human LPA1 ( pIC50 = 0.98 μM ); mouse LPA1 ( pIC50 = 0.73 μM )
LPA₁ receptor (IC₅₀ = 0.025 µM for human LPA₁, IC₅₀ = 0.023 µM for mouse LPA₁)
Selective against LPA₂, LPA₃, LPA₄, and LPA₅ receptors (IC₅₀ > 5 µM) [1]

AM095 suppresses the calcium flux that LPA causes in CHO cells that have been transfected with mouse or human LPA1. For human or mouse LPA1-transfected CHO cells, the IC50 for AM095 antagonism of LPA-induced calcium flux is 0.025 and 0.023 μM, respectively[1]. Compared to vehicle control, AM095 reduces LPA-induced vasorelaxation by approximately 90% at 10 μM[2]. AM095 inhibits human A2058 melanoma cells (IC50=233 nM) and mouse LPA1 (IC50=778 nM)-driven chemotaxis of CHO cells[3].

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AM095 is a potent and selective LPA₁ antagonist that inhibits LPA-induced calcium flux in CHO cells expressing human or mouse LPA₁ receptors with IC₅₀ values of 0.025 µM and 0.023 µM, respectively. It shows no significant activity against other LPA receptors up to 5 µM. [1]

AM095 antagonism of LPA1 and significantly reduces bleomycin induced skin fibrosis [1] AM095 is well tolerated at the doses tested in rats and dogs following oral and intravenous dosing. It also has a high oral bioavailability and a moderate half-life. LPA-stimulated histamine release is dose-dependently reduced by AM095. AM095 reduces the bleomycin-induced rises in inflammatory cell infiltration, collagen, and protein in bronchalveolar lavage fluid. In a mouse unilateral ureteral blockage model, AM095 reduces kidney fibrosis[3].

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In a bleomycin-induced dermal fibrosis mouse model, oral administration of AM095 (30 mg/kg twice daily) significantly attenuated increases in dermal thickness and hydroxyproline content (a marker of collagen). Both preventive (starting at onset of bleomycin) and therapeutic (starting 7 or 14 days after bleomycin) regimens showed efficacy. [1]

In assays, both hLPA1/CHO and mLPA1/CHO cells are used. Protease inhibitors, 10 mM HEPES, pH 7.4, 1 mM dithiothreitol, and approximately 20 mL of ice-cold membrane buffer are added to a cell pellet of hLPA1/CHO or mLPA1/CHO cells. The cells are sonicated, and the cell lysate is centrifuged for 10 minutes at 4°C at 2000 rpm. Further centrifuging of the supernatant is done for 70 minutes at 4°C at 25,000 rpm. Using a Potter-Elvehjem tissue grinder, the membrane pellet is resuspended in 5 mL of ice-cold membrane buffer and homogenized. With the Bradford Protein Assay Kit, the final protein concentration is calculated. To 25 to 40 μg of hLPA1/CHO or mLPA1/CHO membranes and 0.1 nM [³⁵S]-GTPηS in buffer (50 mM HEPES, 0.1 mM NaCl, 10 mM MgCl₂, 50 μg/mL saponin, pH 7.5) containing 0.2% fatty acid-free human serum albumin and 5 μM GDP, known amounts of AM095 (diluted in dimethyl sulfoxide) or vehicle (dimethyl sulfoxide) are added. The capacity of AM095 to impede GTPγS binding stimulated by 900 nM LPA (18:1) is measured in order to assess LPA1 antagonist activity. As an alternative, the capacity of AM095 to promote GTPηS binding in the absence of LPA is assessed in order to assess agonist effects. Membranes are harvested onto glass filter binding plates and three times washed with cold buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, and 10 mM MgCl₂ using a Brandel 96-tip cell harvester after reactions are incubated for 30 minutes at 30°C. After plates are dried, a Packard TopCount NXT microplate scintillation counter is used to measure cpm.

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Calcium flux assays were performed using CHO, HEK, or B103 cells transiently or stably expressing human or mouse LPA receptors. Cells were loaded with a calcium-sensitive dye, treated with test compounds for 30 minutes, then stimulated with LPA. Calcium mobilization was measured using a fluorescence plate reader. Inhibition curves were generated to calculate IC₅₀ values. [1]

In vitro, AM095 was a potent LPA₁ receptor antagonist because it inhibited GTPγS binding to Chinese hamster ovary (CHO) cell membranes overexpressing recombinant human or mouse LPA₁ with IC₅₀ values of 0.98 and 0.73 μM, respectively, and exhibited no LPA₁ agonism. In functional assays, AM095 inhibited LPA-driven chemotaxis of CHO cells overexpressing mouse LPA₁ (IC₅₀= 778 nM) and human A2058 melanoma cells (IC₅₀ = 233 nM)[3].

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CHO cells stably expressing human or mouse LPA₁ were used to assess antagonist activity via calcium flux. Cells were cultured in 96-well plates, serum-starved, loaded with dye, treated with compound, and stimulated with LPA. Calcium mobilization was measured to determine inhibition. [1]

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]

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C57Bl/6 mice were injected subcutaneously with bleomycin (10 µg/ml, 100 µl) once daily for 28 days to induce dermal fibrosis. AM095 (30 mg/kg) or vehicle (sterile water) was administered by oral gavage twice daily on weekdays and once daily on weekends. Treatment began either at the start of bleomycin injections (preventive) or 7 or 14 days after (therapeutic). Skin samples were collected at day 28 for histological and biochemical analysis. [1]

In in vivo experiments, we demonstrated that AM095: 1) possesses high oral bioavailability and a moderate half-life, and is well-tolerated in rats and dogs after oral and intravenous administration within the tested dose range; 2) dose-dependently reduces LPA-stimulated histamine release; 3) attenuates bleomycin-induced increases in collagen, protein, and inflammatory cell infiltration in bronchoalveolar lavage fluid; and 4) alleviates renal fibrosis in a mouse model of unilateral ureteral obstruction. Despite its antifibrotic activity, AM095 had no effect on normal wound healing after incision and excision in rats. These data suggest that AM095 is an LPA₁ receptor antagonist with good oral exposure and antifibrotic activity in rodent models. [3] In C57Bl/6 mice, after oral administration of AM095 (30 mg/kg at 0 and 8 hours), Cmax was 6200 nM (28 µg/ml), Cmin was 170 nM (0.08 µg/ml), and AUC was 118.7 µg·hr/ml. Plasma concentrations were higher than the IC50 of LPA1 throughout the 24-hour period. [1]

[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 multi-organ fibrosis. We recently discovered that lysophosphatidic acid (LPA) is involved in the pathogenesis of pulmonary fibrosis. This study aimed to investigate the role of LPA and its two receptors, LPA₁ and LPA₂, in skin fibrosis in an SSc mouse model. Methods: Wild-type (WT), LPA₁ knockout (KO), and LPA₂ knockout mice were subcutaneously injected with bleomycin or phosphate-buffered saline (PBS) once daily for 28 days. Dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA) or phosphorylated Smad2-positive cells were measured in the bleomycin and PBS-injected groups, respectively. In another experiment, we administered a novel selective LPA₁ antagonist, AM095, or its carrier to C57BL/6 mice via gavage after 28 consecutive days of bleomycin or PBS injections. AM095 or carrier treatment was initiated simultaneously with bleomycin and PBS injections, or 7 or 14 days post-injection, and continued until the end of the experiment. We measured dermal thickness and collagen content at the injection site. Results: LPA₁-KO mice showed significant resistance to bleomycin-induced increases in dermal thickness and collagen content, while LPA₂-KO mice were as susceptible as wild-type mice. In LPA₁-KO mice, the bleomycin-induced increase in dermal α-SMA+ and phosphorylated Smad2+ cells was eliminated. Pharmacological antagonism of LPA₁ using AM095, regardless of whether a prophylactic or both treatment regimens were employed, significantly reduced bleomycin-induced skin fibrosis. Conclusion: These results suggest that LPA/LPA₁ pathway inhibition may be a viable new therapy for systemic sclerosis (SSc), and that LPA₁ is an attractive pharmacological target in skin fibrosis. [1]

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Lysophosphatidic acid (LPA) has been shown to participate in various cardiovascular functions, but its potential role in vascular tone control remains unclear. This study shows that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1-3 receptors) can dilate intact mouse thoracic aortas, and their maximum dilatory effect (Emax) values are similar (53.9% and 51.9% of phenylephrine-induced precontraction, respectively), but the half-maximal effective concentrations (EC50) of LPA and VPC31143 for induced vasodilation are different (400 nM and 15 nM, respectively). Mechanical removal of the endothelium or gene knockout of endothelial nitric oxide synthase (eNOS) not only weakens the vasodilatory effect of LPA or VPC31143, but also converts it into vasoconstriction. Freshly isolated mouse aortic endothelial cells express LPA1, LPA2, LPA4, and LPA5 transcripts. LPA1,3 antagonist Ki16425, LPA1 antagonist AM095, and LPA1 gene knockout (but not LPA2 gene knockout) all eliminated LPA-induced vasodilation. Inhibition of the phosphatidylinositol 3-kinase-protein kinase B/Akt pathway by Watermanin or MK-2206 did not affect the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edefosine (but not PLCε gene knockout) eliminated LPA-induced vasodilation, indicating that other PLC enzymes besides PLCε mediated the response. In summary, this study identified LPA as an endothelium-dependent vasodilator whose mechanism of action involves LPA1, PLC, and eNOS. [2] AM095 is a novel, orally bioavailable selective LPA1 antagonist that alleviates skin fibrosis in a bleomycin-induced scleroderma mouse model. It can reduce the accumulation of myofibroblasts and phosphorylation of Smad2, suggesting that it inhibits the activation of the TGF-β pathway. This study indicates that LPA_sub>1 is a potential drug target for the treatment of fibrotic diseases such as scleroderma. [1]

C₂₇H₂₄N₂O₅

456.49

456.168

C, 71.04; H, 5.30; N, 6.14; O, 17.52

1228690-36-5

AM095; 1345614-59-6

46213949

White to off-white solid powder

1.3±0.1 g/cm3

641.9±55.0 °C at 760 mmHg

342.0±31.5 °C

0.0±2.0 mmHg at 25°C

1.629

4.94

2

6

8

34

666

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

LNDDRUPAICPXIN-GOSISDBHSA-N

InChI=1S/C27H24N2O5/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)/t18-/m1/s1

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

AM095; AM 095; AM-095; AM095 free 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)

DMSO: ~67.3 mg/mL (~147.4 mM)

Solubility in Formulation 1: ≥ 2.25 mg/mL (4.93 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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