Gardos Channel (Ca²⁺-activated K⁺ channel (IC50 = 11 nM)

It was demonstrated that ICA-17043 blocked Gardos channels in mouse (C57 black) RBCs with an IC50 of 50±6 nM. In human red blood cells, ICA-17043 inhibits the rise in cellular hemoglobin concentration in a concentration-dependent way [1].

---

A prominent feature of sickle cell anemia is the presence of dehydrated red blood cells (RBCs) in circulation. Loss of potassium (K(+)), chloride (Cl(-)), and water from RBCs is thought to contribute to the production of these dehydrated cells. One main route of K(+) loss in the RBC is the Gardos channel, a calcium (Ca(2+))-activated K(+) channel. Clotrimazole (CLT), an inhibitor of the Gardos channel, has been shown to reduce RBC dehydration in vitro and in vivo. In this study, researchers have developed a chemically novel compound, ICA-17043, that has greater potency and selectivity than CLT in inhibiting the Gardos channel. ICA-17043 blocked Ca(2+)-induced rubidium flux from human RBCs with an IC(50) value of 11 +/- 2 nM (CLT IC(50) = 100 +/- 12 nM) and inhibited RBC dehydration with an IC(50) of 30 +/- 20 nM. [1]

Following ICA-17043 (10 mg/kg, oral) formulation, there was a significant reduction in gardos channel activity evaluated on days 11 and 21. This reduction was accompanied by an increase in erythrocyte K+ content, but no change in Na+ content. After 11 days of formulation, ICA-17043 (10 mg/kg twice day) significantly increased Hct in SAD mice [1]. Senicapoc (30 mg/kg, face) 48 hours after allergen challenge in sheep decreases eosinophil counts, proliferation, and airway hyperresponsiveness [2].

---

In a transgenic mouse model of sickle cell disease (SAD), treatment with ICA-17043 (10 mg/kg orally, twice a day) for 21 days showed a marked and constant inhibition of the Gardos channel activity (with an average inhibition of 90% +/- 27%, P <.005), an increase in RBC K(+) content (from 392 +/- 19.9 to 479.2 +/- 40 mmol/kg hemoglobin [Hb], P <.005), a significant increase in hematocrit (Hct) (from 0.435 +/- 0.007 to 0.509 +/- 0.022 [43.5% +/- 0.7% to 50.9% +/- 2.2%], P <.005), a decrease in mean corpuscular hemoglobin concentration (MCHC) (from 340 +/- 9.0 to 300 +/- 15 g/L [34.0 +/- 0.9 to 30 +/- 1.5 g/dL], P <.05), and a left-shift in RBC density curves. These data indicate that ICA-17043 is a potent inhibitor of the Gardos channel and ameliorates RBC dehydration in the SAD mouse.[1]

Receptor binding study[1]
A greater understanding of the potential side effect profile of ICA-17043 can be obtained by identifying which common receptors the compound interacts with in vitro. Receptor binding activity of ICA-17043 at a concentration of 10 μM was determined on 30 common receptors: adenosine A1 and A2,α1 and α2-adrenergic,β1-adrenergic, NE uptake,angiotensin (AT1),benzodiazepine, bradykinin(B2),cholecystokinin (CCK),dopamine D1 and D2,dopamine uptake, endothelin (ETA),42 γ-aminobutyric acid (GABA), N-methyl-D-aspartate (NMDA),histamine H1, muscarinic,neurokinin, neuropeptide Y, nicotinic (N central), opiate, phencyclidine (PCP), serotonin (nonselective),serotonin (5-HT1B) and serotonin (5-HT2A), serotonin uptake,sigma opioid, glucocorticoid, and vasopressin-1 (V1) receptors.
ICA-17043 (at 10 μM) was incubated with membrane/cell preparations containing each of the above receptors and a corresponding high-affinity radioligand. Following incubation, membrane/cell fragments were filtered, and bound radioactivity was measured to determine the degree of displacement of radioligand by ICA-17043.

In vitro effects of ICA-17043 on red blood cells[1]
Measurement of inhibition of Ca2+-activated Rb efflux in human and mouse RBCs.[1]
Heparinized human blood was obtained from Biological Specialty. The blood samples were processed within 48 hours after being withdrawn. The whole blood was initially diluted 1:1 with Modified Flux Buffer (MFB), consisting of 140 mM NaCl, 5 mM KCl, 10 mM Tris (tris(hydroxymethyl)aminomethane), 0.1 mM EGTA (ethyleneglycoltetraacetic acid) (pH = 7.4). The blood was centrifuged at 1000 rpm, and the pellet comprised primarily of RBCs was washed 3 times with MFB. The cells were then loaded with 86Rb+ by incubating the washed cells with86Rb+ at a final concentration of 0.185 MBq/mL (5 μCi/mL) in MFB for at least 3 hours at 37°C. After loading with86Rb+, the RBCs were washed 3 times with chilled MFB. The cells were then incubated for 10 minutes with test compound (ICA-17043) at concentrations that ranged from 1 nM to 10 000 nM. Efflux of 86Rb+ was initiated by raising intracellular calcium levels in the RBCs with the addition of CaCl2 and A23187 (a calcium ionophore) to final concentrations of 2 mM and 5 μM, respectively. After 10 minutes of incubation at room temperature, the RBCs were pelleted in a microcentrifuge, and the supernatant was removed and counted in a Wallac MicroBeta liquid scintillation counter. The described protocol is a modification of the protocol for measurement of Gardos channel inhibition in RBCs as previously published by Brugnara et al. The percentage of inhibition and IC50 values were calculated with the use of the Origin software logistic function.

---

Measurement of inhibition of Ca2+-activated dehydration of human RBCs.[1]
Heparinized human blood was centrifuged at 2000 rpm, and the resulting pellet was washed 3 times with MFB as described previously. The RBC pellet was resuspended in MFB to which CaCl2 had been added to a final calcium concentration of 2 mM. Test compound, at increasing concentrations, was incubated with an aliquot of cells for 10 minutes at room temperature. A23187 (a calcium ionophore) was then added to a final concentration of 50 μM, followed by a 15-minute incubation at room temperature. The cells were then pelleted by using a microcentrifuge, Quenching Buffer (140 mM NaCl, 5 mM KCl, 10 mM Tris, 5 mM EGTA, and 0.1% bovine serum albumin [BSA], pH = 7.4) was immediately added to each of the cell pellets, which were then vortexed to resuspend the cells. The resuspended RBCs were then washed 3 times with Quenching Buffer. A blood analyzer (H2 Technicon) was used to measure the distributions of cell hemoglobin concentrations and to calculate the percentage of cells with hemoglobin concentrations above 410 g/L (41 g/dL).

Effects of ICA-17043 on SAD mice.[1]
Transgenic Hbbsingle/single SAD1 (SAD) female and male mice between 3 and 6 months of age, weighing 25 to 30 g, were used for this study. The SAD mice were divided into 2 groups, and either vehicle (n = 6) or ICA 17043 (10 mg/kg) (n = 6) was administered orally by gavage twice daily. C57B6/2J mice were used as controls (wild-type mice). Hematologic parameters were evaluated at baseline and after 11 and 21 days of therapy. Blood sampling and vehicle administration have previously been shown not to affect the blood parameters measured in this study.

---

Effects of ICA-17043 on SAD mice under chronic hypoxia conditions.[1]
In a separate study, after 15 days of dosing (10 mg/kg orally by gavage, twice a day), the SAD mice were maintained at 8% oxygen for 48 hours. Oxygen pressure inside the enclosed cage was monitored by an oxygen electrode. Hematologic parameters, RBC density patterns, Gardos channel activity, and RBC cation content were examined before and after 48 hours of hypoxic exposure. Control mice (C57B6/2J and vehicle-treated SAD mice) were also exposed to 48 hours of an 8% oxygen atmosphere.

---

Measurements of hematologic data and RBC cation content.[1]
A total of 200 μL blood was drawn at specific time intervals from each ether-anesthetized mouse by retroorbital venipuncture into heparinized microhematocrit tubes. The samples were evaluated for Rb+ flux measurements (as described in “Measurements of Ca2+-activated Rb+ influx in SAD mouse red cells”), determination of RBC phthalate density distribution curves, cell morphology studies, determination of RBC cation content, and other hematologic parameters. The hemoglobin concentration was determined by spectroscopic measurement of the cyanmet derivative. The hematocrit (Hct) was determined by centrifugation in a microhematocrit centrifuge. The cells were washed 3 times with phosphate-buffered saline (PBS; 330 mOsm) at 25°C. Density distribution curves and median RBC densities (D50) were obtained according to Danon and Marikovsky by using phthalate esters in microhematocrit tubes.

---

Measurements of Ca2+-activated Rb+ influx in SAD mouse red cells.[1]
Whole blood was incubated for 30 minutes at room temperature in the presence of 1 mM ouabain, 10 μM bumetanide, and 20 mM Tris-Mops, pH 7.4. While stirring, the ionophore A23187 was added to a final concentration of 80 μM. After 6 minutes of incubation at 22°C, Rb+ (in the form of RbCl) was added to the cell suspension (time = 0) to a final concentration of 10 mM and incubated at 37°C. Aliquots were obtained after 0, 2, 3, and 5 minutes and transferred to 2 mL medium containing 150 mM NaCl and 15 mM EGTA, pH 7.4 at 4°C. The samples were washed 3 times at 4°C with the same solution, followed by lysis in 1.5 mL 0.02% Acationox. After centrifugation of the lysate for 10 minutes at 3000g, the Rb+ content was measured in the supernatant by atomic absorption spectrophotometry.

---

Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K(Ca)3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.[2]

[1]. ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice. Blood. 2003 Mar 15;101(6):2412-8.

[2]. K(Ca)3.1 channel-blockade attenuates airway pathophysiology in a sheep model of chronic asthma. PLoS One. 2013 Jun 24;8(6):e66886.

Senicapoc (ICA-17043) is a novel Gardos channel blocker. It is being investigated for the treatment of sickle cell disease.
Senicapoc is an orally bioavailable inhibitor of the calcium-activated potassium channel KCa3.1 (Gardos channel; KCNN4; IK-1; SK4), with potential anti-inflammatory and immunomodulatory activities. Upon oral administration, senicapoc targets, binds to and inhibits KCa3.1. KCa3.1 regulates membrane potential and calcium signaling in a variety of cells including erythrocytes, activated T and B cells, macrophages, microglia, vascular endothelium, epithelia, and proliferating vascular smooth muscle cells and fibroblasts. The inhibition of KCa3.1 may prevent erythrocyte dehydration in sickle cell disease, reduce fluid secretion, fluid retention and inflammation in the lungs in acute respiratory distress syndrome (ARDS), and reduce inflammation and immune responses in various other diseases such as neuroinflammation in Alzheimer's disease. Senicapoc exhibits good brain penetration.

---

Drug Indication
Investigated for use/treatment in anemia (sickle cell) and asthma.

C20H15F2NO

323.3360

323.112

C, 74.29; H, 4.68; F, 11.75; N, 4.33; O, 4.95

289656-45-7

216327

White to yellow solid powder

1.25g/cm3

460.7ºC at 760mmHg

232.4ºC

1.14E-08mmHg at 25°C

1.594

4.484

1

3

4

24

397

0

SCTZUZTYRMOMKT-UHFFFAOYSA-N

InChI=1S/C20H15F2NO/c21-17-10-6-15(7-11-17)20(19(23)24,14-4-2-1-3-5-14)16-8-12-18(22)13-9-16/h1-13H,(H2,23,24)

2,2-bis(4-fluorophenyl)-2-phenylacetamide

ICA-17043; ICA 17043; Senicapoc; 289656-45-7; 2,2-bis(4-fluorophenyl)-2-phenylacetamide; ICA-17043; MFCD09027349; Senicapoc (USAN); Senicapoc [USAN]; TS6G201A6Q; ICA17043

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 : ~50 mg/mL (~154.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.73 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 (7.73 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.73 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.

---

Solubility in Formulation 4: 2.5 mg/mL (7.73 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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