USE OF SENICAPOC FOR TREATMENT OF STROKE

Abstract

Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. The calcium activated potassium channel K.sub.Ca3.1 is expressed selectively in the injured CNS by microglia, and K.sub.Ca3.1 function has been implicated in proinflammatory activation of microglia. K.sub.Ca3.1 is further implicated in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Senicapoc, an investigational drug with a proven safety profile and shown to cross the blood-brain barrier, is a potent and selective K.sub.Ca3.1 inhibitor that intervenes in the inflammation cascade that follows ischemia/reperfusion, and is a potential treatment for stroke.

Claims

1-11. (canceled)

12. A method of treating ischemia/reperfusion brain injury, comprising the inhibition of calcium activated potassium channel K.sub.Ca3.1 on microglia in the brain, by administering to a patient experiencing ischemia/reperfusion brain injury an amount of senicapoc sufficient to inhibit the calcium activated potassium channel K.sub.Ca3.1 in brain microglia where the inhibition of microglia is sufficient to achieve a 50% inhibition in the release of nitric oxide from microglia in the brain when the senicapoc is administered to a patient.

13. A pharmaceutical composition for the treatment of ischemia/reperfusion brain injury comprising inhibiting a calcium activated potassium channel K.sub.Ca3.1 on microglia in the brain, with a composition containing at least one excipient and an amount of senicapoc sufficient to inhibit the calcium activated potassium channel K.sub.Ca3.1 in brain microglia where the inhibition of microglia is sufficient to achieve a 50% inhibition in the release of nitric oxide from microglia in the brain when the senicapoc is administered to a patient.

14. A method of treating ischemia/reperfusion brain injury, comprising the inhibition of calcium activated potassium channel K.sub.Ca3.1 on microglia in the brain, by administering to a patient experiencing ischemia/reperfusion brain injury an amount of senicapoc sufficient to inhibit the calcium activated potassium channel K.sub.Ca3.1 in brain microglia where the inhibition of microglia is sufficient to achieve a 50% inhibition in the release of interleukin 1β from microglia in the brain when the senicapoc is administered to a patient.

Description

DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic showing key neuroimmune pathways and interactions between cells of the CNS in ischemic inflammation and the points of senicapoc activity.

[0018] FIG. 2. Effect of K.sub.Ca3.1 inhibition on NO and regulation of Ca.sup.++ signaling. All downstream effects in microglia (shown in green) and cerebrovascular endothelial cells (shown in red) are attenuated by senicapoc electrophysiology.

[0019] FIG. 3A and FIG. 3B. K.sub.Ca3.1 electrophysiology in rat microglial cells exposed to control or senicapoc. K.sup.+ 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.sup.+. Senicapoc dose dependently inhibited a significant part of the K.sup.+ current.

[0020] FIG. 4A and FIG. 4B. Effect of K.sub.Ca3.1 inhibition on NO and IL-1β 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 lipopolysaccharide (LPS) (3 EU/ml) and incubated for a total of 24 hours. Senicapoc inhibited the production of NO (as measured by its metabolite, nitrite) with an EC.sub.50 of 0.9 nM as shown in a representative experiment (n=3). (B) Primary rat cortical microglia were incubated with LPS (3 EU/ml) for 24 hours to induce expression of IL-1β. Senicapoc dose dependently inhibited IL-1β release from primary microglia with an IC.sub.50 of 1.3 nM (n=3).

DETAILED DESCRIPTION

[0021] Stroke is the leading cause of serious long-term disability and the fifth leading cause of death in the United States. Treatment options for stroke are limited and have limited efficacy. Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. Interfering with the inflammation cascade after stroke holds the promise to modulate stroke outcome. The calcium activated potassium channel K.sub.Ca3.1 is expressed selectively in the injured CNS by microglia. K.sub.Ca3.1 function has been implicated in pro-inflammatory activation of microglia and there is recent literature suggesting that this channel is important in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Accordingly, senicapoc, a K.sub.Ca3.1 inhibitor, may intervene in the inflammation cascade that follows ischemia/reperfusion and limit the damage caused by stroke or other ischemic injury.

[0022] Senicapoc attenuates pro-inflammatory responses in microglia (reducing release of cytokines and nitric oxide) and in epithelial cells attenuating ischemia-induced disruption of the blood-brain barrier (BBB) (FIG. 1) (Staal et al., Neurochem Res. 2017). By modulating elements of the microglial and epithelial cells response to ischemia, senicapoc may influence the neural environment indirectly in a number of ways, for example by enhancing white matter integrity as shown in FIG. 1.

[0023] The ability of senicapoc to inhibit the potassium channel K.sub.Ca3.1 is discussed in detail in our co-pending patent application PCT/US17/57930, filed Oct. 23, 2017.

[0024] K.sub.Ca3.1 is highly expressed on microglia in vitro (Kaushal et al., 2007). The effect of senicapoc was evaluated on microglial K.sup.+ currents elicited by either depolarizing steps (FIG. 3A) or a voltage ramp protocol (FIG. 3B) using automated patch clamp analysis. Senicapoc dose dependently (10, 100, 300 and 1000 nM) inhibited the microglial K.sup.+ current although not completely (FIG. 3A) with an IC.sub.50 of 10 nM. This value is in close agreement with the IC.sub.50 value (10 nM) generated by patch-clamp studies on CHO-K.sub.Ca3.1 cells. Some residual K.sup.+ current still remained which was most likely not K.sub.Ca3.1-sensitive (Kettenmann et al., 2011).

[0025] To show the inhibition of nitric oxide and IL-1β, 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 dependently inhibited the release of NO from LPS-treated microglia with an average IC.sub.50 of 39 nM (FIG. 4A), 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-1β. 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-1β and the release of the liberated IL-1β (another 30 minutes). Senicapoc dose dependently inhibited IL-1β release from primary microglia with an IC.sub.50 of 15 nM (FIG. 4B).

[0026] Unlike TRAM-34, senicapoc has no known off target effects at concentrations that block K.sub.Ca3.1 (Staal et al., 2017). It also does not suffer from metabolic instability or effects on cytochrome P450. Most importantly, senicapoc has been tested in humans in clinical trials without any significant side effects. The finding that senicapoc is also CNS penetrant opens up its use for CNS indications.

[0027] While many devastating neurological and perhaps psychiatric diseases could be potentially treated by senicapoc, the studies of selective inhibitor of K.sub.Ca3.1 with high potency and good CNS penetration as a treatment for stroke lay a foundation for the use of senicapoc on stroke patients.

[0028] Finding a treatment for stroke that can be given beyond the narrow therapeutic window of current treatments would be a major advance. The data on efficacy of the K.sub.Ca3.1 inhibitor, TRAM-34, outside of this narrow therapeutic window suggests that inhibition of K.sub.Ca3.1 could become a promising treatment strategy in acute stroke. The potent and selective K.sub.Ca3.1 inhibitor senicapoc (IC.sub.50 of 11 nM) was initially developed for the treatment of sickle cell anemia (Ataga et al., 2006; Ataga et al., 2011; Ataga et al., 2008; Ataga and Stocker, 2009). The drug was well tolerated in Phase 1 clinical trials in both healthy volunteers and in patients with sickle cell disease (Ataga et al., 2006; Ataga et al., 2011). In a double-blind placebo controlled Phase 2 study, senicapoc (at 10 mg/day) reduced hemolysis and significantly increased hematocrit and hemoglobin levels in patients with sickle cell disease (Ataga et al., 2008). In a subsequent Phase 3 trial, senicapoc was tested for its effects on vaso-occlusive pain crisis (Ataga et al., 2011). However, despite properly engaging erythrocyte K.sub.Ca3.1, reducing hemolysis and increasing hemoglobin and hematocrit levels, senicapoc had no effect on pain outcome measures and the trial was terminated (Ataga et al., 2011). While this was disappointing, it is important to point out that the drug did what it was supposed to do on a molecular and cellular level. The clinical trial failed because the outcome measure chosen were distal to the proposed mode of action and perhaps not completely dependent on this mechanism.

[0029] Importantly, senicapoc has been shown to cross the blood-brain barrier. While the peripheral pharmacokinetics of senicapoc have been described in detail (McNaughton-Smith et al., 2008), the ability of senicapoc to cross the blood-brain barrier has only recently been reported (Staal et al., 2017). After 10 mg/kg oral dosing in rats, senicapoc achieved free plasma concentrations of 17 and 65 nM and free brain concentrations of 37 and 136 nM at one and four hours post-dose, respectively. Cerebrospinal fluid (CSF) concentrations were determined to be 25 and 121 nM at one and four hours post-dosing which are in-line with the free brain concentrations. These data suggest that senicapoc achieves CNS concentrations greater than its IC.sub.50 value for K.sub.Ca3.1 channels (11 nM) and thus should be sufficient to inhibit it (McNaughton-Smith et al., 2008). Furthermore, senicapoc achieves free brain concentrations several fold higher than TRAM-34.

[0030] In the same study, senicapoc's selectivity was assessed in a screen of ˜70 additional neuronal drug targets (50 neuronal receptors, 8 enzymes, 5 transporters and 7 ion channels) (Staal et al., 2017). None of the targets tested was inhibited by senicapoc at 1 μM, providing additional evidence that senicapoc is selective for K.sub.Ca3.1 channels. In vivo, senicapoc was tested in the chronic constriction injury model of neuropathic pain (Bennett and Xie, 1988). Senicapoc dose dependently (10, 30 and 100 mg/kg p.o.) attenuated the mechanical hypersensitivity induced by the peripheral nerve injury, although only the highest dose was significant (Staal et al., 2017). Furthermore, in contrast to reported locomotor effects in kcnn4.sup.−/− mice (Lambertsen et al., 2012) that have no functional K.sub.Ca3.1, the authors did not observe any significant impact of senicapoc on locomotor activity (Staal et al., 2017). While the study does not shed light on the cell types in the CNS that express K.sub.Ca3.1, it clearly demonstrates that senicapoc was efficacious in ameliorating pain behaviors in rats with peripheral nerve injury and these conclusions were supported by the free drug concentrations attained in plasma, brain and CSF.

[0031] Numerous active cellular processes and complex cellular interactions contribute to the resolution of post-ischemic inflammation. senicapoc ameliorated pain behaviors in a model of neuropathic pain (Staal et al., 2017). Since experimental surgery-related inflammation is resolved seven days after the animals are tested, it supported the hypothesis that the efficacy was mediated by inhibition of K.sub.Ca3.1 on microglia in the spinal cord or brain rather than peripheral immune cells. In addition to the prior studies in rats, we report here the ability of senicapoc to penetrate the CNS in mice (see Table 1). The data were similar to those in rats with senicapoc reaching higher levels in brain than plasma and showing a similar t.sub.1/2 demonstrating that senicapoc readily crossed the blood brain barrier and achieved concentrations well above the IC.sub.50. Based on the rat and mouse pharmacokinetic data CNS penetrance in humans seems promising.

[0032] To address in vivo side effects of senicapoc, the most relevant being sedation in pain models, the effect of the drug on rat locomotor activity was tested (Staal et al., 2017). The results showed that senicapoc did not alter activity at doses required for efficacy in the chronic constriction injury model of neuropathic pain. The data suggests that K.sub.Ca3.1 inhibition has few adverse effects. The significance of these pre-clinical findings is enhanced by the human clinical trials that demonstrated that senicapoc is safe and has a low incidence of side effects.

[0033] Based on the animal studies, the major drawback to both TRAM-34 and senicapoc is the short half-life (see Table 1). In contrast to the preclinical studies in rodents, clinical trials showed an unexpected t.sub.1/2 of 23 days in humans. This raises the question whether senicapoc covalently binds to plasma proteins whose t.sub.1/2 is approximately 21 days which is significantly longer than that of the unbound drug. It is important to note that the potential covalent protein binding, should not impact the ability of senicapoc to penetrate the CNS, although it would make dose titration more complex.

[0034] To date, the only CNS disease model in which senicapoc has been evaluated is the chronic constriction injury model of neuropathic pain (Staal et al., 2017).

TABLE-US-00001 TABLE 1 Pharmacokinetics of TRAM-34, NS6180 and senicapoc *IC.sub.50 reported are for human KCa3.1 expressed in recombinant cells. All data are for compounds dosed p.o. TRAM-34 NS1680 Senicapoc Senicapoc Molecular 345 323 323 Weight (g/mol) Species Rat Mouse Dose (mg/kg) 10 10 10 10 T½ (hours) ~2 3.8 1 3.1 T.sub.max (hours) 0.5-1   4 0.33 In-vitro IC.sub.50 (nM)* 20 9.4 hu 12 hu (Wulff et al 2000) C.sub.max-Total Plasma ~2500 186 2,400 709 (nM) (Staal et al., 2017) Brain ~2500 17,000 (Staal et al., 2017) % Plasma 2 2.7 1.5 unbound (Staal et al., 2017) C.sub.max- Brain 0.8 0.8 (nM) (Staal et al, 2017) Unbound Plasma 50 65 C.sub.max- Brain 50 136 Unbound (nM) CSF 121 Reference Chen Strøbæk McNaughton- Previously et al., et al, Smith, 2008 unpublished 2011 2013 data JCBM hu: tested in vitro in the human receptor.

Compounds and Formulations

[0035] In an embodiment, senicapoc may be formulated as an oral pharmaceutical formulation product for use in the prevention or treatment of stroke or ischemic injury as provided herein.

[0036] 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.

[0037] Senicapoc has previously been developed exclusively as an oral formulation, but for stroke patients, an intravenous formulation may be desirable.

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