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R243 (R-243) is a novel and potent inhibitor of CCR8 signaling with antinociceptive and anti-inflammatory activity. It inhibits CCR8 signaling and chemotaxis, inhibits CCL1-induced Ca2+ flux and CCL1-driven peritoneal macrophages aggregation. It also reduced secretion of cytokines such as TNF-α, IL-6, and most strikingly IL-10 from WT PMφ (peritoneal macrophages), but not BMMφ (bone marrow-derived macrophages).
| Targets |
CCR8 (chemokine receptor 8). [1]
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| ln Vitro |
CCL1-induced Ca2+ flow and CCL1-driven peritoneal macrophage accumulation are antagonistic to CCR8 in R243 [1]. R243 reduces the amount of TNF-α, IL-6, and IL-10 that wild-type peritoneal macrophages (WT PMφ) secrete [1]. After lipopolysaccharide (LPS) treatment, R243-treated WT PMφ demonstrated reduction of NF-κB signaling and c-jun N-terminal kinase activity compared to WT PMφ [1].
R243 inhibited CCL1-induced Ca2+ flux in CCR8-expressing CHO cells at concentrations ≥1 μM, with significant inhibition observed. [1] R243 (up to 5 μM) did not affect cell viability in CCR8-CHO cells after 18 hours of culture, and no cytotoxicity was observed in mouse PMφ treated with 5 μM R243. [1] In the CIMA (chemokine-induced macrophage aggregation) assay, R243 suppressed CCL1-induced cell aggregation between mouse mesothelial cells and PMφ in a dose-dependent manner, with suppression observed at concentrations as low as 0.2 μM; at 5 μM, inhibition was comparable to that seen in CCR8-deficient cells. [1] R243 inhibited chemotaxis of PMφ and bone marrow-derived macrophages (BMMφ) induced by CCL1 and CCL2. It also inhibited CCL3-induced chemotaxis in PMφ at higher concentrations, and showed a trend toward inhibition of CCL5-induced chemotaxis in PMφ but did not inhibit CCL5-induced chemotaxis in BMMφ. [1] R243 treatment (5 μM) of WT PMφ reduced LPS-induced cytokine secretion (TNF-α, IL-6, and particularly IL-10) similar to CCR8 deficiency, but had relatively low effect on BMMφ. R243 did not affect Pam3CSK4-induced IL-10 production, unlike CCR8 deficiency. In CCR8-deficient PMφ, R243 showed no additional effect on cytokine production. Synthesized R243 with the same structure demonstrated identical suppressive effects in CIMA and LPS-triggered cytokine production assays. [1] R243 treatment did not alter the expression level of CCR8 or the intracellular localization of LPS and TLR4, and did not affect internalization of the LPS-TLR4 complex. [1] R243 treatment suppressed LPS-induced phosphorylation of JNK and IκBα, and showed a trend of decreased phosphorylation of c-Jun, but did not suppress phosphorylation of ERK, Akt, or p38 in PMφ. [1] |
| ln Vivo |
R243 (0.1-1 mg/kg; intraperitoneal injection; once; male Swiss mice) therapy decreased CCL1-induced analgesia in a dose-dependent manner [2].
R243 (0.1–1 mg/kg, i.p.) dose-dependently inhibited the thermal analgesia induced by subcutaneous administration of CCL1 (10 μg/kg) in mice, as measured by the unilateral hot plate test. Significant blockade was observed at 0.3 and 1 mg/kg. The administration of 1 mg/kg R243 alone did not alter basal withdrawal latencies. In contrast, intrathecal injection of 10 μg of R243 did not modify the analgesic effect evoked by systemic CCL1. These results demonstrate that the analgesic effect of CCL1 is mediated by peripheral CCR8 receptors rather than spinal CCR8 receptors. [2] |
| Cell Assay |
For Ca2+ influx assay, mouse CCR8-DsRed-expressing CHO cells were stimulated with 50 ng/mL of CCL1, and Ca2+ flux was measured in 96-well plates using a FLIPR CA4 Assay Kit and a microplate reader. R243 inhibition was assessed by comparing fluorescence signal values to cells treated with CCL1 + 0.1% DMSO (vehicle). [1]
For cell viability, CCR8-CHO cells were cultured with 1 or 5 μM R243 for 18 hours, and the number of living cells was measured by WST assay. Mouse PMφ were cultured for 24 hours with indicated concentrations of R243, harvested with trypsin/EDTA, and living cells were counted using trypan-blue exclusion. [1] For CIMA assay, mouse mesothelial cells were cultured in a 24-well dish until confluent. Naive mouse PMφ were added and incubated with CCL1 (5 ng/mL) with or without R243 for 24 hours at 37°C. Cell aggregate formation was quantified as aggregation area using images captured by a microscope with a CCD camera and analyzed with NIH ImageJ software. [1] For chemotaxis assay, fluorescently labeled PMφ and BMMφ were stimulated with 10 ng/mL of CCL1, CCL2, CCL3, or CCL5, with or without R243 at indicated concentrations. Migrated cells were quantified by fluorescence level and shown as percent of chemokine-induced migration relative to random migration (=100%). [1] For cytokine production, PMφ and BMMφ from WT or CCR8-deficient mice were stimulated with TLR ligands (e.g., LPS 100 ng/mL) in RPMI1640 medium with 1% FBS for 24 hours, with or without R243 at indicated concentrations or 0.1% DMSO as vehicle. Culture supernatant was used for cytokine ELISA. For some experiments, PMφ were stimulated with LPS for 24 hours in the presence of signaling inhibitors (SN50, U0126, SB203580, SP600125). [1]For phosphoprotein detection, WT PMφ, CCR8-deficient PMφ, or WT PMφ treated with R243 were stimulated with LPS (100 ng/mL) for indicated times, and each phosphoprotein was quantified using a multiplex phosphoprotein detection assay. [1] |
| Animal Protocol |
Animal/Disease Models: Male Swiss mice (7-9 weeks old) were injected with CCL1[2]
Doses: 0.1 mg/kg, 0.3mg/kg, 1 mg/kg Route of Administration: intraperitoneal (ip) injection; Experimental Results:CCL1 (10 μg/kg ; 1 h; sc)-induced analgesia was dose-dependently inhibited. R243 was dissolved in DMSO at 10 mg/ml and further diluted in distilled water to a maximal final DMSO concentration of 10%. For systemic administration, R243 was injected intraperitoneally (i.p.) at doses of 0.1, 0.3, or 1 mg/kg in a volume of 10 ml/kg, 30 minutes before nociceptive testing. For spinal administration, R243 was injected intrathecally (i.t.) at a dose of 10 μg in a volume of 5 μl at the level of L5-L6 in mice anesthetized with isoflurane (3%), also 30 minutes before testing. Male Swiss mice (7–9 weeks old) were used. The unilateral hot plate test (49°C) was employed to measure thermal withdrawal latencies. [2] |
| Toxicity/Toxicokinetics |
R243 at ≤5 μM did not affect cell viability in CCR8-CHO cells after 18 hours of culture, and absence of cytotoxicity in mouse PMφ with R243 treatment at 5 μM was confirmed. [1]
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| References |
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| Additional Infomation |
R243 is described as a selective CCR8 antagonist. Its ability to block CCL1-induced analgesia when administered systemically, but not spinally, supports the conclusion that systemic CCL1 acts through peripheral CCR8 receptors to produce its antinociceptive effect. The compound was used at doses that did not affect basal nociceptive thresholds in mice. [2]
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| Molecular Formula |
C21H27NO4
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|---|---|
| Molecular Weight |
357.443386316299
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| Exact Mass |
357.194
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| Elemental Analysis |
C, 70.56; H, 7.61; N, 3.92; O, 17.90
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| CAS # |
688352-84-3
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| PubChem CID |
2962888
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| Appearance |
White to off-white solid powder
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| LogP |
3.9
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
499
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C2CC3CC1CC(C2)(C3)OCCN4CC5=CC6=C(C=C5OC4)OCO6
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| InChi Key |
JJMLDSFDOODCIR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H27NO4/c1(2-26-21-8-14-3-15(9-21)5-16(4-14)10-21)22-11-17-6-19-20(25-13-24-19)7-18(17)23-12-22/h6-7,14-16H,1-5,8-13H2
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| Chemical Name |
7,8-Dihydro-7-[2-(tricyclo[3.3.1.1(3,7)]dec-1-yloxy)ethyl]-6H-1,3-dioxolo[4,5-g][1,3]benzoxazine
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| Synonyms |
R-243 R243 R 243
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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 : ~125 mg/mL (~349.71 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.82 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.82 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7977 mL | 13.9884 mL | 27.9767 mL | |
| 5 mM | 0.5595 mL | 2.7977 mL | 5.5953 mL | |
| 10 mM | 0.2798 mL | 1.3988 mL | 2.7977 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05670483 | Recruiting | Device: Use Of APRV mode of Ventilation
Device: Use of conventional Synchronized Intermittent Mandatory Ventilation (SIMV) volume control mode |
Morbid Obesity Pulmonary Atelectasis Pulmonary Complication |
Ain Shams University | 2022-12-03 | Not Applicable |
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