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 is a significant unmet medical need affecting over 100 million Americans and costing over $500 billion in revenue (Institute of Medicine, 2011; Holmes, 2016). Despite multi-billion dollar markets for pain management in major markets such as the U.S., Europe, and Japan, currently available therapies are poorly effective, carry significant risks of misuse, and/or have low tolerability and safety; the number of patients required for treatment (i.e., the number of patients needed to achieve 50% or greater pain relief) ranges from 4 to 10 (Nightingale, 2012; Finnerup et al., 2015). Between 2009 and 2014, 33 potential drugs for treating neuropathic pain (a chronic pain caused by damage to the somatosensory system, which is particularly difficult to treat) were discontinued in clinical trials, highlighting the urgent need to identify new targets and develop new therapeutic agents to treat pain (Knezevic et al., 2015). NaV1.7 is a voltage-gated sodium channel sensitive to tetrodotoxin (TTX) that modulates action potential firing in nociceptor neurons of the peripheral nervous system (Klugbauer et al., 1995; Cummins et al., 1998; Black et al., 2012; Dib-Hajj et al., 2013). Sodium ion influx through the NaV1.7 channel generates a ramp current that enhances threshold depolarization stimuli, bringing them to the threshold required to trigger action potentials in sensory neurons. Furthermore, NaV1.7 is involved in the rising phase 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 is also a highly attractive target for developing therapies for chronic pain due to its well-validated human genetics. Loss-of-function mutations result in congenital analgesia (Cox et al., 2006; Goldberg et al., 2007), while gain-of-function mutations result in pain in primary erythromelalgia, paroxysmal severe pain disorder, and small fiber neuropathy (Yang et al., 2004; Fertleman et al., 2006; Faber et al., 2012). Furthermore, in mice, knockout of the NaV1.7 gene in specific neuronal populations reduces pain behavior following inflammation and nerve injury (Nassar et al., 2004; Minett et al., 2012), while systemic knockout of NaV1.7 at birth recreates the congenital analgesia phenotype in humans (Gingras et al., 2014). NaV1.7 has been a focus of research, with the aim of finding inhibitors for the treatment of chronic pain (Bagal et al., 2014; Sun et al., 2014a; de Lera Ruiz and Kraus, 2015; Emery et al., 2016; Vetter et al., 2017). In these studies, sulfonamide small molecule antagonists have emerged as highly effective and selective compounds that can block NaV1.7, while their blocking efficacy against homologous sodium channels (including NaV1.5 expressed in the heart) is significantly reduced. The mechanism involves binding to a novel binding pocket located within the fourth voltage sensor domain, stabilizing NaV1.7 in an inactive conformation, thereby preventing the channel from effectively cycling to a resting/closed state and reducing its subsequent opening probability (McCormack et al., 2013; Sun et al., 2014b; Ahuja et al., 2015; Alexandrou et al., 2016; Focken et al., 2016; Theile et al., 2016). This chemotype exhibits weaker inhibitory effects on rat NaV1.7 channels, attributed to amino acid sequence differences in this region (McCormack et al., 2013; Alexandrou et al., 2016; Focken et al., 2016). The specific interaction of sulfonamide compounds with the fourth voltage sensor domain distinguishes their binding sites from those of previously reported toxin-derived peptides, local anesthetics, and TTX (de Lera Ruiz and Kraus, 2015). This article details the extensive in vitro, ex vivo, and in vivo preclinical pharmacological characterization of the novel sulfonamide NaV1.7 antagonist AMG8379 and its significantly reduced-activity enantiomer AMG8380 (as a negative control). AMG8379 is a potent and selective NaV1.7 inhibitor that blocks action potential firing in peripheral nerve fibers and has shown efficacy in a transformable mouse model of pruritus and pain, while the less active enantiomer AMG8380 does not exhibit these biological effects at similar plasma concentrations. [1]

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AMG8379 is a potent and selective small-molecule sulfonamide antagonist that antagonizes recombinant NaV1.7 channels in heterologous cells from human and mouse (but not rat) sources, as well as native NaV1.7 channels in mouse dorsal root ganglion (DRG) neurons. AMG8379 inhibited the firing of mechanically and thermally induced C-fiber action potentials in mouse skin-nerve specimens and blocked various NaV1.7-dependent behaviors, including histamine-induced scratching, capsaicin-induced licking, and UVB-induced thermal hyperalgesia. The enantiomer AMG8380 showed significantly reduced activity…[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.)