hNav1.7 (IC50 = 907 nM); mNav1.7 (IC50 = 387 nM)

Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses [1].

AMG8379 (30-100 mg/kg; oral) reduces capsaicin-induced nociceptive responses [1]. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels[1].

AMG8379 and AMG8380 Antagonist Activity [1]
AMG8379 and AMG8380 represent a pair of atropisomeric quinolone sulfonamides discovered in our efforts to identify an isoform-selective NaV1.7 inhibitor (Fig. 1A). Discovery and optimization of drug-like properties for this chemical class is described in a separate report (Graceffa et al., 2017). AMG8379 potently inhibited recombinant human and mouse, but not rat, NaV1.7 channels expressed in heterologous HEK293 cells when evaluated by automated patch-clamp electrophysiology on a PatchXpress...

Animal/Disease Models: C57Bl/6 male mice [1]
Doses: 100 mg/kg
Route of Administration: Oral
Experimental Results: Failed to demonstrate statistically significant effects on scratching behavior.

[1]. Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel NaV1.7. J Pharmacol Exp Ther. 2017 Jul;362(1):146-160.

Chronic pain represents a critical unmet medical need that afflicts over 100 million Americans with an economic cost over $500 billion (Institute of Medicine, 2011; Holmes, 2016). Despite the multi-billion dollar pain market in the major markets of the United States, Europe, and Japan, current available therapies lack robust efficacy, carry significant abuse potential, and/or suffer from low tolerability and safety; numbers needed to treat, corresponding to the number of individuals receiving a treatment for one individual to achieve 50% or greater pain relief, range between 4 and 10 for current therapies (Nightingale, 2012; Finnerup et al., 2015). Between 2009 and 2014, 33 potential drugs for neuropathic pain, a form of chronic pain arising from damage to the somatosensory system that is particularly recalcitrant to treatment, were discontinued in clinical trials, highlighting the urgent need to both identify new targets as well as develop new therapeutic agents for treatment of pain indications (Knezevic et al., 2015).
NaV1.7 is a tetrodotoxin (TTX)-sensitive voltage-gated sodium ion channel that regulates action potential firing in nociceptor neurons within the peripheral nervous system (Klugbauer et al., 1995; Cummins et al., 1998; Black et al., 2012; Dib-Hajj et al., 2013). Sodium influx through NaV1.7 generates ramp currents that can boost subthreshold depolarizing stimuli toward the threshold required to trigger sensory neuron action potentials, and NaV1.7 also contributes to the upstroke of nociceptor action potentials (Cummins et al., 1998; Rush et al., 2007; Alexandrou et al., 2016). In addition to its expression profile and role in action potential initiation, NaV1.7 represents a particularly compelling target for the development of chronic pain therapies due to its robust human genetic validation. Loss-of-function mutations lead to congenital insensitivity to pain (Cox et al., 2006; Goldberg et al., 2007), whereas gain-of-function mutations lead to pain in primary erythromelalgia, paroxysmal extreme pain disorder, and small-fiber neuropathy (Yang et al., 2004; Fertleman et al., 2006; Faber et al., 2012). Furthermore, in mice, genetic knockout of NaV1.7 in specific neuronal populations mitigates pain behavior after both inflammatory and nerve injury insults (Nassar et al., 2004; Minett et al., 2012), whereas global knockout of NaV1.7 from birth recapitulates the human phenotype of congenital insensitivity to pain (Gingras et al., 2014).
NaV1.7 has been the intense focus of research activities to identify inhibitors for therapeutic development for chronic pain (Bagal et al., 2014; Sun et al., 2014a; de Lera Ruiz and Kraus, 2015; Emery et al., 2016; Vetter et al., 2017). Among these endeavors, sulfonamide-based small molecule antagonists have emerged as potent and selective compounds that block NaV1.7 with greatly reduced potency against homologous sodium channels, including NaV1.5, expressed in the heart. Mechanistically, block is achieved by engaging a novel binding pocket located within the fourth voltage-sensor domain and stabilizing NaV1.7 in an inactivated conformation, thereby preventing channels from efficiently recycling to the resting/closed state and reducing availability for subsequent opening events (McCormack et al., 2013; Sun et al., 2014b; Ahuja et al., 2015; Alexandrou et al., 2016; Focken et al., 2016; Theile et al., 2016). Poor inhibition of rat NaV1.7 channels by this chemotype is attributed to amino acid sequence differences in this region (McCormack et al., 2013; Alexandrou et al., 2016; Focken et al., 2016). Specific interaction with the fourth voltage-sensor domain differentiates the sulfonamide binding site from previously described NaV binding sites for toxin-derived peptides, local anesthetics, and TTX (de Lera Ruiz and Kraus, 2015). Here, we describe an extensive in vitro, ex vivo, and in vivo preclinical pharmacological characterization of the novel sulfonamide NaV1.7 antagonist AMG8379 and its significantly less active enantiomer AMG8380, which serves as a negative control. AMG8379 represents a potent and selective NaV1.7 inhibitor that blocks action potential firing in peripheral nerve fibers and displays efficacy in translatable mouse models of both itch and pain, whereas the less active enantiomer AMG8380 is devoid of these biologic effects at comparable plasma concentrations.[1]

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AMG8379 is a potent and selective small molecule sulfonamide antagonist of human and mouse, but not rat, recombinant NaV1.7 channels in heterologous cells and native NaV1.7 channels in mouse DRG neurons. AMG8379 inhibited mechanically and thermally induced C-fiber action potential firing in a mouse skin-nerve preparation and blocked multiple NaV1.7-dependent behaviors, including histamine-induced scratching, capsaicin-induced licking, and UVB-induced thermal hyperalgesia. The significantly less...[1]

C25H16CLF2N3O5S

543.93

543.046

C, 55.21; H, 2.97; Cl, 6.52; F, 6.99; N, 7.73; O, 14.71; S, 5.89

1642112-32-0

AMG8379;1642112-31-9;(Rac)-AMG8379;1641574-26-6

117737142

Typically exists as solid at room temperature

4.8

1

9

6

37

969

0

COC1=C(C=C(C(=C1)C2=CC(=CC(=C2)Cl)F)F)N3C4=C(C=CC3=O)C=C(C=C4)S(=O)(=O)NC5=NOC=C5

HXQNEKJQBGXFAG-UHFFFAOYSA-N

InChI=1S/C25H16ClF2N3O5S/c1-35-23-12-19(15-8-16(26)11-17(27)9-15)20(28)13-22(23)31-21-4-3-18(10-14(21)2-5-25(31)32)37(33,34)30-24-6-7-36-29-24/h2-13H,1H3,(H,29,30)

1-[4-(3-chloro-5-fluorophenyl)-5-fluoro-2-methoxyphenyl]-N-(1,2-oxazol-3-yl)-2-oxoquinoline-6-sulfonamide

AMG-8380; AMG8380; (Rac)-AMG8379; 1642112-31-9; 1641574-26-6; 1-[4-(3-chloro-5-fluorophenyl)-5-fluoro-2-methoxyphenyl]-N-(1,2-oxazol-3-yl)-2-oxoquinoline-6-sulfonamide; 1642112-32-0; AMG 8380; AMG8,380

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)

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

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.)