Radioprotectin-1 is a powerful agonist of LPA2 with an EC50 of 5 pM and acts as a complete agonist of the human ortholog of LPA2 [1]. Radioprotectin-1 (0-3 μM; 15 min) substantially decreases apoptosis generated by gamma radiation and the radiomimetic medication doxorubicin in cells expressing LPA2 endogenously or after transfection [1].

Radioprotectin-1 is a powerful agonist of LPA2 with an EC50 of 5 pM and acts as a complete agonist of the human ortholog of LPA2 [1]. Radioprotectin-1 (0-3 μM; 15 min) substantially decreases apoptosis generated by gamma radiation and the radiomimetic medication doxorubicin in cells expressing LPA2 endogenously or after transfection [1].

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Radioprotectin-1 (RP-1) is a specific agonist for the LPA2 GPCR subtype, with an EC50 of 25 nM at the murine LPA2 receptor, comparable to LPA 18:1 (EC50 = 32 nM) as determined by the TGFα-shedding assay. It does not activate or inhibit other LPA receptor subtypes (LPA1, LPA3, LPA4, LPA5, LPA6). [1]
RP-1 (10 µM) significantly reduced caspase 3/7 activity (apoptosis) induced by 15 Gy γ-irradiation in mouse embryonic fibroblasts (MEF) transduced with human LPA2 (LPA2-DKOMEF), but was ineffective in vector-transduced control MEF cells. [1]
In rat intestinal epithelial cells (IEC-6), RP-1 (1.0 or 3.0 µM) administered 30 minutes post-treatment reduced caspase 3/7 activity induced by the radiomimetic drug Adriamycin (1.7 µM) by approximately 50%. [1]
RP-1 (10 µM) treatment 30 minutes after 15 Gy γ-irradiation significantly decreased the percentage of γ-H2AX-positive cells (indicative of DNA double-strand breaks) in LPA2-DKOMEF cells compared to vector control MEFs, as measured by flow cytometry 4 hours post-irradiation. [1]
Clonogenic survival assays in IEC-6 cells showed that treatment with 10 µM RP-1, administered 2 hours post-irradiation, significantly increased the surviving fraction after exposure to 2-10 Gy γ-irradiation compared to vehicle-treated controls. [1]
Quantitative RT-PCR analysis on murine intestinal enteroids showed that treatment with 1 µM RP-1 for 24 hours increased transcript abundance of LPA1, LPA2, and LPA3 receptors in non-irradiated cultures. Furthermore, exposure to 5 Gy γ-irradiation specifically increased Ipar2 (LPA2) transcript levels, and this increase was further enhanced by RP-1 treatment within 30 minutes post-irradiation. [1]
In small intestinal enteroid cultures derived from Lgr5-EGFP-CreER;Tdtomato^flox mice, pre-treatment with 1 µM RP-1 for 24 hours prior to 5 Gy γ-irradiation significantly increased enteroid survival rates compared to untreated irradiated controls on days 5 and 6 post-irradiation. [1]
Lineage tracing in the aforementioned enteroid cultures using 4-hydroxytamoxifen (4-OHT) to induce Tdtomato expression in Lgr5⁺ cells revealed that pre-treatment with 1 µM RP-1 preserved the Lgr5⁺ intestinal stem cell (ISC) population 3 days post-irradiation. An average of 11.6 ± 0.6 Tdtomato⁺ cells per 50 µm crypt length were counted in RP-1 pre-treated, irradiated cultures, comparable to non-irradiated controls, whereas no Tdtomato⁺ cells were detected in irradiated, vehicle-treated cultures. [1]

A highly effective and selective agonist of the mouse LPA2 GPCR is radioprotectin-1 [1]. The C57BL/6 Mouse Mortality Rate (GI-ARS) is decreased by subcutaneous injection of radioprotectin-1 (0.1 mg/kg, 0.3 mg/kg; every 12 hours; for 3 days) [1]. In Lgr5+ stem cells, radioprotectin-1 specifically activates the upregulated LPA2 GPCR to produce its effects of radioprotection and radiomitigation [1].

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In a murine total body irradiation (TBI) model of Hematopoietic Acute Radiation Syndrome (HE-ARS), C57BL/6 mice were exposed to 9.0 Gy γ-irradiation (LD_80/30). Subcutaneous administration of Radioprotectin-1 (RP-1) at doses of 0.1 mg/kg or 0.3 mg/kg, initiated 26 ± 2 hours post-irradiation and given every 12 hours for 3 days, significantly improved 30-day survival compared to vehicle controls. Survival rates were 65% (0.1 mg/kg) and 55% (0.3 mg/kg) versus 25% for vehicle. [1]
In a murine partial body irradiation (PBI) model of Gastrointestinal Acute Radiation Syndrome (GI-ARS) with 15% bone marrow sparing, mice were exposed to 16.0 Gy γ-irradiation. Subcutaneous administration of RP-1 at 0.1 mg/kg or 0.3 mg/kg on the same schedule (starting 26 ± 2 hours post-irradiation) significantly improved 30-day survival. Survival rates were 85% (0.1 mg/kg) and 70% (0.3 mg/kg) versus 45% for vehicle. [1]

Receptor activation for all six murine LPA GPCR subtypes was measured using the transforming growth factor alpha (TGFα) shedding assay. Briefly, HEK293 cells were transiently transfected with plasmids encoding individual LPA receptors, an alkaline phosphatase-conjugated TGFα (AP-TGFα), and the appropriate chimeric Gα proteins. Transfected cells were plated, allowed to adhere, and then stimulated with serial dilutions of Radioprotectin-1 (RP-1) or LPA (18:1) for one hour. The conditioned medium was then transferred to a new plate, and both the cell plate and medium plate were incubated with para-nitrophenylphosphate (p-NPP) substrate. Alkaline phosphatase activity released into the medium, reflecting receptor activation, was quantified by measuring absorbance at 405 nm. Receptor-specific activation was calculated by subtracting the response in mock-transfected cells from the response in receptor-transfected cells. For LPA4, LPA5, and LPA6 transfections, a dual LPA1/3 antagonist was included to block interference from endogenously expressed receptors. [1]

Cell Viability Assay [1]
Cell Types: MEF cells, IEC-6 cells
Tested Concentrations: 0 μM, 0.1 μM, 0.3 μM, 1.0 μM, 3 μM
Incubation Duration: 15 minutes
Experimental Results: Doxorubicin by gamma radiation and radiomimetic chemotherapy.

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Radiation/Drug-Induced Apoptosis Assay: Mouse embryonic fibroblasts (MEFs; LPA2-DKOMEF or vector-DKOMEF) or rat intestinal epithelial cells (IEC-6) were plated and serum-starved. For radiation induction, cells were exposed to 15 Gy γ-irradiation. For drug induction, cells were treated with 1.7 µM Adriamycin. Radioprotectin-1 (RP-1) or vehicle was added at specified times post-insult (e.g., 30 minutes or 1 hour). Apoptosis was quantified 6 hours (for MEFs) or 24 hours (for IEC-6) later using a Caspase-Glo 3/7 assay, which measures luminescence from caspase-3/7 activity. [1]
γ-H2AX Immunofluorescence and Flow Cytometry: LPA2-DKOMEF and vector-DKOMEF cells were plated, serum-starved, and pre-treated with RP-1 (10 µM), LPA (10 µM), or vehicle for 15 minutes before exposure to 15 Gy γ-irradiation. Fresh medium containing the compounds was replaced after irradiation. Four hours post-irradiation, cells were trypsinized, fixed, permeabilized, and stained with a fluorescently labeled antibody against phosphorylated histone H2AX (Ser139, γ-H2AX). The percentage of γ-H2AX-high positive cells was analyzed by flow cytometry. [1]
Clonogenic Survival Assay: IEC-6 cells were plated at low density and serum-starved. The following day, cultures were exposed to 2-10 Gy γ-irradiation. Two hours post-irradiation, the medium was replaced with fresh serum-free medium containing 10 µM RP-1 or vehicle. The treatment medium was replenished daily. After 3 days of incubation, cells were fixed with methanol, stained with crystal violet, and colonies (defined as groups of >50 cells) were counted manually. The surviving fraction was calculated relative to the plating efficiency of non-irradiated controls. [1]
Enteroid Culture and Survival Analysis: Small intestinal crypts were isolated from mice, embedded in Matrigel, and cultured in specialized medium to form enteroids. After 6 days of culture, enteroids were pre-treated with 1 µM RP-1, 1 µM LPA, or vehicle for 24 hours. The medium was then replaced, and cultures were exposed to 5 Gy γ-irradiation. Cultures were maintained with regular medium changes, and the number of surviving (intact, phase-bright) enteroids was counted daily for up to 6 days post-irradiation. Survival was expressed as a percentage relative to the number present on the day of irradiation. [1]
Lineage Tracing in Enteroids: Enteroids were derived from Lgr5-EGFP-CreER;Tdtomato^flox transgenic mice. After the standard pre-treatment and irradiation protocol, 4-hydroxytamoxifen (4-OHT, 1 µM) was added to the culture medium on post-irradiation day 2 or 3 to induce Cre-mediated recombination and permanent Tdtomato expression in Lgr5⁺ cells and their progeny. Enteroids were fixed 24 hours after 4-OHT addition (on day 3 or 4 post-irradiation), immunostained for GFP (to identify Lgr5⁺ cells), and counterstained with Hoechst. Confocal microscopy was used to image the enteroids. Tdtomato⁺ cells within the bottom 50 µm of the crypt region were manually counted from multiple crypts per biological replicate. [1]
Quantitative RT-PCR for LPA Receptor Transcripts: Intestinal enteroids were cultured, pre-treated with 1 µM RP-1 or vehicle for 24 hours, and then exposed to 5 Gy γ-irradiation or sham irradiation. Total RNA was isolated from enteroids 30 minutes post-irradiation using a micro-scale RNA extraction kit. cDNA was synthesized, and quantitative PCR was performed using TaqMan gene expression assays specific for mouse Lpar1, Lpar2, Lpar3, and Gapdh (housekeeping gene). Relative gene expression was calculated using the 2^(-ΔΔCt) method. [1]

Animal/Disease Models: 8-10 weeks old C57BL/6 female mice, subjected to total body irradiation (TBI) [1]
Doses: 0.1 mg/kg, 0.3 mg/kg
Route of Administration: subcutaneous injection; once every 12 hrs (hrs (hours)); for 3 days
Experimental Results: Mortality of C57BL/6 mice was diminished in HE-ARS and GI-ARS models.

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Hematopoietic ARS (HE-ARS) Model: Eight to ten-week-old female C57BL/6 mice were acclimated and then subjected to total body irradiation (TBI) with 9.0 Gy γ-rays from a Cs-137 source at a dose rate of approximately 0.82 Gy/min. Beginning 26 ± 2 hours after irradiation, mice received subcutaneous injections of Radioprotectin-1 (RP-1) at 0.1 mg/kg or 0.3 mg/kg, or vehicle (0.1% Ethanol, 2% Propanediol in PBS). Injections were administered every 12 hours for a total of 3 days. Mice were provided with gel food packs starting on day 4 and had ad libitum access to sterilized chow and water. Animal health and survival were monitored twice daily for 30 days. [1]
Gastrointestinal ARS (GI-ARS) Model: Eight to ten-week-old female C57BL/6 mice were anesthetized and placed in a plexiglass restrainer such that their legs below the knee were shielded with lead, sparing approximately 15% of the bone marrow. They were then subjected to partial body irradiation (PBI) with 16.0 Gy γ-rays. The drug treatment regimen (RP-1 at 0.1 or 0.3 mg/kg or vehicle, administered subcutaneously every 12 hours for 3 days starting 26 ± 2 hours post-irradiation), supportive care, and survival monitoring were identical to the HE-ARS model. [1]

[1]. The LPA2 receptor agonist Radioprotectin-1 spares Lgr5-positive intestinal stem cells from radiation injury in murine enteroids. Cell Signal. 2018 Nov;51:23-33.

Radioprotectin-1 (RP-1), chemically named 5-chloro-2-(N-(4-(1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)butyl)sulfonyl)benzoic acid, is a synthetic non-lipid agonist that specifically targets lysophosphatidic acid receptor 2 (LPA2). It was developed from a series of sulfonylbenzoic acid (SBA) compounds through structure-activity relationship (SAR) studies. [1] The mechanism of RP-1’s radioprotection and radiorelief is believed to be the specific activation of LPA2 GPCRs, which are upregulated in cells, especially Lgr5⁺ intestinal stem cells, after radiation exposure. LPA2 activation is known to recruit anti-apoptotic signaling complexes and participate in enhanced DNA damage repair, as evidenced by the reduction of γ-H2AX foci. [1] RP-1 has shown efficacy in protecting the body from radiation damage in both the hematopoietic system and the gastrointestinal tract, making it a potential candidate drug for alleviating acute radiation syndrome (ARS). It is particularly effective in protecting the Lgr5+ intestinal stem cell population, which is crucial for epithelial regeneration. [1] The study noted that the efficacy of RP-1 varies significantly across species, with a much higher affinity for human LPA2 homologs (EC50 = 5 pM) than for mouse homologs (EC50 = 25 nM) despite high sequence homology. This highlights the importance of cross-species testing of ligands. [1] In addition to radiation protection, the paper also noted that, based on the known biological properties of the LPA2 receptor, substances such as RP-1 that activate LPA2 may have therapeutic potential in other diseases, such as preventing nonsteroidal anti-inflammatory drug-induced gastric erosion, alleviating asthma, and suppressing secretory diarrhea. [1]

C23H19CLN2O6S

486.924763917923

486.065

1622006-09-0

77461257

White to light yellow solid powder

3.7

2

7

8

33

845

0

ClC1C=CC(=C(C(=O)O)C=1)S(NCCCCN1C(C2=CC=CC3=CC=CC(C1=O)=C23)=O)(=O)=O

OVJUYQCJIFWMBI-UHFFFAOYSA-N

InChI=1S/C23H19ClN2O6S/c24-15-9-10-19(18(13-15)23(29)30)33(31,32)25-11-1-2-12-26-21(27)16-7-3-5-14-6-4-8-17(20(14)16)22(26)28/h3-10,13,25H,1-2,11-12H2,(H,29,30)

5-chloro-2-[4-(1,3-dioxobenzo[de]isoquinolin-2-yl)butylsulfamoyl]benzoic 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 : ~125 mg/mL (~256.72 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.27 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 (4.27 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 (4.27 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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 (Please use freshly prepared in vivo formulations for optimal results.)