[0001] This invention pertains to treatment of pain with senicapoc. BACKGROUND

[0002] Existing treatments for neuropathic pain provide effective relief to only 1 in 4 patients (Attal et al., 2010; Finnerup et al., 2015). The majority of these treatments are aimed at targets expressed by neurons of the somatosensory system (e.g. opioid receptors and the α2δ subunit 1 and 2 of voltage gated calcium channels). Expression of these targets in other areas of the CNS are believed to underlie their propensity to cause side effects such as sedation, euphoria and addiction (Finnerup et al., 2015). Recent studies have shown that non-neuronal targets expressed by activated immune cells also contribute to the establishment and maintenance of tactile allodynia in rodent models of neuropathic pain (Ren and Dubner, 2010; Scholz and Woolf, 2007; Zhuo et al., 2011). The clinical relevance of these findings was demonstrated by a recent PET study that showed an increase in PBR28 binding in the thalamus of patients with chronic low back pain (Loggia et al., 2015). PBR28 is a ligand for the translocator protein (TSPO) that increased expression in activated microglia, the CNS resident immune cells (Rupprecht et al., 2010). Collectively, the studies suggest that microglial activation may be a

mechanism common to neuropathic pain models as well as patients with chronic or neuropathic pain. Targeting microglia might provide novel therapeutic options for patients with these debilitating diseases.

[0003] Mechanistically, immune cell receptor activation results in elevations of intracellular Ca ²⁺ concentrations which can subsequently stimulate diverse physiological responses including migration, proliferation, phagocytosis as well as production and release of cytokines, chemokines, prostanoids and reactive oxygen and nitrogen species such as nitric oxide (NO) (Hanisch, 2013). Many studies in models of neuropathic pain have demonstrated that inhibition of these immune cell receptors in the CNS (presumably on microglia) blocks the physiological sequelae of immune cell activation and ultimately pain associated behaviors (Abbadie et al.; Grace et al., 2014; Marchand et al., 2005). In microglia, one important regulator of intracellular Ca ²⁺ concentration is the intermediate conductance calcium-activated potassium channel Kc _a 3.1 (Dale et al., 2016). The K ⁺ efflux resulting from the opening of this channel leads to the hyperpolarization of microglia which in turn facilitates Ca ²⁺ influx resulting in the increased and sustained stimulation of various physiological responses in vitro (Kaushal et al., 2007; Khanna et al., 2001; Schilling et al., 2004).

[0004] The contribution of Kc _a 3.1 to neuropathological processes in vivo has been investigated in models of multiple sclerosis and spinal cord injury using Kc _a 3.1 knock out animals as well as the Kc _a 3.1 inhibitor, TRAM-34. The studies demonstrated that inhibition or loss of Kc _a 3.1 function led to a reduction in lesion size as well as a reduction in cytokine levels in the CNS (Bouhy et al., 2011; Reich et al., 2005). Earlier studies in mouse models of traumatic brain injury also demonstrated that Kc _a 3.1 inhibitors were neuroprotective although cytokines and other markers were not measured (Mauler et al., 2004; Urbahns et al., 2005; Urbahns et al., 2003).

[0005] Accordingly, we evaluated whether inhibition of Kc _a 3.1 alleviates pain behaviors of rats with peripheral nerve injury using senicapoc, a potent, CNS penetrant inhibitor with improved stability and selectivity vs TRAM-34 (Dale et al., 2016; Schilling and Eder, 2004, 2007). Senicapoc is an experimental drug described in U.S. Patent 6,288, 122. Senicapoc has previously been investigated for the treatment of sickle cell anemia and malaria (Ataga et al. 2008; Tubman et al. 2016).

SUMMARY OF THE INVENTION

[0006] Senicapoc reduces Kc _a 3.1 mediated K ⁺ currents and reduced the release of nitric oxide (NO) and interleukin-ΐβ (IL-Ιβ) from cultured rat primary microglia, and reduces pain behaviors of rats across multiple models. Moreover, senicapoc has been shown to penetrate the blood-brain barrier. [0007] Thus, in an embodiment, a method is provided of treating chronic, neuropathic, visceral as well as inflammatory pain in patients with these conditions by the

administration of an effective amount of senicapoc.

[0008] In an embodiment, a pharmaceutical composition is provided containing senicapoc for use in the treatment of chronic, neuropathic, visceral or inflammatory pain in a mammal. The mammal may be a human, or other mammal, such as a pet or livestock.

[0009] In an embodiment, the use of senicapoc is disclosed in the manufacture of a medicament for the treatment of chronic, neuropathic, visceral as well as inflammatory pain. In an embodiment, senicapoc is provided for use in reducing chronic, neuropathic, visceral as well as inflammatory pain in patients.

DESCRIPTION OF THE DRAWINGS

[0010] Fig. 1. Kc _a 3.1 electrophysiology. K ⁺ currents were recorded from primary microglia by using either (A) depolarizing steps or (B) a voltage ramp protocol. In both paradigms, the currents reversed at 0 mV which was the equilibrium potential for K ⁺ . Senicapoc dose dependency inhibited a significant part of the K ⁺ current.

[0011] Fig. 2. Effect of Kca3.1 inhibition on NO and IL-Ιβ release from primary microglia. (A) Primary rat cortical microglia were pre-treated with vehicle or senicapoc at concentrations indicated for 30 minutes followed by addition of LPS (3EU/ml) and incubated for a total of 24 hours. Senicapoc inhibited the production of NO (as measured by its metabolite, nitrite) with an EC50 of 0.9 nM as shown in a representative experiment (n=3). (B) Primary rat cortical microglia were incubated with LPS (3EU/ml) for 24 hours to induce expression of IL-Ιβ. Senicapoc dose dependency inhibited IL-Ιβ release from primary microglia with an IC50 of 1.3 nM (n=3).

[0012] Figure 3. Effect of senicapoc on locomotor activity. In order to evaluate the potential CNS side effects of senicapoc, rats were dosed at 30 and 100 mg/kg and assessed in the locomotor activity assay 4 hours later. Senicapoc had no effect on the total distance the rats traveled at either 30 or 100 mg/kg, p.o. (n=3). Data were analyzed by one-way ANOVA. None of the differences were significant.

[0013] Figure 4. Time course and efficacy of senicapoc in the CCI model. Following nerve injury, CCI rats displayed a marked reduction in the 50% paw withdrawal threshold (g) in response to von Frey stimulation of the injured hind paw compared with their Pre- Op baseline and sham rats. (A) This mechanical allodynia was evident from as early as Day 3 post surgery and was maintained throughout the duration of the experiment. Data are mean +/- S.E.M. n=8 in sham group and n=l 1 in the CCI cohort. *P<0.05 vs sham (two-way repeated measure ANOVA followed by Bonferroni's post test). (B) In a second cohort of rats, the 50% paw withdrawal threshold (g) in response to von Frey stimulation of the injured hind paw was determined in CCI rats (n=12/group) dosed with either vehicle, senicapoc (SEN) or gabapentin (GBP). The efficacy of senicapoc (10, 30 and 100 mg/kg, p.o.) was evaluated at its Tmax, 4 hours after dosing. For vehicle and gabapentin (100 mg/kg), thresholds were determined at 2 hours after administration of 100 mg/kg. Gabapentin significantly reversed the mechanical hypersensitivity of nerve injured rats (66% reversal). The Kc _a 3.1 inhibitor (senicapoc) also reversed the tactile allodynia at 100 mg/kg (48% reversal). Data were analyzed by one-way ANOVA followed by Fisher's post-hoc comparison. * p<0.0001 vs vehicle.

DETAILED DESCRIPTION

[0014] The data in this study demonstrates that senicapoc is a potent blocker of the potassium channel Kc _a 3.1 in rat microglia. Moreover, we found that IL-Ιβ and NO release from cultured microglia can be regulated by senicapoc. These findings confirm that these cells express Kc _a 3.1 and that inhibition of the channel regulates the release of these effector molecules. Senicapoc had an IC50 of 10 nM in electrophysiological experiments and an IC50 of 15 nM and 39 nM in experiments inhibiting the release of IL- 1β and NO. Previous studies have demonstrated that the Kc _a 3.1 inhibitor TRAM-34 can block the release of IL-Ιβ and reactive oxygen species by microglia (Kaushal et al., 2007; Khanna et al., 2001). TRAM-34 however, is not stable and inhibits other non-selective cation channels (Dale et al., 2016; Schilling and Eder, 2004; Wulff and Castle, 2010). Another pharmacological agent, charybdotoxin, was used to assess the role of calcium activated potassium channels, however, this toxin is not selective for Kc _a 3.1 as it also inhibits K _v 1.3, K _v 1.2 and K _Ca l. l (de Novellis et al., 2012).

[0015] In view of the finding that senicapoc is a selective, potent antagonist of the potassium channel Kc _a 3.1 in rat microglia, a method is provided of treating chronic, neuropathic, visceral as well as inflammatory pain in humans by the administration of senicapoc to a patient having chronic, neuropathic, visceral as well as inflammatory pain. In an embodiment, the use of senicapoc is provided in the manufacture of a medicament for the treatment of chronic, neuropathic, visceral as well as inflammatory pain. In an embodiment, an oral dosage form of senicapoc is provided for use in reducing chronic, neuropathic, visceral as well as inflammatory pain in patients with chronic, neuropathic, visceral as well as inflammatory pain. In an embodiment, the oral dosage form may be a tablet, capsule, or powder for dissolution or suspension in a drinkable liquid. In an embodiment, an injectable dosage form of senicapoc is provided for use in reducing chronic, neuropathic, visceral as well as inflammatory pain in patients with chronic, neuropathic, visceral as well as inflammatory pain

[0016] Notably, when senicapoc was screened in a commercially available profiling panel at a concentration approximately 1000-fold higher than the measured IC50 at Kc _a 3.1, appreciable binding was only observed for melatonin 1A, μ- and κ-opioid receptors. Subsequent functional testing in vitro confirmed that the IC ₅ o's obtained for senicapoc at each receptor subtype were in the low micromolar range. Accordingly, at the doses tested here in vivo senicapoc does not achieve sufficiently high free concentrations in plasma, spinal cord or brain to engage these receptors (Table 1).

[0017] Moreover, given that senicapoc had no significant impact on rat locomotor activity when tested up to 100 mg/kg, it is likely that the reversal of injury-induced nociceptive behaviors in CCI rats (see below) did not occur merely via indiscriminate actions on motor circuits. This is important as therapeutically-relevant doses of opioids and gabapentinoids are known to impair locomotor activity in rats and to cause sedation in patients, albeit a head to head comparison of the effects of senicapoc with gabapentin on locomotor activity was not tested in the current study. The lack of significant effect at the low doses may indicate that complete inhibition of Kc _a 3.1 may be required for efficacy.

[0018] Cultured microglia have been demonstrated to highly express Kc _a 3.1 but these findings have not been extended to tissue given the specificity issues with available antibodies (Dale et al., 2016; Lambertsen et al., 2012). Indeed, despite testing of several anti-Kc _a 3.1 antibodies, microglial specific staining in unperturbed CNS tissue has not been shown (Lambertsen et al., 2012). Some reports have suggested that Kc _a 3.1 is expressed on neurons (Bouhy et al., 2011; Engbers et al., 2012; Grundemann and Clark, 2015) but these findings remain controversial (Chen et al., 2011; D'Alessandro et al., 2013; Lambertsen et al., 2012). In injured tissues however, Chen et al did demonstrate KCa3.1 staining, suggesting that expression of the channel on microglia is increased to detectable levels only upon CNS injury. As microglial activation has been well documented in models of stroke and neuropathic pain, it can be inferred that Kc _a 3.1 expression may be increased in the spinal cord of rats with peripheral nerve injury.

Furthermore, they are also well known to participate in inflammatory responses that lead to inflammatory pain. While inflammatory pain can often be treated with generic drugs such as the nonsteroidal anti-inflammatory drugs (NSAIDS) many of these drugs can have significant side effects including gastrointestinal bleeding. Furthermore,

inflammation often accompanies many chronic and neuropathic pain conditions and contribute to the overall pain.

[0019] In addition to microglia (in vitro), Kc _a 3.1 is also highly expressed in peripheral immune cells. Many studies have demonstrated the presence of peripheral immune cells such as macrophages and T cells in somatosensory nerves, dorsal root ganglia as well as the CNS following peripheral nerve injury in rodents (Marchand et al., 2005). While these immune cells are well known to express Kc _a 3.1, their role in nociceptive pain processing is not as well established as for neurons or microglia. Compounds and Formulations

[0020] Senicapoc (ICA- 17043, MedChem Express, Monmouth Junction, NJ) was dissolved at 100 mM in DMSO and diluted in media for in vitro studies. For in vivo studies, senicapoc was formulated with 20% 2-Hydroxypropyl-P-cyclodextrin

(Kleptose®, Roquette, Lestrem, France) as a suspension at 10, 30 and 100 mg/kg.

Gabapentin (Toronto Research Chemicals, Toronto, ON, Canada) was dissolved in saline at 100 mg/mg. All drug solutions were prepared the day of experiments. Acetonitrile (ACN), dimethyl sulfoxide (DMSO), isopropyl alcohol (IP A) and formic acid were purchased from Sigma-Aldrich (St. Louis, MO).

CHO-K1 cells expressing recombinant human Kc _a 3.1

[0021] CHO-K1 cells stably expressing human Kc _a 3.1 (Chantest, Cleveland, OH) were grown in T75 tissue culture flasks to 70-80% confluence (Ham's F12K and 10% Fetal Bovine Serum, Thermo Fisher). On the day of the experiment, the cells were washed with Dulbeco's Phosphate buffered saline, lifted with 2 ml of Detachin™ (Genlantis, San Diego, CA) and centrifuged at 250 x g for 2 minutes. The supernatant was removed and the cells were washed and re-suspended in Qpatch extracellular solution to achieve a final cell density of ~3xl0 ⁶ cells/ml.

QPatch electrophysiology

[0022] Whole-cell patch-clamp experiments were carried out on a QPatch-16 automated electrophysiology platform (Sophion Biosciences, Paramus, NJ). (Jenkins et al., 2013). Kc _a 3.1 channels were activated by including 10 μΜ free Ca ²⁺ in the internal patch pipette solution. Following establishment of the whole-cell configuration, cells were held at -80 mV. Kc _a 3.1 current was elicited by a voltage protocol that held at -80mV for 100 ms then stepped from -90 mV to +90 mV for 600 ms in 20 mV increments. For dose response experiments, Kc _a 3.1 current was measured at 0 mV. The external solution contained (in mM): 140 NaCl, 10 HEPES, 4 KC1, 1 MgCh, 2 CaCh 10 Glucose (pH 7.4). The internal patch pipette solution contained (in mM): 110 K-gluconate, 34 KC1, 1 MgCh, 5 EGTA, 10 HEPES, 4.86 CaChto achieve free Ca ²⁺ concentration of 10 μΜ (pH 7.2). [Ca ²⁺ ]i was calculated according to WEBMAX STANDARD software,

http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm. Currents were measured using the Sophion QPatch software and exported to Microsoft Excel and Prism

(GraphPad, San Diego, CA) for further analysis.

Animals

[0023] All animal studies were conducted in accordance with the recommendations set forth in the Guide for the Care and Use of Laboratory Animals (2011) and with approval of the facility Institutional Animal Care and Use Committee. All rats received food and water ad libitum and were maintained on a 12 h light/dark cycle. For microglial cultures, E14 timed-pregnant Sprague Dawley rats were ordered (Crl:SD, Charles River, Kingston, NY). For pharmacokinetic and locomotor studies, 150-175 g male Sprague Dawley rats (Crl:SD, Charles River) were purchased. For the chronic constriction injury model (CCI), twenty-five -150 g male Sprague Dawley rats were used (Envigo, Indianapolis, IN). Rats were allowed to acclimate for 6 - 7 days prior to any in vivo procedure.

Primary microglial cultures

[0024] Rat primary microglia were prepared and cultured as described by Moller et al (Moller et al., 2000). Mixed glial cultures were maintained in T150 flask (Falcon - Corning, Glendale, AZ) in Dulbecco's modified Eagle medium (DMEM)-GlutaMax (Gibco - Thermo Fischer Scientific, Waltham, MA) containing 4.5 g/1 of D-glucose and supplemented with 10% low endotoxin (0.06 EU/ml) heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (P/S) (Gibco). Cultures were grown in a humidified incubator at 37°C under 5% CO ₂ for 10-14 days at which time they were harvested by tapping the flasks and collecting the

microglia-containing medium. Microglia were pelleted by centrifugation at 276 x g for 5 min, re-suspended in DMEM/10%FBS/PS medium and plated at desired density in poly- d-lysine (PDL)-coated plates (BioCoat - Corning). Purity was assessed by labeling with the microglial maker CD1 lb, which identified >95% of cells as microglia. Microglial IL-Ιβ release

[0025] Rat primary microglia were primed with 3 EU/mL of ultra-pure

Lipopolysaccharide (control standard endotoxin (CSE) Associates of Cape Cod,

Falmouth, MA). After 3.5 hours, vehicle or senicapoc were added and allowed to incubate for 30 minutes. Finally, BzATP (ImM; Sigma- Aldrich, St. Louis, MO) was added to activate P2X7 receptors and trigger release of IL-Ιβ (BzATP also inhibits other receptors such as P2X4 receptors). Cell free supernatants were assayed for IL-Ιβ using a custom rat cytokine assay (N45IA-1; MesoScale Discovery, Rockville, MD). IC50S were determined using Prism (Graphpad, San Diego, CA)

Microglial NO release (measurement of nitrite)

[0026] Rat primary microglia were pre-incubated with vehicle or senicapoc for 30 minutes. Next, 3 EU/ml of ultra-pure Lipopolysaccharide (CSE, Associates of Cape Cod) was added to induce iNOS expression and NO synthesis. After a total incubation time of 24 hours, media was collected, spun down to remove cells and assayed for nitrite (the stable breakdown product of NO) using Griess Reagent (Promega, Madison WI).

Electrophysiology recordings in primary microglia

[0027] Primary rat microglia were plated at a density of 300,000/35 mm dish for 24 hours. Whole cell patch clamp recordings were performed using an EPC9 patch-clamp amplifier and PatchMaster software (HEKA Instruments Inc.). The cell's membrane potential was held at -60 mV. External solution contained (in mM): 140 NaCl, 4 KC1, 1 MgCh, 1.8 CaCli, 10 HEPES, 10 Glucose, pH 7.4. Patch solution contained (in mM): 140 CsCh, 11 EGTA, 2 MgCl ₂ , 10 HEPES, pH 7.2. Drugs were applied by gravity and selected with a capillary tube placed in close proximity to the recording dish. BzATP (Sigma- Aldrich, St. Louis, MO) was applied for 10 seconds.

Determination of rat plasma, brain and spinal cord exposure and free concentration determination

[0028] Male Sprague-Dawley rats (Crl:SD, Charles River) weighing 200-350 g were dosed to determine tissue exposure at 10 mg/kg p.o. (n=3) for screening pharmacokinetics (PK), and at 10, 30 and 100 mg/kg p.o. for the CCI studies (n=3 from each treatment group taken from the last arm of study). In order to confirm the pharmacokinetics of senicapoc, animals were subjected to deep anesthesia with ketamine to obtain CSF via cisternal puncture at 1 h and 4 h post dosing with senicapoc. The rats were then decapitated and trunk blood was collected to in K3EDTA containing tubes. Plasma samples were generated by centrifugation (3500 x g), and stored frozen at -20° C until bioanalysis. The brains were harvested immediately following blood collection and stored frozen until bioanalysis at -20° C. For CCI animals, the plasma and brain harvests as described for the screening PK, although the tissue collection was initiated after the behavioral assessment was completed, approximately 4 hours 15 minutes after dosing. For spinal column harvest, a 20-gauge needle on a 10 ml syringe filled with cold saline was inserted into the spinal cord. The saline was used to flush the spinal column out. The spinal column tissues were then stored frozen at -20 ^° C prior to exposure measurements. Total senicapoc tissue concentrations were determined at Primera Analytical Solutions Corporation (Princeton, NJ) as described in next section.

Bioanalysis of plasma, brain and spinal cord tissues

[0029] Senicapoc concentrations in plasma, brain and spinal cord samples were determined using an LC-10ADVP (Schimadzu) coupled with mass spectrometry (AB Sciex 4000). Prior to analysis, the brain and spinal cord samples were thawed at room temperature, weighed and homogenized using a homogenizing solution

(IPA/H2O/DMSO; 30:50:20) at a ratio of 3/1 (ml/g) to generate tissue homogenates. An aliquot of 50 μΐ plasma, or 50 μΐ brain and spinal cord homogenates is mixed with 150 μΐ of a DMSO/ACN (20:80) solution that contains an internal standard (50 ng/ml). The diluted plasma, brain and spinal cord sample is then centrifuged (-500 g for 15 min at 10°C), supernatant is removed and then injected (10 μΐ) onto LC/MS/MS system for analysis. An Atlantis T3 column 2.6 μ CI 8, 50 x 2.1 mm (Waters, MA) was used for analytical separation of the acid. A 6-minute mobile phase gradient was employed with mobile phase A (0.1% formic acid in water) ramping down from 99% to 1% (0-4 minutes), and then ramping back up to 99% (4- 6 minutes), while ramping up solvent B (1 % formic acid in ACN), from 1% to 99% (0-4 minutes), and then ramping down to 1% (4-6 minutes). Spectra was acquired in positive SRM mode with the parent mass of 324 and a daughter ion of 228.

Determination of plasma and brain free fraction

[0030] The unbound plasma fraction (UBP) or unbound brain fraction (UBBr) of senicapoc were determined in vitro utilizing an equilibrium dialysis method modified slightly from a previously described method (Kalvass and Maurer, 2002). Briefly, naive plasma and brain tissues were homogenized in 3x and 4x w/v in homogenization buffer (50:30:20 H ₂ O:IPA:DMSO), respectively. Senicapoc (10 μΜ) stock solution in DMSO was added to the naive plasma or brain homogenates and subsequently dialyzed against 0.01 M phosphate buffer for 2.5 h with a semipermeable membrane (MW cutoff 2000 Da) using a 96-well HTDialysis Teflon block apparatus (Gales Ferry, CT) incubated at 37 ^° C in 5% CO ₂ . Following equilibration, the buffer sample was fortified with a fixed volume of blank tissue homogenate and the tissue sample was fortified with a fixed volume of buffer. Protein precipitation was performed with ice cold acetonitrile in the presence of an analytical internal standard. The supernatants were quantified for senicapoc using LC-MS/MS as described earlier. The senicapoc "Fraction unbound" was calculated by dividing the peak area response (peak area of analyte/peak area of internal standard) in the buffer compartment by the peak area response in the tissue compartment followed by correcting the dilution factor using the equation suggested by Watson et al. (Watson et al., 2009).

Chronic constriction injury (CO) surgery and behavioral testing

[0031] Peripheral nerve injury was performed according to the method of Bennett and Xie

(Bennett and Xie, 1988), with paw withdrawal thresholds to Von Frey filament stimulation measured as described previously (Chaplan et al., 1994; Tal and Bennett,

1994). [0032] In the first cohort of nerve injured rats, hindpaw mechanical hypersensitivity was evident by day 7 and maintained for the duration of the experiment (see Figure 4A). Pharmacological studies with gabapentin and senicapoc were performed on a second cohort of CCI rats with established hind paw mechanical hypersensitivity between days 14-63 post injury. Experiments were run using a simple cross-over design. Tactile hypersensitivity of rats was evaluated one day prior to drug testing. Drugs were allowed to wash out for one week at which time the mechanical hypersensitivity was reassessed. Rats were then randomly re-assigned to groups prior to the next arm of the study being run. In the final arm, tissue was collected from 3 animals from each group immediately after behavioral assessment of allodynia. Dosing of drugs, assessment of mechanical thresholds and collection of tissues were performed by individual experimenters all blinded to treatments.

[0033] All CCI data are presented as mean +/- S.E.M. and were analyzed using ANOVA followed by Fisher's PLSD post-hoc analysis (Stat View, Cary, NC) when appropriate.

Rat locomotor activity

[0034] Male Sprague-Dawley rats (Charles River) weighing 225-275 g on the test day were divided into groups of 8 animals per treatment. Senicapoc was administered p.o. at doses indicated 4 hours prior to the start of locomotor activity testing. Following the pre- treatment time, rats were placed into 50 x 25 x 20 cm cages lined with crushed corn cob bedding (Bed-o'Cobs ¼ inch, Andersons Lab Bedding, Maumee, OH, USA). The cages were positioned in a Smart Frame Cage Rack (Kinder Scientific, Poway, CA, USA) outfitted with an infrared beam array (7X x 15Y). Rats were allowed to move freely within the cage for 1 hour with beam breaks continuously recorded using

MotorMonitor™ (Kinder Scientific,). Time course and cumulative data were analyzed with Prism 4 (GraphPad, La Jolla, CA). One-way ANOVA with Tukey's Multiple Comparison Post-Hoc Test was used for statistical analysis. Off Target screen of senicapoc

[0035] Screening of senicapoc in vitro against a commercial panel of 50 neuronal receptors, 8 enzymes, 5 transporters was contracted out to CEREP (Celle-Levescault, France http ://www. cerep.fr/cerep/users/pages/ProductsS ervices/pharmacoetAD ME. asp) and 7 Kc _a 3.1 -relevant ion channels was contracted out to Chantest (Cleveland, OH) respectively.

Senicapoc inhibits K ⁺ currents in rat primary microglia

[0036] Kc _a 3.1 is highly expressed on microglia in vitro (Kaushal et al., 2007). The effect of senicapoc was evaluated on microglial K ⁺ currents elicited by either depolarizing steps (Figure 1A) or a voltage ramp protocol (Figure IB) using automated patch clamp analysis. Senicapoc dose dependency (10, 100, 300 and 1000 nM) inhibited the microglial K ⁺ current although not completely (Figure 1A) with an IC50 of 10 nM. This value is in close agreement with the IC50 value (10 nM) generated by patch-clamp studies on CHO-Kc _a 3.1 cells. Some residual K ⁺ current still remained which was most likely not Kc _a 3.1 -sensitive (Kettenmann et al., 2011)

Inhibition of Kc _a 3.1 by senicapoc blocks the release of NO and IL-Ιβ from primary rat microglia

[0037] Primary rat cortical microglia were incubated with either vehicle or senicapoc for 30 minutes prior to the addition of vehicle or ultrapure LPS (3 EU/ml) to stimulate iNOS expression and NO release. After 24 hours, media was assayed for nitrite (stable metabolite of NO). Senicapoc dose dependency inhibited the release of NO from LPS- treated microglia with an average IC50 of 39 nM (Figure 2A), in agreement with previous studies (Kaushal et al., 2007; Khanna et al., 2001). Primary rat cortical microglia were also treated with LPS (3 EU/ml, 3 hours) to stimulate the production of pro-IL-Ιβ. Next, vehicle or senicapoc were added and incubated for an additional 30 minutes followed by the addition of BzATP (1 mM) to activate P2X7 receptors and trigger the activation of caspase 1, its cleavage pro-IL-Ιβ and the release of the liberated IL-Ιβ (another 30 minutes). Senicapoc dose dependency inhibited IL-Ιβ release from primary microglia with an IC50 of 15 nM (Figure 2B).

Pharmacokinetics of senicapoc

[0038] The peripheral pharmacokinetics of senicapoc in rats has been evaluated previously (McNaughton-Smith et al., 2008). Senicapoc had oral bioavailability of 51% with maximum plasma concentration (Cmax) achieved at 4 hours (Tmax) post dose;

however, CNS penetrance had not been evaluated. To improve our understanding of the utility of senicapoc as a tool compound to block Kc _a 3.1 on microglia in the CNS, we measured the levels of senicapoc in the brain, CSF and plasma of rats at 1 and 4 hours after administration. At 10 mg/kg oral dose, senicapoc had a good free brain to free plasma ratio of 2.2 and 2.1 at 1 and 4 h post dose, respectively. Senicapoc showed good systemic distribution with average free plasma concentrations of 17 and 65 nM at 1 and 4 hours, respectively (Table 1). The average free brain concentrations achieved were 37 nM and 136 nM at 1 and 4 hours, respectively. The average measured CSF concentrations were 25 and 121 nM at 1 and 4 hours respectively, similar to what was determined for free brain, indicating the likely establishment of CNS distribution equilibrium of senicapoc by 1-hour post dose. Free brain concentrations at 10 mg/kg were

approximately 4- and 14-fold higher at 1 and 4 hours post dose than the IC50 (10 nM) generated in by patch-clamp studies in microglia. Furthermore, at 1 and 4 hours, the free brain concentrations were 8- and 10- fold higher than the IC50 (15 nM) of senicapoc in the primary microglial IL-Ιβ release assay. This also demonstrates that there is sufficient Kc _a 3.1 target coverage of senicapoc in the CNS.

Table 1. Senicapoc Plasma, Brain and Spinal Cord Exposure and Free Concentration Profile in Rat Screening Pharmacokinetics (PK) and Constriction Injury (CCI) Model

Pharmacological Activity of Senicapoc in Rodents

The potency of Senicapoc in reducing acute and tonic pain, neuropathic pain,

inflammatory pain, post-operative pain and visceral pain was evaluated by comparing semicopac to other well known pain medications such as Morphine, Gabapentin,

Indomethacin and (-)U50 488 H. Senicapoc lOOmg/kg was compared to Morphine 4mg/kg for treatment of acute and tonic pain, neurophatic pain and post-operative pain, to Gabapentin lOOmg/kg for treatment of neurophatic pain, to Indomethacin 30mg and lOmg/kg for treatment of inflammatory pain and to (-)U50 488 H 3mg/kg for treatment of visceral pain. The results show that Senicapoc treated animals have lower sensitivity to in models of chronic, neuropathic, visceral as well as inflammatory pain 240 min after treatment (Table 2). Depending on the test, the percentage of activity was calculated from the mean value of the vehicle-treated animals and compared to naive animals, control paw or cut-off value.

Table 2. Rodent Efficiency Testing

es rom e o ec s or ca a a ase .

Off Target and Side Effect Profile of senicapoc

[0039] Senicapoc was screened in vitro at 10 μΜ against a commercially available panel of 50 neuronal receptors, 8 enzymes, 5 transporters by CEREP and 7 ion channels by Chantest. Of these drug discovery relevant targets senicapoc inhibited only melatonin 1A receptors and μ- and κ-opioid receptors more than 50%. The IC50S for these targets were confirmed in a full dose response curve and determined to be 1.7, 12 and 19 μΜ respectively, well above the IC50 values of senicapoc on Kc _a 3.1 channels, i.e. 10 nM. To assess any overt sedating effects, a frequent side effect of current pain medications, senicapoc was evaluated in the locomotor activity assay at 30 and 100 mg/kg p.o.

Senicapoc did not significantly affect the locomotor activity of rats at either dose tested (Fig- 3).

Time course of CCI model of peripheral nerve injury and dose dependent efficacy of senicapoc

[0040] To test whether Kc _a 3.1 plays a role in the maintenance of mechanical

hypersensitivity following peripheral nerve injury we first determined the onset as well as the duration of hind paw mechanical hypersensitivity in rats with chronic constriction injury (CCI) of the sciatic nerve (CCI). Mechanical hypersensitivity was evident by day 7 and maintained for the duration of the experiment (see Figure 4A). Senicapoc (10, 30 and 100 mg/kg, p.o.) was tested in the same cohort of CCI rats with the positive control gabapentin (100 mg/kg, p.o.), using a simple cross-over design. Kc _a 3.1 inhibition with senicapoc at 100 mg/kg significantly reversed the mechanical hypersensitivity of nerve injured rats by 48%. The positive control, gabapentin, also significantly reduced the tactile allodynia by 66% (Figure 4B).

[0041] Interestingly, a significantly lower tissue exposure profile of senicapoc was observed in CCI animals in comparison to naive rats (Table 1). The difference may be attributed to changes in pharmacokinetics of senicapoc in animals with CCI. However, there was sufficient free drug in CNS tissues (brain and spinal cord) with tissue to plasma disposition ratios similar to naive animals providing free drug concentrations ~11-fold higher than the in vitro IC50 (10 nM) from patch clamp studies in agreement with the observed reversal of tactile allodynia at 100 mg/kg (Table 1).

CONCLUSIONS

[0042]

[0043] Senicapoc reduced Kc _a 3.1 mediated K ⁺ currents and reduced the release of NO and IL-Ιβ from cultured rat primary microglia. In vivo we have shown that senicapoc significantly reverses neuropathic hypersensitivity in CCI rats with efficacy comparable to gabapentin. Importantly, senicapoc did not impair locomotor function in rats. This data suggests that selective inhibition of Kc _a 3.1 by senicapoc will be a novel and effective treatment for neuropathic pain.

DOSAGE FORMS

[0044] Senicapoc may be formulated as an oral or injectable pharmaceutical formulation product for use in chronic, neuropathic, visceral as well as inflammatory pain.

[0045] Oral dosage forms include tablets, capsules, and powders for dissolution or suspension in a drink. Such tablets and capsules may be formulated by any of various methods known in the art, and may include at least one excipient.

[0046] Injectable forms may be formulated for intramuscular or intravenous use. Such injectable formulations may be formulated by any of various methods known in the art, and may include at least one excipient.

REFERENCES

Guide for the Care and Use of Laboratory Animals (2011) 8th ed, Washington (DC).

Abbadie, C, Bhangoo, S., De Koninck, Y., Malcangio, M., Melik-Parsadaniantz, S., White, F.A., 2009. Chemokines and pain mechanisms. Brain Res Rev 60, 125-134.

Ataga KI, Smith WR, De Castro LM, Swerdlow P, Saunthararajah Y, Castro O, Vichinsky E, Kutlar A, Orringer EP, Rigdon GC, Stocker JW, (2008) Efficacy and safety of the Gardos channel blocker, senicapoc (I CA- 17043), in patients with sickle cell anemia, Blood 111:3991-3997; doi: https://doi.org/10.1182/blood-2007-08-110098

Attal, N., Cruccu, G., Baron, R., Haanpaa, M., Hansson, P., Jensen, T.S., Nurmikko, T., 2010. EFNS guidelines on the pharmacological treatment of neuropathic pain: 2010 revision. Eur J Neurol 17, 1113-e 1188.

Bennett, G.J., Xie, Y.K., (1988). A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87-107.

Bouhy, D., Ghasemlou, N., Lively, S., Redensek, A., Rathore, K.I., Schlichter, L.C., David, S., (2011). Inhibition of the Ca(2)(+)-dependent K(+) channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury. J Neurosci 31, 16298- 16308.

Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., (1994). Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53, 55-63.

Chen, Y.J., Raman, G., Bodendiek, S., O'Donnell, M.E., Wulff, H., (2011). The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke. J Cereb Blood Flow Metab 31, 2363-2374.

DAlessandro, G., Catalano, M., Sciaccaluga, M., Chece, G., Cipriani, R., Rosito, M., Grimaldi, A., Lauro, C, Cantore, G., Santoro, A., Fioretti, B., Franciolini, F., Wulff, H., Limatola, C, (2013). KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo. Cell Death Dis 4, e773.

Dale, E., Staal, R.G., Eder, C, Moller, T., (2016). KCa 3.1-a microglial target ready for drug repurposing? Glia 64, 1733-1741.

de Novellis, V., Luongo, L., Guida, F., Cristino, L., Palazzo, E., Russo, R., Marabese, I., DAgostino, G., Calignano, A., Rossi, F., Di Marzo, V., Maione, S., (2012). Effects of intra- ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity. Eur J Pharmacol 676, 41-50.

Engbers, J.D., Anderson, D., Asmara, H., Rehak, R., Mehaffey, W.H., Hameed, S., McKay, B.E., Kruskic, M., Zamponi, G.W., Turner, R.W., (2012). Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells. Proc Natl Acad Sci U S A 109, 2601-2606.

Finnerup, N.B., Attal, N., Haroutounian, S., McNicol, E., Baron, R., Dworkin, R.H., Gilron, I., Haanpaa, M., Hansson, P., Jensen, T.S., Kamerman, P.R., Lund, K., Moore, A., Raja, S.N., Rice, A.S., Rowbotham, M, Sena, E., Siddall, P., Smith, B.H., Wallace, M, (2015). Pharmacotherapy for neuropathic pain in adults: a systematic review and metaanalysis. Lancet Neurol 14, 162-173.

Grace, P.M., Hutchinson, M.R., Maier, S.F., Watkins, L.R., (2014). Pathological pain and the neuroimmune interface. Nat Rev Immunol 14, 217-231.

Grundemann, J., Clark, B.A., (2015). Calcium- Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation. Cell Rep 12, 1715-1722.

Hanisch, U.K., (2013). Functional diversity of microglia - how heterogeneous are they to begin with? Front Cell Neurosci 7, 65.

Jenkins, D.P., Yu, W., Brown, B.M., Lojkner, L.D., Wulff, H., (2013). Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators. Assay Drug Dev Technol 11, 551-560.

Kalvass, J.C., Maurer, T.S., (2002). Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharmaceutics & drug disposition 23, 327-338.

Kaushal, V., Koeberle, P.D., Wang, Y., Schlichter, L.C., (2007). The Ca2+-activated K+ channel KCNN4/KCa3.1 contributes to microglia activation and nitric oxide-dependent neurodegeneration. J Neurosci 27, 234-244.

Kettenmann, H., Hanisch, U.K., Noda, M., Verkhratsky, A., (2011). Physiology of microglia. Physiol Rev 91, 461-553.

Khanna, R., Roy, L., Zhu, X., Schlichter, L.C., (2001). K+ channels and the microglial respiratory burst. Am J Physiol Cell Physiol 280, C796-806.

Lambertsen, K.L., Gramsbergen, J.B., Sivasaravanaparan, M., Ditzel, N., Sevelsted- Moller, L.M., Olivan-Viguera, A., Rabjerg, M., Wulff, H., Kohler, R., (2012). Genetic KCa3.1 -deficiency produces locomotor hyperactivity and alterations in cerebral monoamine levels. PLoS One 7, e47744.

Loggia, M.L., Chonde, D.B., Akeju, O., Arabasz, G., Catana, C, Edwards, R.R., Hill, E., Hsu, S., Izquierdo-Garcia, D., Ji, R.R., Riley, M., Wasan, A.D., Zurcher, N.R., Albrecht, D.S., Vangel, M.G., Rosen, B.R., Napadow, V., Hooker, J.M., (2015). Evidence for brain glial activation in chronic pain patients. Brain 138, 604-615.

Marchand, F., Perretti, M., McMahon, S.B., (2005). Role of the immune system in chronic pain. Nat Rev Neurosci 6, 521-532.

Mauler, F., Hinz, V., Horvath, E., Schuhmacher, J., Hofmann, H.A., Wirtz, S., Hahn, M.G., Urbahns, K., (2004). Selective intermediate-/small-conductance calcium-activated potassium channel (KCNN4) blockers are potent and effective therapeutics in experimental brain oedema and traumatic brain injury caused by acute subdural haematoma. Eur J Neurosci 20, 1761-1768. McNaughton-Smith, G.A., Burns, J.F., Stocker, J.W., Rigdon, G.C., Creech, C, Arrington, S., Shelton, T., de Franceschi, L., (2008). Novel inhibitors of the Gardos channel for the treatment of sickle cell disease. J Med Chem 51, 976-982.

Moller, T., Hanisch, U.K., Ransom, B.R., (2000). Thrombin-induced activation of cultured rodent microglia. J Neurochem 75, 1539-1547.

Reich, E.P., Cui, L., Yang, L., Pugliese-Sivo, C, Golovko, A., Petro, M., Vassileva, G., Chu, I., Nomeir, A.A., Zhang, L.K., Liang, X., Kozlowski, J.A., Narula, S.K., Zavodny, P. J., Chou, C.C., (2005). Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice. Eur J Immunol 35, 1027-1036.

Ren, K., Dubner, R., (2010). Interactions between the immune and nervous systems in pain. Nat Med 16, 1267-1276.

Rupprecht, R., Papadopoulos, V., Rammes, G., Baghai, T.C., Fan, J., Akula, N., Groyer, G., Adams, D., Schumacher, M., (2010). Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov 9, 971- 988.

Schilling, T., Eder, C, (2004). A novel physiological mechanism of glycine-induced immunomodulation: Na+-coupled amino acid transporter currents in cultured brain macrophages. J Physiol 559, 35-40.

Schilling, T., Eder, C, (2007). TRAM-34 inhibits nonselective cation channels. Pflugers Arch 454, 559-563.

Schilling, T., Stock, C, Schwab, A., Eder, C, (2004). Functional importance of Ca2+- activated K+ channels for lysophosphatidic acid-induced microglial migration. Eur J Neurosci 19, 1469-1474.

Scholz, J., Woolf, C. J., (2007). The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10, 1361-1368.

Tal, M., Bennett, G.J., (1994). Extra-territorial pain in rats with a peripheral mononeuropathy: mechano-hyperalgesia and mechano-allodynia in the territory of an uninjured nerve. Pain 57, 375-382.

Venee N. Tubman, Pedro Mejia, Boris E. Shmukler, Amy K. Bei, Seth L. Alper, James R. Mitchell, Carlo Brugnara, and Manoj T. Duraisingh (2016). The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity, Antimicrob Agents Chemother. Jan; 60(1): 613-616; Published online 2015 Dec 31. Prepublished online 2015 Oct 12. doi: 10.1128/AAC.01668-15

Urbahns, K., Goldmann, S., Kruger, J., Horvath, E., Schuhmacher, J., Grosser, R., Hinz, V., Mauler, F., (2005). IKCa-channel blockers. Part 2: discovery of cyclohexadienes. Bioorganic & medicinal chemistry letters 15, 401-404.

Urbahns, K., Horvath, E., Stasch, J.P., Mauler, F., (2003). 4-Phenyl-4H-pyrans as IK(Ca) channel blockers. Bioorganic & medicinal chemistry letters 13, 2637-2639. Watson, J., Wright, S., Lucas, A., Clarke, K.L., Viggers, J., Cheetham, S., Jeffrey, P., Porter, R., Read, K.D., (2009). Receptor occupancy and brain free fraction. Drug metabolism and disposition: the biological fate of chemicals 37, 753-760.

Wulff, H., Castle, N.A., (2010). Therapeutic potential of KCa3.1 blockers: recent advances and promising trends. Expert Rev Clin Pharmacol 3, 385-396.

Zhuo, M., Wu, G., Wu, L.J., (2011). Neuronal and microglial mechanisms of neuropathic pain. Mol Brain 4, 31.