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| 25mg |
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| 100mg |
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AM095 free acid is a novel, potent and selective LPA1 receptor antagonist that inhibited GTPγS binding to Chinese hamster ovary (CHO) cell membranes overexpressing recombinant human or mouse LPA1 with IC50 of 0.98 and 0.73 μM, respectively. It did not show agonism for LPA1. Bioactive phospholipid lysophosphatidic acid (LPA) communicates via the LPA1-6 family of G protein-coupled receptors, which consists of at least six receptors. The LPA type 1 receptor (LPA1) is widely distributed throughout tissues and is involved in the regulation of numerous physiological and pathological cellular processes.
| Targets |
human LPA1 ( pIC50 = 0.98 μM ); mouse LPA1 ( pIC50 = 0.73 μM )
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| ln Vitro |
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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| ln Vivo |
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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| Enzyme Assay |
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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| Animal Protocol |
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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| References |
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| Molecular Formula |
C₂₇H₂₄N₂O₅
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|---|---|
| Molecular Weight |
456.49
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| Exact Mass |
456.17
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| Elemental Analysis |
C, 71.04; H, 5.30; N, 6.14; O, 17.52
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| CAS # |
1228690-36-5
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| Related CAS # |
AM095; 1345614-59-6
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| Appearance |
Solid powder
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| SMILES |
CC1=NOC(=C1NC(=O)O[C@H](C)C2=CC=CC=C2)C3=CC=C(C=C3)C4=CC=C(C=C4)CC(=O)O
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| InChi Key |
LNDDRUPAICPXIN-GOSISDBHSA-N
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| InChi Code |
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
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| Chemical Name |
2-[4-[4-[3-methyl-4-[[(1R)-1-phenylethoxy]carbonylamino]-1,2-oxazol-5-yl]phenyl]phenyl]acetic acid
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| Synonyms |
AM095; AM 095; AM-095; AM095 free acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~67.3 mg/mL (~147.4 mM)
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1906 mL | 10.9531 mL | 21.9063 mL | |
| 5 mM | 0.4381 mL | 2.1906 mL | 4.3813 mL | |
| 10 mM | 0.2191 mL | 1.0953 mL | 2.1906 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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