| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 50mg | |||
| Other Sizes |
| Targets |
NPFF1 receptor antagonist (IC50 = 0.41 μM, against 1 μM NPFF-induced inhibition of forskolin-stimulated cAMP accumulation in HEK-293 cells expressing NPFF1 receptor) [2]
NPFF2 receptor antagonist (IC50 = 0.41 μM, against 1 μM NPFF-induced inhibition of forskolin-stimulated cAMP accumulation in HEK-293 cells expressing NPFF2 receptor) [2] |
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| ln Vitro |
In Neuro 2A cells, RF9 (10 μM) pretreatment totally inhibited NPFF-induced neurite development [2].
In vitro, RF9 dose-dependently reversed the inhibitory effect of 1 μM NPFF on forskolin-stimulated cAMP accumulation in HEK-293 cells stably expressing the NPFF1 receptor, with an IC50 value of 0.41 (0.24-0.69) μM. [2] In HEK-293 cells stably expressing the NPFF2 receptor, RF9 also reversed the effect of 1 μM NPFF on forskolin-stimulated cAMP accumulation, with an IC50 value of 0.41 (0.24-0.69) μM. [2] In Neuro 2A cells, which endogenously express the NPFF2 receptor, treatment with 1 μM NPFF significantly induced neurite outgrowth (24.05 ± 1.19% of cells with neurites), and this effect was completely blocked by pretreatment with 10 μM RF9 (12.86 ± 0.77% of cells with neurites, P < 0.001). At the same dose, RF9 alone did not alter neurite outgrowth (12.41 ± 0.53% of cells with neurites). [2] |
| ln Vivo |
Heroin-induced delayed hyperalgesia and tolerance are avoided when RF9 (0.1 mg/kg, subcutaneous injection) is administered concurrently with heroin [1]. MAP and heart rate did not alter significantly after receiving RF9 (10 μg) infusion alone. On the other hand, the NPFF-induced increases in heart rate and MAP are dramatically inhibited when RF9 is delivered in conjunction with NPFF [1].
In the mouse tail-flick test, supraspinal (i.c.v.) administration of RF9 (10 nmol) alone did not alter tail-flick latency. [2] In the carrageenan-induced inflammatory pain model, spinal (i.t.) pre-administration of RF9 (10 nmol) significantly antagonized the anti-allodynic effects induced by the NPFF2 agonist dNPA, as well as by the branched peptidomimetics EKR and RKE (P < 0.001), but had no effect on morphine-induced anti-allodynia. [2] In antinociceptive tolerance studies, i.c.v. administration of RF9 (20 nmol) 5 min prior to morphine (4 nmol, i.c.v.) once daily for 8 days significantly reversed the development of morphine antinociceptive tolerance from day 6 to day 8 (P < 0.001). However, RF9 did not alter the antinociceptive effects of EKR (20 nmol, i.c.v.) or RKE (20 nmol, i.c.v.) during the testing days. [2] |
| Enzyme Assay |
The in vitro functional activity of RF9 towards NPFF receptors was evaluated using a cAMP accumulation assay in HEK-293A cells stably expressing the NPFF1 or NPFF2 receptor. Cells were cultured in DMEM with 10% FBS and 0.1% antibiotics. After 24 h, cells were incubated with 1 mM IBMX in serum-free medium at 37°C for 10 min. Then, cells were treated with various concentrations of RF9 and 5 μM forskolin, and cultivated at 37°C for 30 min. To evaluate antagonistic properties, the ability of RF9 to reverse the effect of 1 μM NPFF on forskolin-induced cAMP accumulation was measured. Cells were lysed using 0.2 M HCl for 30 min and then neutralized with 10 M NaOH. cAMP levels were determined using a competition protein kinase A (PKA) binding assay. [2]
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| Cell Assay |
Neurite outgrowth assay was performed in Neuro 2A cells. Cells were cultured in DMEM with 10% FBS and 1% antibiotics, and plated onto 12-well plates. After 24 h, cells were incubated with 1 μM NPFF in the absence or presence of 10 μM RF9 in serum-free medium at 37°C for 18 h. Cells with neurites of equal to or longer than two cell body lengths were counted under a phase contrast microscope. For each well, randomly chosen areas containing about 300 cells were scored. [2]
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| Animal Protocol |
Animal/Disease Models: Rat[1].
Doses: 0.1 mg/kg. Route of Administration: 0.3 mg/kg heroin or saline was injected subcutaneously (sc) (sc) 30 minutes before basal nociceptive threshold in rats. Experimental Results: Against delayed heroin-induced hyperalgesia and related tolerance. Animal studies were performed in male Kunming mice. For intracerebroventricular (i.c.v.) administration, drugs were injected into the lateral ventricle at a fixed volume of 4 μl at a constant rate of 10 μl/min, followed by 1 μl saline to flush the catheter. For intrathecal (i.t.) injection, a 25 μl microsyringe was inserted between L5 and L6 vertebrae, and drugs were injected into the subarachnoid space with a volume of 5 μl at a rate of 10 μl/min. For intraperitoneal (i.p.) injection, mice received a total volume of 10 ml/kg. RF9 was dissolved in physiological saline. In the tail-flick test, RF9 (10 nmol, i.c.v.) was administered 5 min prior to agonists. In the carrageenan-induced inflammatory pain model, RF9 (10 nmol, i.t.) was administered 5 min prior to agonists. In tolerance development studies, RF9 (20 nmol, i.c.v.) was treated 5 min before morphine (4 nmol, i.c.v.), EKR (20 nmol, i.c.v.) or RKE (20 nmol, i.c.v.) once a day for 8 days, and antinociceptive effects were measured daily using the tail-flick test. [2] |
| References |
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| Additional Infomation |
RF9 is a dipeptide composed of L-arginine and L-phenylalanine residues, wherein the hydrogen atom on the α-amino nitrogen atom of the arginine residue is replaced by a 1-adamantane carbonyl group. RF9 has been reported as a potent and selective neuropeptide FF (NPFF) receptor antagonist, but recent studies have found it to be an agonist of both the NPFF1R and kisspeptin receptors (KISS1R). It functions similarly to Arg-Ala.
RF9 (CAS#: 876310-60-0) is a potent and selective neuropeptide FF (NPFF) receptor antagonist. It has been reported to prevent opioid-induced hyperalgesia and tolerance. In the present study, RF9 was used as a reference NPFF receptor antagonist to investigate the pharmacological properties of novel branched peptidomimetics EKR and RKE. RF9 completely blocked NPFF2 receptor-mediated neurite outgrowth of Neuro 2A cells, and reversed the development of morphine tolerance. These findings confirm the important role of the NPFF system in opioid-mediated tolerance. [2] |
| Molecular Formula |
C26H38N6O3
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|---|---|
| Molecular Weight |
482.63
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| Exact Mass |
482.3
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| CAS # |
876310-60-0
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| Related CAS # |
RF9 hydrochloride
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| PubChem CID |
53320361
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| Appearance |
White to off-white solid powder
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| LogP |
4.325
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
35
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| Complexity |
777
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1C2CC3CC1CC(C2)(C3)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC4=CC=CC=C4)C(=O)N
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| InChi Key |
UMKHUSRDQFQHAK-RNJMTYCLSA-N
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| InChi Code |
InChI=1S/C26H38N6O3/c27-22(33)21(12-16-5-2-1-3-6-16)31-23(34)20(7-4-8-30-25(28)29)32-24(35)26-13-17-9-18(14-26)11-19(10-17)15-26/h1-3,5-6,17-21H,4,7-15H2,(H2,27,33)(H,31,34)(H,32,35)(H4,28,29,30)/t17?,18?,19?,20-,21-,26?/m0/s1
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| Chemical Name |
N-[(2S)-1-[[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]adamantane-1-carboxamide
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| Synonyms |
RF-9 RF9 RF 9
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 (~259.00 mM)
H2O : ~16.67 mg/mL (~34.54 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.31 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.31 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10 mg/mL (20.72 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0720 mL | 10.3599 mL | 20.7198 mL | |
| 5 mM | 0.4144 mL | 2.0720 mL | 4.1440 mL | |
| 10 mM | 0.2072 mL | 1.0360 mL | 2.0720 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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