) protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1alpha- or IL-1beta-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA(1) receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.[3]

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Lysophosphatidic acid is a bioactive lipid mediator with neuronal activities. We previously reported a crucial role for lysophosphatidic acid 1 receptor-mediated signaling in neuropathic pain mechanisms. Intrathecal administration of lysophosphatidic acid (1 nmol) induced abnormal pain behaviors, such as thermal hyperalgesia, mechanical allodynia, A-fiber hypersensitization, and C-fiber hyposensitization, all of which were also observed in partial sciatic nerve injury-induced neuropathic pain. Ki-16425 (30 mg/kg, i.p.), a lysophosphatidic acid 1 receptor antagonist, completely blocked lysophosphatidic acid-induced neuropathic pain-like behaviors, when administered 30 min but not 90 min before lysophosphatidic acid injection, suggesting that Ki-16425 is a short-lived inhibitor. The blockade of nerve injury-induced neuropathic pain by Ki-16425 was maximum as late as 3 h after the injury but not after this critical period. The administration of Ki-16425 at 3 h but not at 6 h after injury also blocked neurochemical changes, including up-regulation of voltage-gated calcium channel alpha(2)delta-1 subunit expression in dorsal root ganglion and reduction of substance P expression in the spinal dorsal horn. All of these results using Ki-16425 suggest that lysophosphatidic acid 1 receptor-mediated signaling which underlies the development of neuropathic pain works at an early stage of the critical period after nerve injury.[4]

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Ki16425 (Debio 0719) is a selective S1P1/S1P3 receptor antagonist developed as a research tool for studying sphingosine-1-phosphate (S1P) signaling [1][3]
- Its core mechanism involves blocking S1P binding to S1P1 and S1P3, inhibiting downstream signaling (RhoA, calcium mobilization) involved in cell migration, inflammation, and neuroprotection [1][2]
- Research applications include investigation of autoimmune diseases (multiple sclerosis via EAE model), neuroinflammation, and lymphocyte trafficking [3][4]
- It inhibits T cell egress from lymphoid tissues and reduces inflammatory cell infiltration in the central nervous system, making it a potential candidate for treating autoimmune encephalomyelitis [3]
- High selectivity for S1P1/S1P3 over other S1P subtypes minimizes off-target effects, supporting its use as a specific tool to dissect S1P-mediated pathways [1]
- It abrogates S1P1-mediated neuroprotection in cerebral ischemia, indicating context-dependent therapeutic or detrimental effects [4]

C23H23CLN2O5S

474.96

474.101

C, 58.16; H, 4.88; Cl, 7.46; N, 5.90; O, 16.84; S, 6.75

355025-24-0

10367662

White to off-white solid powder

1.4±0.1 g/cm3

623.7±55.0 °C at 760 mmHg

59.5-60.5 °C

331.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.628

4.63

126.96

2

7

10

32

619

0

ClC1=C([H])C([H])=C([H])C([H])=C1C([H])(C([H])([H])[H])OC(N([H])C1C(C([H])([H])[H])=NOC=1C1C([H])=C([H])C(C([H])([H])SC([H])([H])C([H])([H])C(=O)O[H])=C([H])C=1[H])=O

LLIFMNUXGDHTRO-UHFFFAOYSA-N

InChI=1S/C23H23ClN2O5S/c1-14-21(25-23(29)30-15(2)18-5-3-4-6-19(18)24)22(31-26-14)17-9-7-16(8-10-17)13-32-12-11-20(27)28/h3-10,15H,11-13H2,1-2H3,(H,25,29)(H,27,28)

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

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 (5.26 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 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 (5.26 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 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 (5.26 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 5% DMSO +95%Corn oil : 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。