Nav1.7 (IC50 = 3 nM)

GNE-131 (Compound 13) had a 0.003±0.001 μM IC50 against human NaV1.7 and moderate clearance in human liver microsomes. It also demonstrated high functional activity against the virus. In vitro metabolic stability and potency of GNE-131 are both good [1].

GNE-131 (Compound 13) was also evaluated for their effects on mouse NaV1.7 using the Qube automated electrophysiology platform, and IC50 values of 0.007 μM (13) and 0.005 μM (25) were measured. Selected analogs were tested in vitro for effects on the hERG potassium channel by automated voltage clamp, and inhibition was generally low (11% ± 2% inhibition at 10 μM test concentration for compound 13). The ADME profile for GNE-131 (Compound 13) is shown in Table 7. All compounds showed moderate to high metabolic stability in liver microsomes, and GNE-131 (Compound 13) also showed high stability in human, rat, and dog hepatocytes. GNE-131 (Compound 13) demonstrated moderate permeability in MDR1 transfected MDCK cells, with 25 showing high potential for P-glycoprotein (Pgp) mediated efflux. The compounds showed no appreciable inhibition of CYP isoforms 3A4, 1A2, 2C9, and 2D6, at concentrations of 10 μM. Plasma protein binding (PPB) was determined by rapid equilibrium dialysis and was generally very high (99.0–99.9% in human, mouse, and rat). Testing of GNE-131 (Compound 13) by equilibrium dialysis using HT Dialysis membranes indicated 99.8% and 99.7% PPB in mouse, respectively.

In vivo clearance of GNE-131 is poor in rats, mice, and dogs. Excellent efficacy of GNE-131 has also been demonstrated in transgenic mouse models of pain [1].

The pharmacokinetic parameters for GNE-131 in mice, rats, and dogs after iv administration are shown in Table 8. GNE-131 showed low total clearance (0.8–7 mL/min/kg), consistent with the low in vitro clearance inferred from stability testing with hepatocytes (Table 7). Volume of distribution was low (<0.7 L/kg) and within the range typically observed for acidic compounds[1].

Similarly, cyclopropyl sulfonamide analog GNE-131 showed significant reduction of nociceptive behavior at the doses tested, with up to 85% reduction at the 30 mg/kg dose and a plasma concentration of 2.8 ± 0.8 μM. The EC50 values for the inhibition of nociceptive behavior by the two adamantyl triazoles 10 and GNE-131 were calculated as 5.8 μM (10) and 0.5 μM ( GNE-131), respectively (Figure 6B)[1] .

Competitive Binding Experiments[1]
Binding reactions were performed in 96-well polypropylene plates at room temperature for 18 h. In a 360 μL assay volume, membranes were incubated with 50 pM [3H]GX-545 and increasing concentrations of test compound (1% DMSO). Nonspecific binding was defined in the presence of 1 μM unlabeled GX-545. Total binding was determined in the presence of DMSO (1%). Reactions were transferred and filtered through 96-well glass fiber/C filter plates presoaked with 0.5% poly(ethylene imine). The filtered reactions were washed 5 times with 200 μL of ice cold buffer containing 0.25% BSA. Bound radioactivity was determined by liquid scintillation counting.

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Metabolic Stability Studies in Liver Microsomes[1]
The oxidative metabolism was evaluated in pooled liver microsomes from CD-1 mice (n = 10), Sprague–Dawley rats (n = 20), cynomolgus monkeys (n = 4), beagle dogs (n = 3), and humans (n = 15). The incubation mixture was prepared for each species in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.5 mg/mL microsomal protein, 1 mM NADPH, and 1 μM of test compound. Reactions were initiated with the addition of NADPH. Samples were incubated at 37 °C, and aliquots were sampled at 0, 20, 40, and 60 min. Reactions were quenched with 95/5 acetonitrile/water (v/v) with internal standard at each time point. Samples were centrifuged at 3000g for 10 min. Supernatant was diluted with water (1:2 ratio), and the percentage of compound remaining was determined by LC–MS/MS using the t = 0 peak area ratio values as 100%. The in vitro Clint and scaled hepatic Clhep were determined as described by Obach et al.

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n Vitro Pgp (MDR1) Transport Assays[1]
Madin–Darby canine kidney (MDCK) cells heterologously expressing human Pgp were used to determine if a compound was a substrate of these transporters. MDR1-MDCKI cells were licensed from the National Cancer Institute. For transport studies, cells were seeded on 12-well Costar Transwell plates 4 days before use (polyester membrane, 0.4 μm pore size; Corning Life Sciences, Lowell, MA) at a seeding density of 1.3 × 105 cells/mL. Compounds were tested at 10 μM in the MDR1 assay in the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions. The compound was dissolved in transport buffer consisting of Hank’s balanced salt solution and 10 mM HEPES. Lucifer Yellow was used as the paracellular marker. The efflux ratio (ER) was calculated as ER = ; standard deviation is not available.

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In Vitro Plasma Protein Binding[1]
RED Device[1]
The extent of protein binding was determined in vitro and in CD-1 mouse, Sprague–Dawley rat, cynomolgus monkey, beagle dog, and human plasma by equilibrium dialysis using the RED (rapid equilibrium dialysis) device. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to plasma for a final concentration of 5 μM of test compound and 1% of DMSO in the plasma. Plasma samples (300 μL) were dialyzed against PBS buffer (500 μL) on a shaking platform in a humidified incubator for 4 h at 37 °C. Following dialysis, buffer and plasma samples were transferred to a 96-well plate. Plasma proteins were precipitated with acetonitrile containing an internal standard. The amount of the parent analyte in the plasma and buffer samples was quantified by LCMS/MS, and percent unbound fraction (fu) was calculated.

HT Dialysis[1]
The extent of protein binding was determined at WuXi Apptec in CD-1 mouse plasma (BioreclamationIVT) by equilibrium dialysis using HT Dialysis plates (model HTD 96b) in a similar fashion as described above, except that a final concentration of 2 μM of test compound was used.

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Cytochrome P450 (CYP) Inhibition Assays[1]
Test compounds were incubated with 0.2 mg/mL human liver microsomes (150 donor pool) together with NADPH cofactor and the specific probe substrate for each CYP tested: CYP1A2 (phenacetin), CYP2C9 (warfarin), CYP2C19 (mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone and midazolam). The reactions were terminated after 30 min by addition of cold acetonitrile/formic acid (94:6 v/v) containing an internal standard. Inhibition was determined through conversion of the probe substrates by CYPs as determined by LCMS/MS analysis. Five concentrations of each test compound were tested (0.1, 1, 5, and 10 μM, as well as a solvent control) to generate IC50 values. IC50 values were obtained from single experiments; SD is not applicable.

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Human Pregnane X Receptor (PXR) Activation Assay[1]
The ability of test compounds to activate PXR was determined by using DPX2 cells, a human HepG2-derived cell line stably integrated with a PXR expression vector plus a luciferase reporter gene. DPX2 cells were treated with test compounds at eight concentrations from 0.046 to 20 μM in duplicate for 24 h at 37 °C in a CO2 incubator. Cell viability was assessed fluorimetrically with CellTiter-Fluor to obtain relative fluorescent units (RFUs). The luciferase activity, which was directly proportional to the extent of PXR activation and accompanying gene transcription in the DPX2 cells was measured using One-Glo to obtain relative luminescent units (RLUs). The PXR (luciferase) activity was normalized with cell viability by dividing the average RLU by the average RFU at each test compound concentration, as well as in the vehicle control. PXR receptor activation fold increase was calculated by dividing the normalized luciferase activity of individual doses by that of the normalized DMSO vehicle control. The final data was expressed as fold activation relative to the vehicle control. Rifampicin (RIF) was used as positive control. The use of eight concentrations of test compounds and RIF allowed for the derivation of EC50 from nonlinear regression analysis of the log dose–response curves. Induction of PXR by test compound was expressed as percentage of activation seen with rifampicin when both compounds were used at a test concentration of 10 μM. Test compounds showing activation <15% of 10 μM RIF would be considered negative.

PatchXpres Automated Voltage Clamp Platform[1]
Macroscopic sodium currents were recorded in the whole-cell configuration. The intracellular solution comprised 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM EGTA (adjusted to pH 7.2 with CsOH), while the extracellular solution comprised 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (adjusted to pH 7.4 with NaOH). Generally, the external sodium was reduced by equimolar replacement with choline. Osmolarity in the internal and external solutions was adjusted to 300 and 310 mOsm/kg with glucose, respectively. Currents were recorded at 40 kHz sampling frequency, filtered at 5 Hz, and analyzed with DataXpress software. Series resistance compensation was applied at 60–80%. Compound inhibition was fitted to the Hill equation Y = [C]h/(IC50h + [C]h) to estimate the half maximal inhibition concentration (IC50 value); where Y is the normalized inhibition relative to the control, [C] the test compound concentration, IC50 the concentration of test compound to inhibit the currents 50%, and h the Hill coefficient. IC50 values generated on the PatchXpress automated voltage-clamp platform are presented as mean ± SD.

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QPatch HT Automated Voltage Clamp Device[1]
Intra- and extracellular solutions and osmolarity were used as described for the PatchXpress system. Data were analyzed with QPatch Assay Software. IC50 values on the QPatch automated voltage-clamp platform were generated from pooled data, for which SD is not applicable.

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Qube384 Automated Voltage Clamp System[1]
A reverse sodium gradient was used during electrophysiological recordings. Specifically, low sodium (1 mM) extracellular and high sodium (120 mM) intracellular solutions were used. These solutions contained the following (mM): low sodium, 1 NaCl, 139 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES; high sodium, 120 NaF, 10 CsCl2, 0.1 CaCl2, 2 MgCl2, 10 HEPES, and 10 EGTA. The external solution was titrated with sodium hydroxide to pH 7.4, while the intracellular solution was titrated with CsOH to pH 7.2. Currents were low pass filtered at 5 kHz and recorded at 25 kHz sampling frequency. Appropriate filters for minimum seal resistance and minimum current size were applied, and series resistance was compensated 100% (routinely membrane resistance >500 MΩ and current magnitude >1 nA at a 0 mV test pulse from a −120 holding potential). Data was collected at room temperature, which corresponds to 27 ± 2 °C at the recording chamber. Every experimental plate contained vehicle controls to quantify and correct for any compound independent “run-down” of currents. Baselines were established after 20 min in vehicle. Maximal inhibition was established by adding 300 nM tetrodotoxin (TTX) to each well at the end of the experiment. Fractional block was calculated from fractional reduction of current amplitude from baseline to maximal block after 20 min of exposure for IC50 determinations or 26 min in kinetic experiments. Data analysis was performed using Analyzer and Prism software. IC50 values on the Qube automated voltage-clamp platform were generated from Hill equation fits to pooled data, for which SD is not applicable.

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Site Directed Mutagenesis of VSD4 Residues[1]
The human NaV1.7 channel was coexpressed with the β1 subunit in a stable HEK293 cell line as described above. Stable HEK293 cell lines with a permanently transfected Y1537A, R1602A, R1605A, or R1608A mutation of hNaV1.7 were also generated, each coexpressed the β1 subunit. hNaV1.7 VSD4 mutants were generated using Agilent’s QuikChange II XL site-directed mutagenesis kit, and all constructs were confirmed by ull-length DNA sequencing. Whole-cell patch clamp recordings of mutant sodium currents were recorded using the Qube384 utomated voltage clamp system using conditions stated above unless otherwise stated. To determine inactivated state block, the membrane potential was maintained at −60 mV (NaV1.7WT and NaV1.7Y1537A) or −75 mV (NaV1.7R1602A, NaV1.7R1605A, and NaV1.7R1608A; see Supporting Information). Once every 10 s, the voltage was stepped back to a very negative (Vhold = −150 mV) voltage for 20 ms and then a test pulse is applied to quantify the drug block. Normalized compound inhibition was fit to the Hill equation. Data analysis was performed using Analyzer and Prism software. Results are presented as mean ± SEM. Statistical comparisons (Student’s t test, one-way ANOVA) were performed using Prism, and P values of <0.05 were considered statistically significant.

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 Metabolic Stability Study in Cryopreserved Hepatocytes[1]
The oxidative and conjugative metabolism were evaluated in cryopreserved hepatocytes from CD-1 mice (n = 10), Sprague–Dawley rats (n = 3), cynomolgus monkey, beagle dog, and humans (n = 10). The cells were seeded at a density of 0.5 × 106 cells/mL; reactions were initiated with the addition of test compound to make the final substrate concentration of 1 μM. Samples were incubated at 37 °C in 5% carbon dioxide with saturating humidity, and aliquots were sampled at 0, 1, 2, and 3 h. Reactions were quenched with acetonitrile containing internal standard at each time point. Samples were centrifuged at 2000g for 10 min. Supernatant was diluted with water (1:2 ratio), and the percentage of test compound remaining was determined by LC/MS/MS. Using the t = 0 peak area ratio values as 100%, the in vitro Clint and scaled hepatic Clhep were determined as previously described, where QLiver is the liver blood flow; standard deviation is not applicable.

Pharmacokinetic Studies in Mice, Rats, and Dogs[1]
FVB Mouse PK[1]
Male FVB mice between 6 and 7 weeks of age with body weight ranging from 23 to 30 g were divided into groups of 3 per route and dose, and were not fasted before dosing. Mice in each group received an oral dose of the test article at 30 mg/kg prepared in 0.5% methyl cellulose with 0.2% Tween 80 (MCT) at a dose volume of 5 mL/kg. Blood samples (15 μL) were collected via tail nick from each animal at 0.25, 0.5, 1, 2, 4, and 6 h postdose and added to 60 μL of EDTA (1.7 mg/mL) water. Test article concentration in each blood sample was determined by a nonvalidated LCMS/MS assay at Genentech. The study for compound 25 was performed at Xenon Pharmaceuticals at a dose of 10 mg/kg using otherwise similar conditions.

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CF-1 Mouse PK[1]
Male CF-1 mice (4–5 weeks old) ranging from 30 to 40 g were were given ad libitum food and water access prior to studies. For iv dosing, three mice were given a single iv dose in 50% poly(ethylene glycol) 400 in phosphate buffered saline and 50% 2-(hydroxpropyl)-β-cyclodextrin in phosphate buffered saline (40:60). Blood samples (approximately 0.04 mL per sample) were collected from each animal into tubes containing K2EDTA at 0.033, 0.167, 0.5, 1, 2, 4, 8, and 24 h after dose administration. For oral dosing, three mice were given a single po dose in 0.5% w/w methyl cellulose and 0.2% v/v tween 80 in deionized water. Blood samples (approximately 0.04 mL per sample) were collected from each animal into tubes containing K2EDTA at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dose administration. Blood was centrifuged for 10 min to harvest plasma. The concentration of test compound in each plasma sample was determined by LCMS/MS assay at Xenon.

Rat PK[1]
Male Sprague–Dawley (SD) rats (9–11 weeks old) ranging from 250 to 300 g were fasted 16 h before oral dose administration. Three rats were given a single iv dose in 10% DMSO, 50% poly(ethylene glycol) 400, and 40% phosphate buffered saline. Blood samples (approximately 0.2 mL per sample) were collected from each animal into tubes containing K2EDTA at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h after dose administration. Blood was centrifuged for 10 min to harvest plasma. The concentration of test compound in each plasma sample was determined by a nonvalidated LCMS/MS assay at Genentech. Pharmacokinetic analysis was performed at Genentech using noncompartmental methods.

Dog PK[1]
Pharmacokinetics of test compound was determined in male Beagle dogs following a single intravenous bolus (iv) administration. Non-naive male Beagle dogs ages ranging from 6 months to 3 years old and weighing between 6 and 12 kg were obtained from Marshall Bioresources (Beijing, China). Animals were not fasted before iv dosing. Blood samples were collected in tubes containing K2EDTA at predose and at 0.033, 0.083, 0.25, 0.5, 1, 3, 6, 9, and 24 h post-iv administration. Blood was centrifuged for 10 min to harvest plasma. The concentration of test compound in each plasma sample was determined by a nonvalidated LCMS/MS assay at Wuxi AppTech, Inc. Pharmacokinetic analysis was performed at Genentech using noncompartmental methods.

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Efficacy Studies in Human NaV1.7 I848T Transgenic Mice[1]
Male FVB hSCN9a Tg used for the experiments were seven to 10 weeks old and weighed 19 to 27 g. The test compound was administered orally in 0.5% w/w methyl cellulose and 0.2% v/v tween 80 in deionized water at a dose volume of 10 mL/kg 2 h before injection of the pain agent. The mice were then acclimatized to the test chamber for 60 min prior to injection of the pain-inducing agent. A volume of 20 μL of 39 μM aconitine was injected subcutaneously into the dorsal region of the left hind paw to induce spontaneous pain. Video data acquisition begins immediately after injection of the pain agent and continues for a total duration of 70 min. The first 30 min of each acquired video was analyzed and scored for the time spent flicking, lifting, biting, or licking the injected paw. At approximately 3–4 h post treatment (dosing of test compound), terminal blood samples via cardiac puncture were collected upon euthanasia of animals in addition to brain tissue. Plasma prepared in EDTA and brain tissue were frozen in liquid nitrogen and stored at −80 °C for PK analysis.

[1]. Design of Conformationally Constrained Acyl Sulfonamide Isosteres: Identification of N-([1,2,4]Triazolo[4,3- a]pyridin-3-yl)methane-sulfonamides as Potent and Selective hNaV1.7 Inhibitors for the Treatment of Pain. J Med Chem. 2018 Jun 14.

The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.[1]

C23H30N4O3S

442.574304103851

442.20386

C, 62.42; H, 6.83; N, 12.66; O, 10.84; S, 7.24

1629063-81-5

91666633

Light yellow to yellow solid powder

4.8

1

6

7

31

780

0

C1(S(NC2N3C(=NN=2)C=C(OCC24CC5CC(CC(C5)C2)C4)C(C2CC2)=C3)(=O)=O)CC1

FPERPEQIXLOVIK-UHFFFAOYSA-N

InChI=1S/C23H30N4O3S/c28-31(29,18-3-4-18)26-22-25-24-21-8-20(19(12-27(21)22)17-1-2-17)30-13-23-9-14-5-15(10-23)7-16(6-14)11-23/h8,12,14-18H,1-7,9-11,13H2,(H,25,26)

N-(7-(Adamantan-1-ylmethoxy)-6-cyclopropyl-[1,2,4]-triazolo[4,3-a]-pyridin-3-yl)cyclo-propanesulfonamide

GNE-131; GNE 131; GNE-131; 1629063-81-5; GNE131; N-[7-(1-adamantylmethoxy)-6-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl]cyclopropanesulfonamide; GNE 131; N-(7-(adamantan-1-ylmethoxy)-6-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)cyclopropanesulfonamide; N-[7-(adamantan-1-ylmethoxy)-6-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl]cyclopropanesulfonamide; CHEMBL4290579; GNE131

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 (~282.44 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.70 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.70 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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