Nav1.8 sodium channel (IC50 = 8 nM)

A-803467 reverses the multidrug resistance mediated by ABCG2 in a selective and significant manner. In cell lines transfected with ABCG2, A-803467 (7.5 μM) considerably amplifies the cytotoxicity of mitoxantrone and topotecan. MX build-up in cells transfected with ABCG2. At varying time intervals, A-803467 (7.5 μM; 0~120 minutes) substantially inhibits the intracellular [3H]-MX efflux from ABCG2-transfected cells. A-803467 increases ABCG2's ATPase activity[1].

---

In this study, researchers investigated the effect of a tetrodotoxin-resistant sodium channel blocker, A-803467 on ABCG2-overexpressing drug selected and transfected cell lines. We found that at non-toxic concentrations, A-803467 could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies demonstrated that A-803467 (7.5 μM) significantly increased the intracellular accumulation of [(3)H]-mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression levels. In addition, A-803467 stimulated the ATPase activity in membranes overexpressed with ABCG2. [1]

---

In this study, researchers report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na(v)1.8 (IC(50) = 8 nM) and was >100-fold selective vs. human Na(v)1.2, Na(v)1.3, Na(v)1.5, and Na(v)1.7 (IC(50) values >or=1 microM).

In male NCR nude mice, A-803467 (35 mg/kg; po) exhibits no discernible toxicity[1]. When A-803467 and topotecan are combined, the growth of tumors in mice implanted with H460/MX20 cells that overexpress ABCG2 is greatly reduced. Restoring the sensitivity of cancers overexpressing the ABCG2 transporter to topotecan is accomplished by -803467, but tumors lacking ABCG2 expression are not significantly affected[1].

---

In a murine model system, combination treatment of A-803467 (35 mg/kg) and topotecan (3 mg/kg) significantly inhibited the tumor growth in mice xenografted with ABCG2-overexpressing cancer cells. Our findings indicate that a combination of A-803467 and ABCG2 substrates may potentially be a novel therapeutic treatment in ABCG2-positive drug resistant cancers. [1]

---

A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain[2].

ABCG2 ATPase assay [1]
The Vi-sensitive ATPase activity of ABCG2 in the membrane vesicles of High Five insect cells was measured as previously described. The membrane vesicles (100 μg protein/ml) were incubated in ATPase assay buffer with or without 0.3 mM vanadate at 37°C for 5 min and then incubated with different concentrations of A-803467 ranging from 0 to 80 μM, topotecan, and MX (0 – 30 μM), at 37°C for 3 min. The ATPase reaction was induced by the addition of 5 mM Mg-ATP, and the total volume was 0.1 mL. After incubation at 37°C for 20 min, the reactions were stopped by loading 0.1 mL of 5% SDS solution. The liberated inorganic phosphate (Pi) was measured as described previously.

---

Pharmacological Selectivity Assays. [2]
The activity of A-803467 (10 μM) was evaluated in assays to assess pharmacological selectivity relative to other cell-surface receptors, ion channels, transport sites, and enzymes including the opioid receptors, and cycloxygenases 1 and 2, by use of standardized assay protocols (CEREP and in-house assays) as described.

Cell viability assay [1]
Cytotoxicity tests and reversal experiments were performed using the MTT colorimetric assay as described previously. Cells were harvested and resuspended in a final concentration of 6 × 103 cells/well for HEK293/pcDNA3.1, HEK/ABCB1, HEK/ABCC10, HEK293/R482, HEK293/R482G and HEK293/R482T cells, and 4 × 103 cells/well for H460 and H460/MX20 cells. Cells were seeded evenly into 96-well plates. To determine the cytotoxicity of A-803467, different concentrations of drug were added into the each well after 24 h of incubation. To determine the reversal capability of A-803467, various concentrations of chemotherapeutic drugs were added into designated wells after 2 h preincubation with A-803467, FTC, verapamil or cepharanthine. After 68 h of drug incubation, MTT reagent (4 mg/mL) was added. The plates were incubated for an additional 4 h, the supernatant was discarded and 100 μl of DMSO were added to dissolve the formazan crystals. Cell viability was measured at a wavelength of 570 nm. All the experiments were repeated at least 3 times, and the mean and standard deviation (SD) values were calculated.

---

[3H]-MX accumulation and efflux assay [1]
Researchers examined the effect ofA-803467 on the intracellular accumulation and efflux of [3H]-MX in ABCG2-overexpressing cells as previous described. Briefly, the cells (5 × 106/cells) were resuspended and incubated in the RPMI 1640 medium in the presence or absence of A-803467 (7.5 μM) or FTC (5 μM) at 37°C for 2 h. Cells were then incubated with 0.01 μM [3H]-MX containing medium for additional 2 h at 37°C, with or without A-803467 (7.5 μM) or FTC (5 μM), and subsequently washed twice with ice-cold PBS. For the accumulation assay, cells were lysed by the 10 mM lysis buffer (pH 7.4, containing 1% Triton X-100 and 0.2% SDS) and then placed in scintillation fluid. For the efflux assay, the suspended cells were then cultured in [3H]-MX free medium, with or without A-803467 (7.5 μM) or FTC (5.0 μM) at 37°C. The aliquots of cells were harvested at the indicated times (0, 30, 60, and 120 min), and then washed with ice-cold PBS and transferred to respective scintillation vials. The radioactivity was measured using the Packard TRI-CARB1 190`A liquid scintillation analyzer.

---

Western blot analysis [1]
Cell lysates were prepared as described previously. Equal amounts of total cell lysates (30 μg protein) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation in a blocking solution (5% milk) for 1 h at room temperature, the membranes were immunoblotted overnight with primary monoclonal antibodies against actin at 1:1000 dilution or ABCG2 at 1:500 dilution at 4°C, and were then further incubated for 2 h at room temperature with horseradish peroxide (HRP)-conjugated secondary antibody (1:1000 dilution). The protein–antibody complex was detected by enhanced chemiluminescence detection system.

---

Immunofluorescence analysis [1]
For immunofluorescence analysis, H460 and H460/MX20 cells were seeded in 24 well plates. Cells were incubated with or without A-803467 (7.5 μM) for 72 h. Thereafter, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature and then rinsed with PBS three times, followed by permeabilization with 1% triton X-100 for 10 min at 4°C. Cells were again washed for three times with PBS, and then blocked with 2 mg/ml of BSA for 1 h at 37°C. Fixed cells were incubated with monoclonal antibody against ABCG2 (BXP 21) (1:50) for 16 h at 4°C, followed by three washes with PBS. The cells were then further incubated with Alexa flour 488 goat anti-mouse IgG (1:60) for 1 h at 37°C. DAPI was used for nuclear counterstaining. Immunofluorescence images were taken with a Nikon fluorescence microscope.

Animal/Disease Models: Nude mice[1]
Doses: 35 mg/kg
Route of Administration: Po
Experimental Results: demonstrated no noticeable toxicity in the male NCR nude mice.

---

Nude mouse MDR xenograft models [1]
The ABCG2-overexpressing NSCLC cell H460/MX20 xenograft mouse models were established as previously explained [41]. H460/MX20 cells (6 × 106) and H460 cells (4 × 106) were injected subcutaneously under the right and left armpit regions of the nude mice, respectively. We performed a pilot study using three different doses of A-803467 (17.5, 35 and 70 mg/kg) and we found that 35 mg/kg dose was effective in increasing the topotecan sensitivity in tumors without significantly increase toxicity, therefore 35 mg/kg dose was used throughout the following study. [1]
The mice were randomized into 4 groups (n = 6) when the tumors attained a mean diameter of 0.5 cm (day 0), and then received treatments as follows: (a) Vehicle (10% N-methyl pyrrolidine (NMP) in PEG-300, p.o., every 2nd and 3rd day; total 12 times), (b) A-803467 diluted in 10% NMP in PEG-300 (35 mg/kg, p.o., every 2nd and 3rd day; total 12 times), (c) Topotecan (3.0 mg/kg, i.p., every 3rd day; total 6 times), and (d) A-803467 (35 mg/kg, every 2nd and 3rd day; total 12 times, given 1 h before topotecan) + topotecan (3.0 mg/kg, i.p., every 3rd day: total 6 times). The body weights of the mice were monitored and the two perpendicular diameters of tumors (A and B) were recorded every 4th day, and tumor volumes (V) were calculated according to the following formula described previously

---

A-803467 was dissolved in 5% DMSO/95% polyethylene glycol (PEG 400) for i.p. administration in a 2 ml/kg injection volume. The compound was injected 30 min before behavioral testing.
Spinal Dorsal Horn Neuronal Electrophysiology. [2]
Electrophysiological recording of spinal dorsal horn neurons was conducted as described. Briefly, neuropathic rats (L5–L6 spinal nerve ligation) were anesthetized with pentobarbital (50 mg/kg, i.p.), and catheters were placed in the left and right external jugular veins. A laminectomy was performed to remove vertebral segments T12-L3. The animals were then secured in a stereotaxic frame. Anesthesia was maintained for the duration of the experiment by a continuous infusion of propofol (8–12 mg/kg/hr, i.v.). Body temperature was kept at 37°C by placing the animals on a circulating water blanket. Platinum-plated stainless steel microelectrodes were used to record the activity of spinal wide dynamic range (WDR) neurons. Spike waveforms were monitored, digitized (32 points), and stored for offline analysis. Baseline spontaneous activity was recorded for 5 min before stimulation. Rats were stimulated three times (5 min apart) before drug administration with a von Frey hair (10 g) applied to the neuronal receptive field for 15 s located on the ipsilateral hindpaw. The mean of three stimulations represented baseline evoked activity. A-803467 (20 mg/kg, i.v.) or vehicle was infused over a 5- to 7-min period, and the von Frey hair was reapplied 35 min after this infusion. For comparison to baseline firing levels, statistical significance was established by using Wilcoxon's matched-pairs test. [2]
Pharmacological Selectivity Assays. [2]
The activity of A-803467 (10 μM) was evaluated in assays to assess pharmacological selectivity relative to other cell-surface receptors, ion channels, transport sites, and enzymes including the opioid receptors, and cycloxygenases 1 and 2, by use of standardized assay protocols (CEREP and in-house assays) as described.

---

Analgesia and Side-Effect Assays. [2]
A-803467 was evaluated in well characterized in vivo models to assess acute, inflammatory, and neuropathic pain. The specific methodologies for these nociceptive assays, models of postoperative and visceral pain, and models of motor performance are described in the SI. Unless otherwise noted, all experimental and control groups contained at least six animals per group, and data are expressed as mean ± SEM. Data analysis was conducted by using analysis of variance and appropriate post hoc comparisons (P < 0.05). ED50 values were estimated by using least-squares linear regression.

[1]. A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo. Oncotarget. 2015;6(36):39276-39291.

[2]. A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A. 2007;104(20):8520-8525.

To further understand the interaction of A-803467 with ABCG2, we performed an ATPase assay using ABCG2 overexpressed membranes. The majority of TKIs that interact with the ABC drug transporters stimulate ATP hydrolysis and the fact that A-803467 stimulates the ATP hydrolysis of ABCG2 in a concentration dependent manner (Fig. 3A) indicates that it behaves similar to other known substrates (Fig. 3B and 3C) of ABCG2 transporter, such as MX and topotecan. These results further prove that A-803467 not only interacts directly with the ABCG2 transporter, but may also be a competitive inhibitor of the transporter.
To identify the molecular interaction of A-803467 with the ABCG2 transporter, docking simulation was performed at various sites of the human ABCG2 homology model. The crystal structure of human ABCG2 transporter is not completely elucidated. Comparing the docking scores shown in Table 4, the most favorable binding site was identified as site-1. Molecular docking of topotecan, a well-known ABCG2 substrate, at the same site of ABCG2 was performed. The docking score of topotecan (−5.57 kcal/mol) is much higher than that of A-803467 (−8.07 kcal/mol). The lower docking score indicates stronger interaction between A-803467 to ABCG2 (Fig. 4). Moreover, molecular structure of A-803467 also exhibited the pharmacophoric features such as hydrophobic groups, aromatic ring centers (phenyl ring and furan ring) and hydrogen bond acceptors that have been reported as essential for ABCG2 inhibition. Overall, this molecular simulation will provide clues to optimize further derivatives of ABCG2 inhibitors. [1]

---

Although lacking analgesic effects on acute thermal pain in normal rats, A-803467 significantly decreased acute mechanical nociception. This effect is in agreement with previous knockout and antisense data demonstrating that knockdown of Nav1.8 was associated with decreased acute mechanical nociception. More than 50% of C fibers and 10% of A fibers express Nav1.8 channels. The Nav1.8-positive C fibers are NGF- or GDNF-responsive, and many of these fibers also express TRPV1, supporting a role in acute thermal nociception. However, the expression of Nav1.8 channels on A-fibers and the data showing that disruption of Nav1.8 function reduces acute mechanical nociception provide evidence that activation of Nav1.8 channels contributes to normal sensitivity to noxious mechanical stimulation.
In summary, the present data demonstrate that A-803467 is a potent and highly selective blocker of Nav1.8 channels and that this compound effectively blocks sensory neuron excitability in vitro and in vivo. Evaluation of A-803467 in a wide range of animal pain models demonstrates that selective blockade of Nav1.8 channels in vivo results in a significant reduction in nociceptive sensitivity after nerve injury and inflammation. [2]

C19H16CLNO4

357.79

357.076

C, 63.78; H, 4.51; Cl, 9.91; N, 3.91; O, 17.89

944261-79-4

16038374

White to off-white solid powder

1.3±0.1 g/cm3

450.6±45.0 °C at 760 mmHg

128-130?C

226.3±28.7 °C

0.0±1.1 mmHg at 25°C

1.611

4.93

1

4

5

25

429

0

O=C(C1=CC=C(C2=CC=C(Cl)C=C2)O1)NC3=CC(OC)=CC(OC)=C3

VHKBTPQDHDSBSP-UHFFFAOYSA-N

InChI=1S/C19H16ClNO4/c1-23-15-9-14(10-16(11-15)24-2)21-19(22)18-8-7-17(25-18)12-3-5-13(20)6-4-12/h3-11H,1-2H3,(H,21,22)

5-(4-chlorophenyl)-N-(3,5-dimethoxyphenyl)furan-2-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.99 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.99 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

View More

Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

---

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