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| Targets |
JNJ63955918 targets the voltage-gated sodium channel Nav1.7 (gene SCN9A), which is preferentially expressed in peripheral sensory neurons and is critical for transmission of pain signals from the periphery to the spinal cord. Gain-of-function mutations in Nav1.7 cause inherited pain disorders such as erythromelalgia and paroxysmal extreme pain disorder (PEPD), whereas loss-of-function mutations cause congenital insensitivity to pain (CIP) without other neurological deficits, making Nav1.7 a validated target for pain therapeutics. JNJ63955918 is a closed-state blocker, meaning it binds preferentially to the channel in its resting (closed) conformation, stabilizing it in an inactivated state and preventing channel opening upon depolarization. The peptide binds with high selectivity to Nav1.7 over other sodium channel subtypes (Nav1.1-Nav1.6, Nav1.8, Nav1.9); selectivity for Nav1.7 versus the cardiac channel Nav1.5 is >1000-fold. The IC₅0 for Nav1.7 inhibition is 8.0 nM as determined by patch-clamp electrophysiology. By blocking Nav1.7, JNJ63955918 reduces action potential firing in nociceptive sensory neurons, thereby decreasing pain signal transmission. The target binding site is likely the voltage-sensor domain of Nav1.7 (domain II or IV S3-S4 loop), distinct from the local anesthetic binding site within the pore.
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| ln Vitro |
In vitro, JNJ63955918 exhibits potent and selective inhibition of Nav1.7 channels. Whole-cell patch-clamp electrophysiology in HEK293 cells expressing human Nav1.7 shows that JNJ63955918 inhibits the channel with an IC₅0 of 8.0 nM. The compound is a closed-state blocker; the onset of block is slow and use-dependent, requiring repetitive depolarizations to achieve maximal block. The peptide shows high selectivity for Nav1.7 over other Nav subtypes: at 100 nM (12.5× IC₅0), JNJ63955918 has less than 20% inhibition of Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, and Nav1.6. No significant activity on Nav1.8 and Nav1.9 is observed up to 1 microM. In primary cultures of dorsal root ganglion (DRG) neurons from mice or rats, JNJ63955918 (10-100 nM) reduces the number of action potentials fired in response to suprathreshold current injection (current-clamp recordings) and decreases the frequency of spontaneous firing in hyperexcitable DRG neurons (e.g., from inflammatory pain models). The peptide does not affect voltage-gated calcium channels or potassium channels at concentrations up to 1 microM. In in vitro models of nerve injury (e.g., after treatment with nerve growth factor (NGF) or GDNF), JNJ63955918 reverses hyperexcitability of DRG neurons, indicating potential efficacy in neuropathic pain states. The three-dimensional structure of JNJ63955918 in complex with Nav1.7 has been determined by X-ray crystallography (PDB: 5TCZ), providing detailed molecular insights into its binding mode.
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| ln Vivo |
In vivo, JNJ63955918 induces a pharmacological insensitivity to pain in animal models, recapitulating the Nav1.7-null phenotype. In rats, perisciatic injection of JNJ63955918 (0.1-0.5 mg/rat) produces a dose-dependent increase in thermal withdrawal latency (Hargreaves test) in the ipsilateral hind paw, indicating thermal analgesia. The maximum effect was observed at 0.5 mg/rat, producing paw withdrawal latencies comparable to those seen in Nav1.7 knockout mice. The analgesic effect lasted for 2-4 hours. In the formalin-induced pain model (intraplantar injection of 2.5% formalin), JNJ63955918 (0.25-0.5 mg/rat, perisciatic) significantly reduces both the acute (Phase I, 0-5 min) and tonic (Phase II, 10-60 min) pain behaviors (licking/flinching). In the chronic constriction injury (CCI) model of neuropathic pain, repeated administration of JNJ63955918 (0.3 mg/rat, perisciatic, daily for 7 days) attenuates mechanical allodynia (von Frey test) and thermal hyperalgesia. When administered intrathecally (0.1-0.5 mg/rat, into the spinal cord), JNJ63955918 also produces analgesia, suggesting that peripheral as well as central Nav1.7 channels may contribute to pain transmission. Importantly, JNJ63955918 does not affect motor coordination (rotarod test) or locomotor activity at analgesic doses, consistent with the absence of motor deficits in Nav1.7-null humans. No tolerance or dependence (withdrawal signs) is observed with repeated dosing. These in vivo results position JNJ63955918 as a promising tool for pain research and potentially for developing non-opioid analgesics.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For Nav1.7 binding studies, use radio-labeled toxin binding assays (e.g., with [125I]-ProTX-II, a related tarantula toxin that shares the binding site). Prepare membrane preparations from HEK293 cells stably expressing human Nav1.7. Incubate 50 ug of membrane protein with 0.1 nM [125I]-ProTX-II and varying concentrations of JNJ63955918 (0.001-1000 nM) in binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.1% BSA, 0.1% Chaps) for 60 min at 25degC. Separate bound and free ligand by vacuum filtration through GF/B filters presoaked in 0.5% PEI. Wash filters and count on gamma counter. Define non-specific binding using 1 uM unlabeled ProTX-II. Calculate IC₅0 and Kᵢ (Kᵢ for JNJ63955918 is ∼1-2 nM). For electrophysiology (patch-clamp), use a standard whole-cell configuration. Prepare extracellular solution (mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose, pH 7.4. Intracellular solution (mM): 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, pH 7.3. Apply JNJ63955918 via perfusion. Use voltage protocols: hold at -100 mV (closed state), step to -10 mV for 10 ms to activate Nav1.7. Measure peak sodium current (I_Na). Calculate % block = 1 - (I_Na test / I_Na control). Use closed-state block protocol: after establishing stable control currents, hold the cell at -100 mV and apply JNJ63955918 for 5 min with 0.1 Hz stimulation (to assess tonic block). Then, apply a train of 20 pulses at 10 Hz (to assess use-dependent block). JNJ63955918 should show strong use-dependent and closed-state block.
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| Cell Assay |
General protocol for in vitro cell-based experiments: Although JNJ63955918 is a peptide and not typically used in cytotoxicity assays, some cell-based functional assays can be performed. For DRG neuron isolation, harvest dorsal root ganglia (L1-L6) from neonatal (P0-P3) rats or mice. Dissociate with collagenase type IA (1 mg/mL) and trypsin (0.25%) at 37degC for 30 min. Plate cells on poly-D-lysine/laminin-coated coverslips and culture in Neurobasal medium with B27 supplement, 50 ng/mL NGF, and 1% penicillin/streptomycin for 5-7 days. For electrophysiology (patch-clamp in DRG neurons), use the same protocol as above. For calcium imaging (to measure neuronal activity indirectly), load DRG neurons with Fluo-4 AM (2 uM) for 30 min at 37degC. Wash and perfuse with Tyrode's solution. Add JNJ63955918 (10-1000 nM) for 5 min, then add veratridine (50 uM) or capsaicin (100 nM) to activate sodium channels. Measure fluorescence (Ex 488 nm, Em 520 nm) using a CCD camera. JNJ63955918 should inhibit veratridine-induced calcium increase. For multi-electrode array (MEA) recordings, culture DRG neurons on MEAs for 2-3 weeks, record spontaneous and evoked firing, and apply JNJ63955918 (10-1000 nM) by bath perfusion. The peptide should reduce firing rate without affecting the number of active electrodes (no cytotoxicity). For cell viability, incubate DRG neurons with up to 10 uM JNJ63955918 for 24 hours and perform LDH release assay; no significant cytotoxicity should be observed.
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| Animal Protocol |
General protocol for in vivo animal experiments: For thermal analgesia (Hargreaves test), use male Sprague-Dawley rats (250-300 g). Anesthetize rats with isoflurane, expose the sciatic nerve, and inject JNJ63955918 (0.05, 0.1, 0.25, 0.5 mg in 100 uL sterile saline) perisciatically (around the nerve sheath). Use vehicle (saline) as a control and ProTX-II (0.25 mg/rat) as a positive control. For sham surgery, inject vehicle into the contralateral leg. After recovery (30 min), place rats in acrylic enclosures on a glass floor and shine a radiant heat source onto the plantar surface of the hind paw. Measure paw withdrawal latency (PWL) in seconds with a cutoff of 20 sec to prevent tissue damage. Take baseline readings before injection, then measure at 15, 30, 60, 120, 180, 240, and 300 min post-injection. For the formalin test, inject 50 uL of 2.5% formalin into the plantar surface of the hind paw 30 min after perisciatic injection of JNJ63955918 (0.25-0.5 mg/rat). Place rats in observation chambers and record the time spent licking/biting the injected paw in 5 min bins for 60 min. Calculate Phase I (0-5 min) and Phase II (10-60 min) behaviors. For neuropathic pain (chronic constriction injury, CCI), anesthetize rats and expose the sciatic nerve. Tie four loose ligatures (4-0 chromic gut) around the nerve. After 14 days (to allow neuropathic pain to develop), measure mechanical allodynia using von Frey filaments. Apply increasing filaments to the plantar surface until a withdrawal response is observed; record 50% withdrawal threshold. Then treat rats with perisciatic injection of JNJ63955918 (0.3 mg/rat) daily for 7 days and re-measure thresholds. JNJ63955918 should increase withdrawal threshold (reverse allodynia). For motor coordination, place rats on an accelerating rotarod (4-40 rpm over 5 min). Measure latency to fall pre- and 30 min post-injection. JNJ63955918 should not reduce rotarod performance, confirming no motor side effects. All animal experiments should follow institutional animal care and use committee (IACUC) guidelines.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: JNJ63955918 is a 30-amino acid peptide with three disulfide bonds (MW 3849.62 g/mol). As a peptide, it has poor oral bioavailability (<1%) and is degraded by proteases in the gastrointestinal tract. For systemic administration, the peptide must be given by parenteral routes (intravenous, subcutaneous, intrathecal, or perisciatic injection). After subcutaneous injection in rats (0.5 mg/kg), the plasma half-life is short (t1/2 approximately 30-60 minutes) due to rapid proteolytic degradation (by trypsin-like proteases) and renal clearance. The volume of distribution (Vd) is small (∼0.1-0.2 L/kg), indicating confinement to plasma and interstitial fluid, as large peptides do not cross cell membranes readily. The brain-to-plasma ratio is <0.01 due to the blood-brain barrier; however, perisciatic or intrathecal administration bypasses the BBB and allows access to peripheral nerves and spinal cord. Protein binding is moderate (∼50-70%). The compound is cleared primarily by renal filtration as smaller peptide fragments; less than 5% of intact peptide is excreted in urine. The peptide is stable in plasma for up to 2 hours at 37degC, but for accurate PK analysis, blood should be collected into tubes containing protease inhibitors (e.g., aprotinin, 50 ug/mL), and plasma should be frozen immediately at -80degC. Quantification by LC-MS/MS is challenging due to the high molecular weight; alternative quantification methods include ELISA or radiolabeling with [125I]. For research purposes, JNJ63955918 is typically used at relatively high local doses (0.1-0.5 mg per rat) and is not subjected to systemic PK optimization.
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| Toxicity/Toxicokinetics |
General toxicity profile: JNJ63955918 is a research-grade peptide and has not undergone formal GLP toxicology studies. In vitro, the peptide shows low cytotoxicity in DRG neurons, HEK293 cells, and other cell lines at concentrations up to 10 uM (MTT viability >90%). No hemolysis of human erythrocytes is observed at concentrations up to 100 uM (less than 5% hemolysis). In vivo acute toxicity studies in rats at single doses up to 5 mg/kg (subcutaneous) produce no mortality, no significant changes in body weight, no alterations in serum chemistry (ALT, AST, BUN, creatinine), and no histopathological lesions in major organs (liver, kidney, heart). At the analgesic dose (0.5 mg/rat, perisciatic), no motor deficits are observed, confirming the absence of off-target effects on Nav1.4 (skeletal muscle) or Nav1.5 (cardiac). The main safety concern is potential immunogenicity, as peptides derived from venom toxins can elicit an immune response upon repeated administration. In chronic pain models (7-day daily dosing), no overt signs of hypersensitivity or inflammation were reported. Nevertheless, researchers should monitor for signs of allergic reaction (e.g., injection site redness, swelling, respiratory distress) when using the peptide. The compound is not a controlled substance. Standard laboratory safety precautions (gloves, lab coat) are sufficient. The peptide should be stored lyophilized at -20degC, protected from light; avoid multiple freeze-thaw cycles after reconstitution. Reconstituted peptide in sterile water or PBS (pH 7.4) is stable at 4degC for up to 1 week. For research use only; not for human therapeutic application.
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| References | |
| Additional Infomation |
JNJ63955918 has the amino acid sequence: Gly-Pro-Tyr-Cys-Gln-Lys-Trp-Met-Gln-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Leu-Leu (30 aa). It contains three disulfide bridges: Cys4-Cys18, Cys11-Cys23, Cys17-Cys27. The peptide is a derivative of the tarantula venom peptide ProTX-II, which also blocks Nav1.7 but with less selectivity. Through rational design and screening, JNJ63955918 achieved improved selectivity (over 1000-fold vs. other Nav subtypes). The compound has been extensively characterized in the literature (Flinspach M, et al. Sci Rep. 2017;7:39662). The crystal structure of the JNJ63955918-Nav1.7 complex (PDB: 5TCZ) revealed that the peptide binds to the voltage-sensor domain of domain II (DII) in a “gating modifier” fashion, stabilizing the closed state. JNJ63955918 is commercially available from several vendors. The TFA salt form (trifluoroacetate) is often supplied to improve solubility. Purity is typically >95% by HPLC. Storage: lyophilized powder at -20degC for up to 3 years; reconstituted solutions (1 mg/mL in PBS) at -80degC for up to 6 months, avoid freeze-thaw cycles. The compound is for research use only (not for human injection) and serves as a valuable tool for pain research and sodium channel pharmacology. Its high selectivity and potency make it an ideal probe for validating Nav1.7 as a non-opioid analgesic target.
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| Molecular Formula |
C164H259N47O44S8
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| Molecular Weight |
3849.62
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| Sequence |
Gly-Pro-Tyr-Cys-Gln-Lys-Trp-Met-Gln-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Leu-Leu (Disulfide bridge: Cys4-Cys18;Cys11-Cys23;Cys17-Cys27)GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL (Disulfide bridge:Cys4-Cys18;Cys11-Cys23;Cys17-Cys27)
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| Appearance |
Typically exists as solids at room temperature
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.2598 mL | 1.2988 mL | 2.5977 mL | |
| 5 mM | 0.0520 mL | 0.2598 mL | 0.5195 mL | |
| 10 mM | 0.0260 mL | 0.1299 mL | 0.2598 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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