Sodium Channels (Naᵥ1.3, Naᵥ1.7, Naᵥ1.8): Ralfinamide blocks Na⁺ currents in depolarized, hyperexcitable rat primary afferent neurons. Affinity for inactivated channels: Ki = 5-10 μM. [1]
Calcium Channels (Caᵥ2.2): Ralfinamide inhibits Ca⁺⁺ currents in DRG neurons, including Caᵥ2.2, with similar potency to Na⁺ channel blockade (Ki = 10 μM, unpublished data cited). [1]
NMDA Receptors (polyamine binding site): Ralfinamide antagonizes NMDA receptors, inhibiting NMDA-evoked currents with IC50 = 8 μM. [1]

Substance P Release Inhibition: Ralfinamide inhibits substance P release from spinal cord synaptosomes in vitro. [1]
Neuroprotection: Ralfinamide protects cultured cortical neurons from NMDA-induced excitotoxicity. [1]

Neuropathic pain is suppressed by ralfinamide (80 mg/kg; oral; twice daily; 7 days) used as a preoperative therapy [1].

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Spontaneous Neuropathic Pain (Autotomy Model): In rats with hindpaw denervation (sciatic and saphenous nerve transection), chronic oral ralfinamide administration significantly suppressed autotomy behavior (self-mutilation of denervated paw), a model of spontaneous neuropathic pain. [1]
Preemptive Analgesic Effect: Preoperative ralfinamide (80 mg/kg, bid for 7 days before nerve section) significantly delayed autotomy onset day (P = 0.009) and suppressed autotomy scores during the drug washout period (days 43-63, P = 0.01), indicating long-lasting analgesia. [1]
Postoperative Analgesic Effect: Postoperative ralfinamide (30 or 60 mg/kg, bid for 42 days after nerve section) significantly suppressed autotomy scores during treatment (AUC d0-42: P = 0.02 for 30 mg/kg, P = 0.01 for 60 mg/kg) and delayed onset (P = 0.006 for 60 mg/kg). The higher dose (60 mg/kg) also significantly reduced the incidence of high autotomy scores on day 42 (P = 0.02). [1]
Combined Pre- and Postoperative Effect: Combined treatment (80 mg/kg pre-op + 30 or 60 mg/kg post-op) significantly suppressed autotomy scores during treatment (P = 0.008 for 30 mg/kg, P = 0.01 for 60 mg/kg) and reduced high autotomy incidence on day 42 (P = 0.007 for 30 mg/kg, P = 0.001 for 60 mg/kg). However, the onset delay seen with single treatments was lost in combined groups. [1]
Long-lasting Analgesia: All ralfinamide treatment groups (pre-op only, post-op only, combined) maintained significant suppression of autotomy scores after treatment cessation (days 43-63), with P values ranging from 0.001 to 0.02, and significantly reduced incidence of high autotomy on day 63 (P = 0.007 to 0.04). [1]
Optimal Regimen: The best overall analgesic effect was achieved with combined pre- and postoperative treatment at the higher postoperative dose (R/R2: 80 mg/kg pre-op + 60 mg/kg post-op), showing suppressed scores during treatment, lowest incidence of high autotomy on day 42 (P = 0.001), and retained suppression after cessation. [1]
Lack of Sedation: At the doses used (30-80 mg/kg, bid), ralfinamide produced no signs of sedation. Rats behaved normally, groomed, and gained weight at normal rates. The higher dose (80 mg/kg) is <13% of the dose affecting rat motor performance in the Rotarod test (TD50 = 470 mg/kg, PO). In mice, oral doses up to 60 mg/kg showed no significant differences in ambulatory or stereotypic motor activity in open-field tests compared to vehicle. [1]

NMDA-evoked currents were studied in cultured cortical neurons to assess the neuroprotective properties of ralfinamide. The drug inhibited these currents with an IC50 of 8 µM and protected neurons from NMDA-induced excitotoxicity [2].
Na⁺ currents were studied in rat primary afferent neurons. Ralfinamide blocked these currents in depolarized, hyperexcitable neurons at concentrations of 10-20 µM. The affinity for inactivated channels was determined to be Ki = 5-10 µM [2].
Ca⁺⁺ currents, including those through CaV2.2 channels, were studied in dorsal root ganglion neurons. Ralfinamide inhibited these currents with a Ki of approximately 10 µM [2].
Substance P release was studied using spinal cord synaptosomes. Ralfinamide inhibited this release in vitro [2].

Animal/Disease Models: 81 adult male SD (SD (Sprague-Dawley)) rats (260-460 g) [1]
Doses: 80 mg/kg (7 days before surgery), 30 mg/kg, 60 mg/kg (after surgery) twice a day times; until the 42nd postoperative day.
Experimental Results: Neuropathic pain was suppressed.

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Animals:** Adult male Sprague-Dawley rats (260-460 g, Harlan, Canada), housed singly to prevent social interactions from affecting autotomy. Diet did not contain casein (known to affect autotomy). Light/dark cycle: lights on 07:00-19:00. [1]
* **Surgery (Neuroma Model):** Rats were deeply anesthetized with isoflurane. Under aseptic conditions, the sciatic nerve was exposed unilaterally, tightly ligated with 5-0 silk just proximal to the trifurcation, and cut 1 mm distal to the ligature. About 3 mm of the tibial, sural, and common peroneal tributaries were excised to prevent regeneration. The saphenous nerve was then exposed medial to the knee, transected, and a 2 mm segment removed to prevent regeneration. Wounds were closed in layers with 4-0 silk and Michel clips. Antibiotic powder and crystalline penicillin (5,000 U, i.m.) were administered. [1]
* **Drug Administration:** Ralfinamide or vehicle (distilled water, 1 mL) was administered by oral gavage twice daily (bid), between 9:00-11:00 and 17:00-19:00. Preoperative treatment: 80 mg/kg for 7 days before surgery. Postoperative treatment: 30 or 60 mg/kg for 42 days after surgery. Groups: NV/V (no pre-op treatment/vehicle pre-op + vehicle post-op), R/V (80 mg/kg pre-op + vehicle post-op), V/R1 (vehicle pre-op + 30 mg/kg post-op), V/R2 (vehicle pre-op + 60 mg/kg post-op), R/R1 (80 mg/kg pre-op + 30 mg/kg post-op), R/R2 (80 mg/kg pre-op + 60 mg/kg post-op). [1]
* **Behavioral Testing (Autotomy):** From surgery day until day 63, rats were observed daily by a blinded experimenter. Autotomy was scored on an 11-point scale: 1 point for removal of at least two nails, +1 point for every half toe injured (max 11 = all five nails and toes injured). Rats reaching score 11 were euthanized by CO₂ inhalation, and their scores retained for analysis. Endpoint parameters: Autotomy Onset Day (AOD, first sign of injury to at least two nails), Area Under the Curve (AUC) of scores for treatment period (d0-42) and washout period (d43-63), and incidence of high autotomy (scores 9-11) on d42 and d63. [1]
* **Denervation Verification:** On d42, sensory status was tested by pinching skin with toothed forceps on plantar/dorsal aspects of both paws. Lack of response on denervated paw confirmed complete denervation. After euthanasia, nerves were exposed and examined under stereomicroscope (25×) to confirm cut ends. [1]
* **Data Analysis:** Group means of AOD and AUC compared with ANOVA + Dunnett's post hoc or t-test (two-tailed). Incidence of high autotomy compared with Fisher exact test. False discovery rate used to control type I error (q < 0.05). P ≤ 0.05 considered significant. [1]

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Animals: Adult male Sprague-Dawley rats (260-460 g, Harlan, Canada), housed singly to prevent social interactions from affecting autotomy. Diet did not contain casein (known to affect autotomy). Light/dark cycle: lights on 07:00-19:00. [1]
Surgery (Neuroma Model): Rats were deeply anesthetized with isoflurane. Under aseptic conditions, the sciatic nerve was exposed unilaterally, tightly ligated with 5-0 silk just proximal to the trifurcation, and cut 1 mm distal to the ligature. About 3 mm of the tibial, sural, and common peroneal tributaries were excised to prevent regeneration. The saphenous nerve was then exposed medial to the knee, transected, and a 2 mm segment removed to prevent regeneration. Wounds were closed in layers with 4-0 silk and Michel clips. Antibiotic powder and crystalline penicillin (5,000 U, i.m.) were administered. [1]
Drug Administration: Ralfinamide or vehicle (distilled water, 1 mL) was administered by oral gavage twice daily (bid), between 9:00-11:00 and 17:00-19:00. Preoperative treatment: 80 mg/kg for 7 days before surgery. Postoperative treatment: 30 or 60 mg/kg for 42 days after surgery. Groups: NV/V (no pre-op treatment/vehicle pre-op + vehicle post-op), R/V (80 mg/kg pre-op + vehicle post-op), V/R1 (vehicle pre-op + 30 mg/kg post-op), V/R2 (vehicle pre-op + 60 mg/kg post-op), R/R1 (80 mg/kg pre-op + 30 mg/kg post-op), R/R2 (80 mg/kg pre-op + 60 mg/kg post-op). [1]
Behavioral Testing (Autotomy): From surgery day until day 63, rats were observed daily by a blinded experimenter. Autotomy was scored on an 11-point scale: 1 point for removal of at least two nails, +1 point for every half toe injured (max 11 = all five nails and toes injured). Rats reaching score 11 were euthanized by CO₂ inhalation, and their scores retained for analysis. Endpoint parameters: Autotomy Onset Day (AOD, first sign of injury to at least two nails), Area Under the Curve (AUC) of scores for treatment period (d0-42) and washout period (d43-63), and incidence of high autotomy (scores 9-11) on d42 and d63. [1]
Denervation Verification: On d42, sensory status was tested by pinching skin with toothed forceps on plantar/dorsal aspects of both paws. Lack of response on denervated paw confirmed complete denervation. After euthanasia, nerves were exposed and examined under stereomicroscope (25×) to confirm cut ends. [1]
Data Analysis: Group means of AOD and AUC compared with ANOVA + Dunnett's post hoc or t-test (two-tailed). Incidence of high autotomy compared with Fisher exact test. False discovery rate used to control type I error (q < 0.05). P ≤ 0.05 considered significant. [1]

Human PK: In healthy male volunteers (n=6/dose), single oral doses of 40, 80, 160, 320 mg ralfinamide showed good linearity in plasma levels. Cmax and AUC increased proportionally with dose (Fig. 2A,B). Blood samples collected at baseline, 20, 40 min, 1, 2, 4, 6, 8, 12, 16, 24, 36, 48 h post-dose. Plasma analyzed by validated LC-MS/MS (LOQ 20 ng/mL). [1]
Rat PK: In male SD rats (n=3/dose), single oral doses of 30, 100, 200 mg/kg ralfinamide showed good linearity in plasma levels. Cmax and AUC increased proportionally with dose (Fig. 2A,B). Blood samples collected from caudal vein at 15, 30, 60 min, 2, 4, 6, 8, 12 h post-dose. Plasma analyzed by LC-MS/MS. Pharmacokinetic parameters processed with WinNonLin 5.1. [1]
Plasma Levels at Study Doses: The doses used in the behavioral study (30, 60, 80 mg/kg) produce plasma levels similar to those achieved in clinical trials with human doses of 40-320 mg. Peak plasma concentrations in rats after single administration at these doses range from 2-5 μM, close to the Ki for channel blockade (5-10 μM) and IC50 for NMDA antagonism (8 μM). [1]
Half-Life: Ralfinamide has a half-life of approximately 2.5 hours at the doses used in this study. [1]
Brain Penetration: Ralfinamide has good brain penetration ability. At an oral dose of 30 mg/kg, rat brain concentration reaches approximately 15 μM, which is within the range that blocks its channel targets. [1]

Safety Margin: The higher dose used in this study (80 mg/kg) is <13% of the dose affecting rat motor performance in the Rotarod test (TD50 = 470 mg/kg, PO). [1]
Mortality: In a preliminary experiment, no deaths occurred in SD rats administered 60 mg/kg ralfinamide bid for 42 days. [1]
Lack of Sedation: Ralfinamide-treated animals showed no signs of sedation, behaved normally, groomed, and gained weight at normal rates. In mice, oral doses up to 60 mg/kg showed no significant differences in ambulatory or stereotypic motor activity in open-field tests compared to vehicle (n=8-10/group). [1]
Cardiotoxicity at High Doses: In electrophysiological studies, increasing systemic ralfinamide above the dose that inhibits spontaneous neuroma firing killed animals by cardiac arrest before blocking action potential initiation or nerve conduction. [1]

[1]. Ralfinamide administered orally before hindpaw neurectomy or postoperatively provided long-lasting suppression of spontaneous neuropathic pain-related behavior in the rat. Pain. 2008 Oct 15;139(2):293-305.

[2]. Effects of ralfinamide in models of nerve injury and chemotherapy-induced neuropathic pain. Eur J Pharmacol. 2018 Mar 15;823:27-34.

Drug Indication
This drug has been studied for the treatment of pain (acute or chronic).

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Background: Ralfinamide (formerly NW-1029) is an α-aminoamide derivative with multiple sites of action for suppressing chronic pain. It was in clinical development for neuropathic pain at the time of this publication. [1]
Mechanism of Action Summary: Ralfinamide has a broad mechanism including: (1) blocking Na⁺ currents in primary afferent neurons (Naᵥ1.3, 1.7, 1.8 subtypes); (2) blocking Ca⁺⁺ currents (including Caᵥ2.2); (3) antagonizing NMDA receptors at the polyamine binding site; and (4) inhibiting substance P release from spinal cord synaptosomes. [1]
Preemptive Analgesia Concept: The study demonstrates that preoperative ralfinamide can prevent or reduce chronic pain development, presumably by blunting "injury discharge" (the train of impulses from freshly injured nerves) or protecting CNS neurons from excitotoxicity. This is the first demonstration of ralfinamide's analgesic effect on spontaneous (non-evoked) chronic pain. [1]
Clinical Relevance: The doses producing analgesia in rats achieved plasma levels similar to those causing analgesia in human pain patients (receiving 40-320 mg ralfinamide twice daily in clinical trials). [1]
Long-lasting Effect: All treatment regimens produced analgesia that outlasted drug administration by at least 3 weeks, suggesting disease-modifying or neuroprotective effects rather than merely symptomatic relief. [1]

C17H19FN2O2

302.3494

302.143

133865-88-0

Ralfinamide mesylate;202825-45-4

5745207

White to off-white solid powder

1.189

479ºC at 760mmHg

243.5ºC

2.46E-09mmHg at 25°C

1.569

3.415

2

4

7

22

346

1

C[C@@H](C(=O)N)NCC1=CC=C(C=C1)OCC2=CC=CC=C2F

BHJIBOFHEFDSAU-LBPRGKRZSA-N

InChI=1S/C17H19FN2O2/c1-12(17(19)21)20-10-13-6-8-15(9-7-13)22-11-14-4-2-3-5-16(14)18/h2-9,12,20H,10-11H2,1H3,(H2,19,21)/t12-/m0/s1

(2S)-2-[[4-[(2-fluorophenyl)methoxy]phenyl]methylamino]propanamide

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 (~413.44 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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