human LPA1 ( pIC50 = 0.98 μM ); mouse LPA1 ( pIC50 = 0.73 μM )
LPA1 (human) (IC50 = 0.025 μM in calcium flux assay)[1]
LPA1 (mouse) (IC50 = 0.023 μM in calcium flux assay)[1]

AM095 is a potent LPA1 receptor antagonist as it inhibits GTPγS binding to the membrane of Chinese hamster ovary (CHO) cells overexpressing recombinant human or mouse LPA1 with IC50 values of 0.98 and 0.73 μM, respectively. AM095 inhibits LPA-driven chemotaxis of CHO cells overexpressing mouse LPA1 (IC50=778 nM) and human A2058 melanoma cells (IC50=233 nM). The IC50 of AM095 in human LPA1 GTPγS binding assay is comparable to our previously published compounds AM966 (IC50=0.98±0.17 μM) and Debio-0719 compound (IC50=0.60±0.04 μM) [1]. AM095 inhibits LPA-induced calcium flux in CHO cells stably transfected with human or mouse LPA1. The IC50 of AM095 antagonizing LPA-induced calcium flux in human or mouse LPA1-transfected CHO cells is 0.025 and 0.023 μM, respectively [2].

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AM095 inhibited LPA-induced calcium flux in CHO cells stably expressing human LPA1 with an IC50 of 0.025 μM, and in CHO cells expressing mouse LPA1 with an IC50 of 0.023 μM. The IC50 for antagonism of LPA-induced calcium flux was >5 μM for CHO, HEK, or B103 cells transfected with LPA2, LPA3, LPA4, or LPA5 receptors (human or mouse), demonstrating selectivity for LPA1.[1]

AM095 has high oral bioavailability and a moderate half-life and is well tolerated at doses tested in rats and dogs after oral and intravenous administration. Following oral administration (10 mg/kg) to rats, AM095 plasma concentrations peaked at 2 hours with a Cmax of 41 μM and then decreased to 10 nM by 24 hours. After intravenous (2 mg/kg) administration, a Cmax of 12 μM was observed within 15 minutes, which also dropped to approximately 10 nM by 24 hours, with a t1/2 of 1.79 hours. In dogs, a single oral dose of 5 mg/kg produces a peak plasma concentration of 21 μM within 15 minutes of administration, which then decreases to 10 nM within 24 hours. In comparison, an intravenous dose of 2 mg/kg resulted in a Cmax of 11 μM within 15 minutes, which decreased to 15 nM within 8 hours, resulting in a t1/2 of 1.5 hours [1].
In C57Bl/6 mice challenged with 28 daily subcutaneous bleomycin injections to induce dermal fibrosis, AM095 administered at 30 mg/kg by oral gavage in a 'preventive' regimen (starting from day 1) attenuated bleomycin-induced increases in dermal thickness by 70% and hydroxyproline content by 52% compared to vehicle-treated mice.[1]
In 'therapeutic' regimens where AM095 administration began on day 7 or day 14 after the onset of bleomycin challenge, dermal thickness increases were attenuated by 85% (day 7 start) and 61% (day 14 start), while hydroxyproline increases were attenuated by 32% (day 7 start) and 26% (day 14 start), respectively.[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 assay for LPA receptor antagonism: Cells stably or transiently transfected with LPA receptors were plated in 96-well Poly-D-Lysine-coated black-wall clear-bottom plates at 20,000-40,000 cells/well and cultured overnight. Cells were then serum-starved overnight (stably expressing cells) or for 4 hours (transient transfectants). On assay day, cells were loaded with FLIPR Calcium 4 dye in HBSS supplemented with 20 mM HEPES, 2 mM probenecid, and 0.3% fatty acid-free HSA for 1 hour at 37°C. Test compounds (25 μl of % DMSO) were added and incubated at room temperature for 30 minutes. LPA (50 μl of 5X stock in HBSS with 20 mM HEPES and 0.3% fatty acid-free HSA) was added after 15 seconds baseline measurement. Final LPA concentrations: 10 nM for LPA1 and LPA3, 30 nM for LPA2 and LPA5, 300 nM for LPA4. Intracellular calcium mobilization was measured using a FLEXstation III. Inhibition curves were generated by plotting percentage inhibition versus log10 compound concentration, and IC50 values calculated by nonlinear regression using sigmoidal dose-response equation.[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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Calcium flux assay in transfected cells: CHO cells stably expressing human or mouse LPA1, human LPA3, and mouse LPA3; HEK cells stably expressing mouse LPA3; and B103 rat neuroblastoma cells transiently expressing human or mouse LPA2, LPA4, or LPA5 using Lipofectamine 2000 were used. Cells were cultured in appropriate media (F12 with 10% FBS and hygromycin B for CHO and HEK; DMEM with 10% FBS and hygromycin B for HEK-mLPA3) and plated as described above. After serum starvation and dye loading, cells were treated with increasing concentrations of AM095 or vehicle, then stimulated with LPA. The fluorescence signal was recorded and IC50 values were calculated.[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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Animal model: Female and male C57Bl/6 mice (6-8 weeks old) were injected subcutaneously with bleomycin (100 μl of 10 μg/ml in PBS) or PBS alone once daily for 28 consecutive days. AM095 was dissolved in sterile water and administered by oral gavage at 30 mg/kg per mouse, twice daily on weekdays and once daily on weekends. Vehicle control received sterile water alone. Three treatment regimens were tested: 'preventive' (starting from day 1, concurrent with bleomycin), and two 'therapeutic' regimens (starting on day 7 or day 14 after bleomycin onset). All treatments continued until the end of the experiment. At day 28, mice were sacrificed and 6 mm punch biopsies of injected skin were collected for histology and hydroxyproline assay.[1]
Additional animal experiments: LPA1 knockout mice (hybrid C57Bl/6/129Sv/J background) and LPA2 knockout mice (BALB/c background) were similarly challenged with bleomycin or PBS for 28 days to assess genetic deletion effects.[1]

In in vivo experiments, we demonstrated that AM095: 1) has 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) reduces LPA-stimulated histamine release in a dose-dependent manner; 3) attenuates the increase in collagen, protein and inflammatory cell infiltration in bronchoalveolar lavage fluid induced by bleomycin; and 4) reduces renal fibrosis in a mouse model of unilateral ureteral obstruction. Although AM095 has antifibrotic activity, it has 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]

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AM095 pharmacokinetics in mice: C57Bl/6 mice received 30 mg/kg by oral gavage at time 0 and 8 hours. Blood was collected at 0, 4, 8, 9, 12, and 24 hours. Plasma concentrations were analyzed by LC-MS/MS. AUC was 118.7 μg*hr/ml. Maximum plasma concentration (Cmax) was 6200 nM (28 μg/ml), and minimum plasma concentration (Cmin) was 170 nM (0.08 μg/ml). Twice daily 30 mg/kg dosing produced plasma concentrations greater than the IC50 against LPA1 throughout the treatment 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 showed that LPA₁-KO mice were significantly resistant 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 for skin fibrosis. [1] Lysophosphatidic acid (LPA) has been shown to participate in various cardiovascular functions, but its potential role in the regulation of vascular tone 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 Watmanin 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 mediate the response. In conclusion, this study identified LPA as an endothelium-dependent vasodilator whose mechanism of action involves LPA1, PLC, and eNOS. [2]

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AM095 sodium (sodium [4'-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl]-acetate) is a selective LPA1 antagonist. It was developed by Amira Pharmaceuticals. In the bleomycin-induced dermal fibrosis model, AM095 reduced myofibroblast accumulation and TGF-β/Smad signaling (as indicated by reduced α-SMA+ and phosphoSmad2+ cells) when administered preventively. The ability of AM095 to attenuate established fibrosis when started after injury suggests its potential as an anti-fibrotic therapy for scleroderma and other fibrotic diseases.[1]

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

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.35 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 20.8 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: ≥ 2.08 mg/mL (4.35 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 20.8 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: ≥ 2.08 mg/mL (4.35 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (10.45 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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