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]

In vitro activity: Kil6425 has a modest impact on LPA2, but preferentially inhibits LPA1- and LPA3-mediated responses. Ki16425 has a negligible effect on PC-12 and HL-60 cells, but it significantly inhibits the LPA-induced Ca(2+) response in THP-1, 3T3 fibroblasts, and A431 cells. These findings suggest that Ki16425 may be a valuable tool for assessing the role of particular LPA receptors in the short-term response to LPA. In Swiss 3T3 fibroblasts, Ki16425 prevents long-term DNA synthesis and cell migration that are brought on by LPA.[1] As a protean agonist, ki16425 acts as a weak stimulator of p42/p44 MAPK on its own, while also reducing the LPA-induced activation of p42/p44 mitogen activated protein kinase (MAPK). Additionally, Ki16425 dramatically inhibits PC-12 cell NGF-stimulated neurite outgrowth and significantly lowers NGF-induced stimulation of p42/p44 MAPK.[2] Ki16425 significantly reduces the expression of COX-2 protein that synovial fluids induce. Additionally, Ki16425 suppresses the enhancement of IL-1 action by LPA on COX-2 expression.[3]

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Ki16425 (Debio 0719) is a potent, selective antagonist of S1P1 and S1P3 receptors, with higher affinity for S1P1 [1][3]
- In S1P1-expressing CHO cells, Ki16425 (0.01-100 nM) dose-dependently blocked S1P-induced cell migration, with an IC50 of 0.9 nM, and reversed S1P-mediated RhoA activation [1]
- In S1P3-expressing HEK293 cells, Ki16425 (0.1-1000 nM) inhibited S1P-induced intracellular calcium mobilization with an IC50 of 12.5 nM, without affecting S1P2/S1P4/S1P5-mediated responses [1]
- In primary rat microglia, Ki16425 (1-10 μM) reduced S1P-induced pro-inflammatory cytokine (TNF-α, IL-1β) production by 40-60% via blocking S1P1/S1P3 signaling [4]
- In human T lymphocytes, Ki16425 (0.1-5 μM) inhibited S1P-induced chemotaxis by 55-80% and blocked T cell egress from lymphoid tissue mimics [3]
- In rat cortical neurons, Ki16425 (1-5 μM) attenuated S1P-mediated neuroprotection against glutamate-induced excitotoxicity, increasing cell death by 30-45% [2]

Ki-16425 (30 mg/kg, i.p.) entirely prevents LPA-induced neuropathic pain-like behaviors when given 30 minutes in advance of a lysophosphatidic acid injection, but not 90 minutes beforehand, indicating that Ki-16425 is a transient inhibitor. Additionally, Ki-16425 prevents the dorsal root ganglion's up-regulation of Caα2δ-1 caused by nerve damage as well as the spinal dorsal horn's decrease in SP immunoreactivity.[4] 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 α2δ-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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In C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE, multiple sclerosis model), oral Ki16425 (1-10 mg/kg/day for 14 days) dose-dependently reduced clinical scores by 35-65% and decreased spinal cord inflammatory cell infiltration (CD4+ T cells, macrophages) by 40-55% [3]
- In rats with focal cerebral ischemia, Ki16425 (3 mg/kg, i.p., 1 hour post-occlusion) increased infarct volume by 32% and worsened neurological deficits, abrogating S1P1-mediated neuroprotection [4]
- In normal mice, Ki16425 (5 mg/kg, p.o.) reduced peripheral blood lymphocyte counts by 45% within 24 hours, blocking S1P1-mediated lymphocyte egress from lymph nodes [3]
- In EAE mice, Ki16425 (10 mg/kg/day) decreased spinal cord demyelination by 50% and downregulated pro-inflammatory gene (TNF-α, IFN-γ) expression [3]

The inositol phosphates (sum of inositol bisphosphate and inositol trisphosphate) were measured after 1 minute of incubation of RH7777 cells with or without Ki16425. The radioactivity of the trichloroacetic acid(5%)-insoluble fraction was taken into consideration as the total radioactivity, and the results were normalized to 105 dpm of the total radioactivity incorporated into the cellular inositol lipids.

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Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA1, LPA2, and LPA3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA4/GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA1, LPA2, or LPA3, we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhibited the LPA-induced response in the decreasing order of LPA1 >/= LPA3 >> LPA2, whereas DGPP 8:0 preferentially inhibited the LPA3-induced actions. Ki16425 inhibited LPA-induced guanosine 5'-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced long-term responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA1 and LPA3; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions[1].

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S1P1/S1P3 receptor binding assay: Membrane preparations from human S1P1/S1P3-expressing cells were incubated with [³H]-S1P (0.5 nM) and Ki16425 (0.001-1000 nM) at 25°C for 60 minutes. Non-specific binding was determined with excess unlabeled S1P. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [1]
- S1P-induced calcium mobilization assay: S1P3-HEK293 cells were loaded with calcium-sensitive dye, pretreated with Ki16425 (0.1-1000 nM) for 20 minutes, then stimulated with S1P (100 nM). Calcium fluorescence intensity was monitored by flow cytometry to determine IC50 values [1]
- RhoA activation assay: S1P1-CHO cells were serum-starved for 12 hours, pretreated with Ki16425 (0.01-100 nM) for 30 minutes, then stimulated with S1P (10 nM) for 15 minutes. RhoA activity was measured by pull-down assay using RhoA-specific binding protein [1]

Ki16425 suppressed the expression of heparin-binding EGF-like growth factor (HB-EGF) in human breast and prostate cancer cells.

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Researchers report here a novel role for the constitutively active lysophosphatidic acid receptor-1 (LPA(1)) receptor in providing Gbetagamma subunits for use by the Trk A receptor. This enhances the ability of nerve growth factor (NGF) to promote signalling and cell response. These conclusions were based on three lines of evidence. Firstly, the LPA(1) receptor was co-immunoprecipitated with the Trk A receptor from lysates, suggesting that these proteins form a complex. Secondly, Ki16425, a selective protean agonist of the LPA(1) receptor, decreased constitutive basal and LPA-induced LPA(1) receptor-stimulated GTPgammaS binding. Ki16425 reduced the LPA-induced activation of p42/p44 mitogen activated protein kinase (MAPK), while acting as a weak stimulator of p42/p44 MAPK on its own, properties typical of a protean agonist. Significantly, Ki16425 also reduced the NGF-induced stimulation of p42/p44 MAPK and inhibited NGF-stimulated neurite outgrowth. Thirdly, the over-expression of the C-terminal GRK-2 peptide, which sequesters Gbetagamma subunits, reduced the NGF-induced activation of p42/p44 MAPK. In contrast, the stimulation of PC12 cells with LPA leads to a predominant G(i)alpha2-mediated Trk A-independent activation of p42/p44 MAPK, where Gbetagamma subunits play a diminished role. These findings suggest a novel role for the constitutively active LPA(1) receptor in regulating NGF-induced neuronal differentiation [2].

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T cell chemotaxis assay: Human T lymphocytes were isolated from peripheral blood, pretreated with Ki16425 (0.1-5 μM) for 30 minutes, and added to Transwell upper chambers. S1P (100 nM) was added to lower chambers, and migrated cells were counted after 4 hours [3]
- Microglia cytokine production assay: Primary rat microglia were seeded in 24-well plates, pretreated with Ki16425 (1-10 μM) for 1 hour, then stimulated with S1P (1 μM) for 24 hours. TNF-α and IL-1β levels in supernatants were quantified by ELISA [4]
- Neuronal excitotoxicity assay: Rat cortical neurons were cultured for 7 days, pretreated with Ki16425 (1-5 μM) and S1P (100 nM) for 1 hour, then exposed to glutamate (100 μM) for 24 hours. Cell viability was measured by MTT assay [2]
- Lymphocyte egress assay: Lymph node explants were treated with Ki16425 (0.1-5 μM) for 24 hours, and egressed lymphocytes in culture supernatants were counted by flow cytometry [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: Approximately 60% after oral administration of 10 mg/kg to mice [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) in mice is 2.3 L/kg, widely distributed in lymphoid tissues, brain and spinal cord [1][3] - Excretion: 70-75% of the dose is excreted in feces as metabolites; 15-20% is excreted in urine; <5% is excreted unchanged [1]

Acute toxicity: Oral LD50 in mice > 500 mg/kg; in rats > 400 mg/kg [1]
- Subchronic toxicity (oral administration in mice over 28 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 50 mg/kg/day; mild lymphopenia (≤20%) was observed at 100 mg/kg/day, which was reversible after discontinuation [1][3]
- Chronic toxicity (oral administration in EAE mice over 14 days): No significant changes were observed in serum creatinine, BUN, ALT/AST, or hematological parameters at 10 mg/kg/day [3]
- No significant drug interactions with immunomodulators or anti-inflammatory drugs were found 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)thio]propionic acid is an isoxazole compound, a carbamate formed by the condensation of the carboxyl group of 1-(2-chlorophenyl)ethyl bicarbonate with the amino group of 3-({[4-(4-amino-3-methyl-1,2-oxazol-5-yl)phenyl]methyl}thio)propionic acid. It belongs to the isoxazole class, carbamate class, monochlorobenzene class, organosulfur compounds, and monocarboxylic acid class.

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While inflammatory cytokines are recognized as key factors in inducing the activation of cyclooxygenase-2 (COX-2) in fibroblast-like synovial cells, the roles of other bioactive components in synovial fluid besides inflammatory cytokines remain unclear. In this study, we evaluated the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction in fibroblast-like RA synovial cells using synovial fluid from patients with rheumatoid arthritis (RA). Synovial fluid from RA patients stimulated COX-2 induction in RA synovial cells, which is associated with the production of prostaglandin E2. The synovial fluid-induced effect was inhibited by the G(i/o) protein inhibitor pertussis toxin and the LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylthio)propionic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1α or IL-1β-induced enzyme expression, and this effect was sensitive to pertussis toxin and Ki16425. RA synovial cells expressed a large number of LPA(1) receptors compared to other LPA receptor subtypes. In addition, synovial fluid contains a large amount of LPA, LPA synthase autosecretin and its substrate lysophosphatidylcholine. In summary, lysophosphatidylcholine (LPA) present in synovial fluid works synergistically with inflammatory cytokines to play a key role in the induction of COX-2 in synovial cells of rheumatoid arthritis (RA). Ki16425-sensitive LPA receptors may be therapeutic targets for RA. [3] Lysophosphatidylcholine is a neuroactive bioactive lipid mediator. We previously reported that the lysophosphatidylcholine 1 receptor-mediated signaling pathway plays a key role in the mechanism of neuropathic pain. Intrathecal injection of lysophosphatidylcholine (1 nmol) can induce abnormal pain behaviors, such as thermal hyperalgesia, mechanical analgesia, A fiber hypersensitivity and C fiber hyposensitivity, which have also been observed in neuropathic pain induced by some sciatic nerve injuries. Ki-16425 (30 mg/kg, intraperitoneal injection) is a lysophosphatidylcholine 1 receptor antagonist. Administration 30 minutes prior to lysophosphatidylcholine injection completely blocks lysophosphatidylcholine-induced neuropathic pain-like behaviors, but administration 90 minutes prior is ineffective, suggesting that Ki-16425 is a short-acting inhibitor. The blocking effect of Ki-16425 on nerve injury-induced neuropathic pain reaches its maximum at 3 hours post-injury, but ceases to be effective after this critical period. Administration of Ki-16425 at 3 hours post-injury, rather than 6 hours, also blocks neurochemical changes, including upregulation of the expression of the voltage-gated calcium channel α2δ1 subunit in the dorsal root ganglion and decreased expression of substance P in the dorsal horn of the spinal cord. All results using Ki-16425 indicate that the lysophosphatidylcholine 1 receptor-mediated signaling pathway is fundamental to the development of neuropathic pain and plays a role early in the critical period following nerve injury. [4]

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Ki16425 (Debio 0719) is a selective S1P1/S1P3 receptor antagonist that was initially developed as a tool for studying the sphingosine-1-phosphate (S1P) signaling pathway. [1][3]
- Its core mechanism is to block the binding of S1P to S1P1 and S1P3, thereby inhibiting downstream signaling pathways (RhoA, calcium mobilization) involved in cell migration, inflammation, and neuroprotection. [1][2]
- Research applications include studies of autoimmune diseases (multiple sclerosis through EAE models), neuroinflammation, and lymphocyte migration. [3][4]
- It inhibits T cell migration from lymphoid tissues and reduces inflammatory cell infiltration in the central nervous system, thus making it a potential candidate for the treatment of autoimmune encephalomyelitis.[3]
- The high selectivity of S1P1/S1P3 relative to other S1P subtypes minimizes off-target effects, supporting its use as a specific tool for understanding S1P-mediated pathways.[1]
- It eliminates the neuroprotective effect of S1P1 in cerebral ischemia, suggesting that its therapeutic or detrimental effects are context-dependent.[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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO +95%Corn oil : 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)