NEUROTOXICITY BY Kvl.3 INHIBITION

Related Applications

[0001] This patent application claims priority to United States Provisional Patent Application No. 61/495,350, filed June 9, 2011, the entire disclosure of which is expressly incorporated herein by reference. Additionally, this application is a continuation-in-part of copending United States Patent Application Serial No. 12/939,912 entitled 4-Phenoxybutoxy-Substituted Heterocycles and Their Use as Inhibitors of the Kvl.3 Lymphocyte Potassium Channel filed November 4, 2010, which claims priority to United States Provisional Patent Application No. 61/258,134 filed November 4, 2009 and is a continuation in part of United States Patent Application Serial No. 12/498,334 entitled 5 -Phenoxyalkoxypsoralens and Methods or Selective Inhibition of the Voltage Gated Kvl.3 Potassium Channel filed July 6, 2009 and now issued as United States Patent No. 8,067,460 which is a continuation of United States Patent Application Serial No. 10/958,997 entitled 5- Phenoxyalkoxypsoralens And Methods For Selective Inhibition Of The Voltage Gated Kvl.3 Potassium Channel filed October 4, 2004 and now issued as United States Patent No. 7,557,138, the entire disclosure of each such application and patent being expressly incorporated herein by reference.

Field of the Invention

[0002] This invention relates generally to the fields of chemistry, pharmacology and medicine and more particularly to the treatment of neurodegenerative diseases, deterring or reducing neuronal damage following ischemic/hypoxic/anoxic events and treatment of other conditions wherein microglia-mediated neurotoxicity occurs.

Statement Regarding Government Support

[0003] This invention was made with Government support under Grant No. R21 AG038910 awarded by the National Institutes of Health. The Government has certain rights in the invention. Background of the Invention

[0004] Pursuant to 37 CFR 1.71(e), this patent document contains material, which is subject to copyright protection. The copyright owner does not object to facsimile reproduction of the entire patent document, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0005] Microglia are non-neural, interstitial cells of mesodermal origin that form part of the supporting structure of the central nervous system in humans and other mammals. Microglia are tissue resident macrophages of the brain. Microglia come in various forms and may have slender branched processes. They are migratory and, when activated (usually by some instigating stimulus), can act as phagocytes, which engulf and remove nervous tissue waste products.

[0006] In various neurodegenerative diseases, damage to nerve cells is believed to occur, at least in part, due to activation of microglia by some instigating stimulus (an "activator"). For example, in Alzheimer's disease (AD), amyloid plaques accumulate between nerve cells (neurons) in the brain. Amyloid is a term, which broadly refers to protein fragments that the body produces normally. Beta amyloid (Αβ) is a protein fragment that comes from an amyloid precursor protein. In healthy brains, these Αβ protein fragments are broken down and eliminated. However, in AD, the Αβ protein fragments aggregate to form hard, insoluble plaques. Aggregated forms of Αβ as well as soluble precursor forms called oligomeric Αβ act as microglial activators. The activated microglia have a beneficial effect of phagocytizing Αβ deposits, but they also have deleterious neuron-damaging effects, such as direct microglial neuron killing and by causing production of neurotoxic nitric oxide (NO) and inflammatory cytokines.

[0007] Microglia also play a roll in causing brain damage following hypoxic or anoxic insults to the brain. Hypoxic or anoxic brain insults may occur due to various causes, including but not limited to ischemic or hemorrhagic strokes, cardiac arrest and resuscitation, carbon monoxide poisoning, trauma, asphyxiation, strangulation, drowning, hemorrhagic shock, inhalant substance abuse ("huffing"), brain edema, iatrogenic disruption of cerebral circulation during surgery or other medical procedures like irradiation, etc.

[0008] Inhibition of certain cellular potassium channels has been proposed as an approach for reducing microglia-mediated neurotoxicity. Potassium channels are encoded by a super-family of 78 genes and are involved in diverse physiological processes ranging from repolarization following neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. The voltage-gated Kvl .3 channel, is expressed in T and B lymphocytes, macrophages and microglia. However, in contrast to stronger immunosuppressants like calcineurin inhibitors and anti-TNF reagents, Kvl .3 inhibitors do not affect the ability of rodents or primates to respond to or to clear bacterial or viral infections and Kvl .3 is therefore regarded as a relatively safe drug target. Recent findings on the role of Kvl .3 in microglia activation in various experimental models have prompted us to study Kvl .3 in microglia as a potential target for AD.

[0009] AD is the most common cause of dementia among people aged 65 and older in all ethnic groups and is one of the most disabling and burdensome health conditions worldwide. AD is currently estimated to affect 4.5 million Americans and its incidence has more than doubled since 1980. Based on the increasing incidence of AD there is an urgent need for new therapeutics that can either prevent AD or slow its progression. All currently FDA-approved drugs for AD, the three acetylcholinesterase inhibitors Aricept, Razadyne, and Exelon, and the N-methyl-D-aspartate receptor antagonist, Namenda, only treat the symptoms of AD and cannot hold its progression.

[0010] It is desirable for therapies aimed at microglia-mediated neurotoxicity to meet the following goals:

(a) reduce the neurotoxic effects of microglia while at the same time maintaining their neuroprotective functions such as phagocytosis of amyloid-beta deposits;

(b) be specific to microglia so that its inhibition does not adversely affect important neuronal or astroglia functions; and

(c) not be broadly immunosuppressive.

In this patent application, Applicants describe compositions and methods for reducing microglia-mediated neurotoxicity in a manner that meets some or all of these goals. Summary of the Inventions

[0011] In accordance with the present invention, there is provided a method for deterring microglia-mediated neurotoxicity in a human or non-human animal subject, said method comprising the step of inhibiting or blocking the intermediate- conductance calcium-activated potassium channel Kvl .3 in microglia. The inhibition or blocking of the Kvl .3 channels may be accomplished by administering to the subject a therapeutically effective amount of a Kvl.3 inhibiting substance. Examples of Kvl .3 inhibiting substances are described in United States United States Patent Nos. 7,557,138 (Wulff et al.) and 8,067,460 (Wulff et al.) and in co-pending United States Patent Application Serial No. 12/939,912, the entire disclosures of which are expressly incorporated herein by reference. Kvl .3 inhibition can cause relatively mild immunosuppression. Thus, the present invention is particularly suited to treatment of diseases, such as AD, that are characterized by Αβ-induced microglial neurotoxicity while not substantially deterring Αβ phagocytosis. Also, as described herein, in addition to symptomatic treatment, the methods of the present invention are effective to slow the onset or progression of those diseases.

[0012] Further in accordance with the present invention, the methods of the present invention may in some embodiments comprise administering to the subject, in an amount that is therapeutically effective to cause microglial Kvl.3 inhibition, a 5- phenoxyalkoxypsoralen compound of the following General Formula 1 :

wherein: n is 1 through 10, cyclic or acyclic and optionally substituted or

unsubstituted; X is O, S, N or C; and

Rl is aryl, heterocyclyl or cycloalkyl and is optionally substituted with one or more substituents selected from alkyl, alkoxy, amino and its alkyl derivatives, acylamino, carboxyl and its alkyl ester, cyano, halo, hydroxy, nitro and sulfonamido groups.

Numerous specific compounds of General Formula 1 are described in the above- incorporated United States Patent Nos. 7,557,138 (Wulff et al.) and 8,067,460 (Wulff et al.). Included among these compounds is 5-(4-Phenoxybutoxy)psoralen (PAP-1) (also sometimes referred to as 4-(4-Phenoxybutoxy)-7H-furo[3,2- g][l]benzopyran-7-on), which has the following structure:

PAP-1

PAP-1 is a highly potent and selective small molecule Kvl .3 blocker. PAP-1 inhibits the Kvl .3 channel with an IC ₅₀ of 2 nM and exhibits excellent selectivity over other ion channels, receptors and transporters. PAP-1 has a half- life of 3 hours in rats and of 6.7 hours in rhesus macaques. PAP-1 is orally bioavailable and has not exhibited long-term toxicity in rodents or primates. As described in greater detail below, PAP-1 reduces Αβ-induced microglia activation and subsequent neurotoxicity in both dissociated and organotypic hippocampal slice cultures, but does not block the ability of microglia to phagocytose Αβ. In pharmacokinetic studies PAP-1 has been shown to cross the blood brain barrier and to reach brain concentrations that equal or slightly exceed plasma-concentrations.

[0013] Still further in accordance with the present invention, the methods of the present invention may in some embodiments comprise administering to the subject, in an amount that is therapeutically effective to cause microglial Kvl.3 inhibition, a 4- phenoxybutoxy-substituted heterocyclic compound having the following General Formula 1 : wherein Ar is selected from the group consisting of: phenyl, napthlalene-l-yl; anthraquinone-l-yl; phenanthrene-9-yl;

quinoline-4-yl; isoquinolin-5-yl; quinazolin-4-yl; 1 ,2-dihydro-N- methyl-quinolin-2-one-4-yl; 2H-[l]benzopyran-2-one-4-yl; 2- phenyl-4H-[ 1 ]benzopyran-4-one-3yl; 2H-[ 1 ]benzopyran-2-one-5- yl; benzofuran-4-yl; furo[2,3-b]quinolin-4(9H)-one-9-yl; 7,8- dimethoxy-furo [2,3 -b] quinoline-4-yl ; furo [2 ,3 -b] quinoline-4-yl ;

psoralen-8-yl; 5,8-dimethoxy-psoralen-4-yl; 5-methoxy-4-methyl- psoralen-8-yl; 9H-xanthene-9-yl; 7-methyl-5H-furo[3,2- g][l]benzopyra-5-one-4-yl; 9-methoxy-7-methyl-5H-furo[3,2- g] [ 1 ]benzopyran-5-one-4-yl; 5H-furo [3 ,2-g] [ 1 ]benzopyran-5 -one- 4-yl; 2-methyl-6,7-methylendioxy-4H-[l]benzopyran-4-one-5-yl;

2,6-dihydro-8-methyl-pyrano[3,2-g][l]benzopyran-2,6-dione-5- yl

and 7H-furo[3,2-g]chromene-7-thione-4-yl.

[0014] Further in accordance with the present invention, the methods of the present invention are in some embodiments carried out by administering a compound of General Formula 1 or of General Formula 2 or any pharmaceutically acceptable salt thereof alone or in combination with one or more pharmaceutically acceptable carriers, excipients and other ingredients commonly used in pharmaceutical preparations for oral, rectal, intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intrathecal, sublingual, bucal, intranasal, trans-mucosal, trans-dermal, topical, other enteral, other parenteral and/or other possible route(s) of administration.

[0015] Further in accordance with the invention, in some embodiments, the inhibition or blockade of voltage-gated potassium channel Kvl .3 may be carried out in a manner that reduces neurotoxic effects of the microglia without preventing beneficial (e.g., phagocytic) effects of the microglia. [0016] Still further in accordance with the invention, the method may be carried out to deter or slow neuron damage in subjects who suffer from a neurodegenerative disease. Some such subjects may have Αβ deposits (such as those suffering from Alzheimer's Disease or who are in the process of developing Alzheimer's Disease) and the inhibition or blockade of the voltage-gated potassium channel Kvl.3 may be carried out in a manner that reduces at least one neurotoxic effect of microglia (e.g., microglia-mediated neuronal killing, microglial production of NO and/or microglial cytokine production) while not preventing microglia from phagocytosing Αβ deposits.

[0017] Still further in accordance with the invention, in some embodiments, the method will be carried out to reduce neural damage in subjects who have suffered or are suffering an ischemic, anoxic or hypoxic conditions, events or insults, such as those who suffer a) ischemic stroke, b) hemorrhagic stroke, c) cardiac arrest and resuscitation, d) carbon monoxide poisoning, e) trauma, f) asphyxiation, g) strangulation, h) drowning, i) hemorrhagic shock, j) inhalant substance abuse or huffing, k) brain edema and 1) iatrogenic disruption of cerebral circulation during a surgery or other medical procedure.

[0018] Still further aspects and details of the present invention will be understood upon reading of the detailed description and examples set forth herebelow.

Brief Description of the Drawings

[0019] Figure 1A is a bar graph showing NF-kB activation in Microglia after 2 hours of treatment with either control (vehicle only), ΑβΟ, ΑβΟ + doxycycline or ΑβΟ + ΡΑΡ-1.

[0020] Figure IB is a bar graph comparing NO production by microglia after 24 hr of treatment with either control (vehicle only), ΑβΟ, ΑβΟ + doxycycline or ΑβΟ + PAP-1.

[0021] Figure 2 is a bar graph comparing microglial cell-associated fluorescence as measured by flow cytometry following 2 hours of pre-treatment with either control (vehicle only), anti-SRA (scavenger receptor A) antibody, doxycycline or PAP-1.

[0022] Figure 3 is a graph comparing the patch clamp response of the cultured microglia to a 500 millisecond pulse following treatment with control (saline only) and Ι μΜ PAP-1. [0023] Figure 4A is a graph showing that AbO and lipopolysaccharides (LPS) significantly increase Kv peak current density (** p<0.01) in microglia in a patch clamp assay.

[0024] Figure 4B is a graph showing the patch clamp response of the cultured microglia to a 500 millisecond pulse following a 48 hour pre-treatment with control (saline).

[0025] Figure 4C is a graph showing the patch clamp response of the cultured microglia to a 500 millisecond pulse following a 48 hour pre-treatment with ΑβΟ.

[0026] Figure 4D is a graph showing the patch clamp response of the cultured microglia to a 500 millisecond pulse following a 48 hour pre-treatment with LPS.

[0027] Figure 4E is a bar graph of qRT-PCR data showing that 24-hr treatment of microglia with AbO and LPS significantly increased the transcript level of Kvl .3 (n = 3,p < 0.05).

[0028] Figure 5 A shows Kv currents from an LPS activated microglial cell in response to voltage-steps from -80 mV to +100 mV in increments of 20 mV.

[0029] Figure 5B shows a Boltzman plot of the data in Figure 5A.

[0030] Figure 5C shows the characteristic use-dependence of Kvl .3 in an LPS activated microglial cell. Currents were elicited every second by stepping the membrane from -80 mV to +40 mV.

[0031] Figure 5D shows the effect of 100 pM of the Kvl .3 blocking peptide ShK- 186 on the microglial Kv current.

[0032] Figure 5E shows the effect of 1 nM of the Kvl .3 blocking peptide MgTX on the microglial Kv current.

[0033] Figure 5F shows the effect of 10 nM of PAP-1 on the microglial Kv current.

[0034] Figure 6 shows that freshly isolated microglia from the brains of 4 and 6 months old transgenic mice, which are widely used as a model for Alzheimer's disease express a higher Kvl .3 current density than microglia from 4 month-old control (wild- type) mice.

[0035] Figure 7 shows that oligomeric ΑβΟ suppresses LTP (lont-term synaptic potentiation) in rat brain slides and that treatment with PAP-1 prevents this suppression of LTP. Detailed Description and Examples

[0036] The following detailed description and the accompanying drawings to which it refers are intended to describe some, but not necessarily all, examples or embodiments of the invention. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The contents of this detailed description and the accompanying drawings do not limit the scope of the invention in any way.

General Methodology Used for Immunohistochemical analysis (IHC) o f microglia activation and Kyl.3 expression

[0037] In the following examples and accompanying drawings, reference is made to various experiments wherein IHC analysis was carried out. In such examples, routinely cryopreserved mouse brain tissue was cryosectioned alternating for IHC and Western blot for evaluating microglia activation. Antibodies were used to 1) IBA-1 to reveal all microglia, 2) CDl lb and SRAl to reveal activated microglia, 3) TNF-a and IL-6 to reveal the classical activation state, 4) IL-4 and IL-13 for the alternative activation state, and 5) Kvl.3. Numbers of microglia in specific brain regions will be quantified using unbiased stereology in sections chosen from specified coordinates. We have successfully used two antibodies, a polyclonal antibody from Sigma and a monoclonal antibody, which was developed by the University of California at Davis/National Institutes of Health NeuroMab Facility (www. Neuromab.com) to detect Kvl .3. Multiplex immunostaining of Kvl .3 and microglia activation markers such as CDl lb or SRA reveal the increased expression of Kvl.3 in activated microglia. Applicants also used unbiased and sensitive laser scanning cytometry (LSC) to analyze multiplex staining. In this automated method, location- and marker- specific quantitative data regarding immunoreactivity, size, and morphological features can be easily linked and compared.

[0038] Where morphometric analysis of dendrites was carried out, Paraffin embedded hippocampus was Golgi-stained and analyzed by Neurolucida utilizing the Sholl method of concentric circles.

[0039] In instances where electrophysiology is used, CDl lb ⁺ microglia were isolated using Percoll separation and anti-CD l ib magnetic beads, attached to poly- lysine coated cover slips and immediately used for whole-cell patch-clamp experiments. This method effectively eliminates contaminating astrocytes and endothelial cells and takes less than 4 hrs. A small aliquot will be fixed for later flow cytometrical analysis for purity of microglia. K l.3 channels will be recorded in the whole-cell mode of the patch-clamp technique. The molecular identity of the currents will be further confirmed by their sensitivity to the Kvl.3 blockers used in the following examples. With this approach we envision that we will be able to study at least 40-50 cells per preparation. In parallel to the electrophysiological experiments we will also determine the expression of Kvl .3 and other Kv-1 family channels like Kvl .5 by qRT-PCR as previously described (48).

Kyl.3 expression is increased in plaque-associated microglia

[0040] Applicants investigated 5XFAD and APPswe/PSlDe9 mice, which are animal models of AD. Hippocampal sections from 5xFAD mice and wild-type (Wt) littermates were stained with anti-Kvl .3 and the amyloid dye FSB. FSB demonstrated the typically small amyloid plaques in 5xFAD mice. In 5xFAD mice, the Kvl .3 stain was coarsely granular in contrast to the finely diffuse stain seen in wt mice. Also, microglia surrounding an amyloid plaque in hippocampal sections from APPswe/PSlDe9 mice were doubly stained with anti-Kvl.3 and CDl lb. Kvl.3 was localized to CD1 lb-immunoreactive activated microglia closely associated with amyloid plaques.

[0041] Separate experiments showed that Kvl .3 antibodies did not stain neurons, astrocytes, or oligodendrocytes, in keeping with its reported expression pattern in the brain.

Kyl.3 blockade inhibits Αβ-induced microglia activation and microglia-mediated neurotoxicity

[0042] Applicants found that the specific Kvl .3 blocker PAP-1 inhibited signs of microglia activation induced by ΑβΟ in cultured mouse microglia, such as proliferation and morphological transformation, as well as NFK-B activation and nitric oxide (NO) production. In addition, PAP-1 also blocked increased microglia release of tumor necrosis factor-a, NFK-B activation, and NO production induced by ίΑβ stimulation. Figures 1A and IB show data regarding ΑβΟ-induced NFK-B and NO, respectively. [0043] Figure 1A is a bar graph showing NF-kB activation in Microglia after 2 hours of treatment with either control (vehicle only), ΑβΟ, ΑβΟ + doxycycline or ΑβΟ + PAP-1. Two (2) hours after administration of the indicated treatment, the mouse brains were sectioned and immunostained with an antibody for p65 of NFKB to mark cells with NFKB activation. Numbers of p65-positive cells per 200 DAPI- labeled cells were determined.

[0044] Figure IB is a bar graph comparing NO production by microglia after 24 hr of treatment with either control (vehicle only), ΑβΟ, ΑβΟ + doxycycline or ΑβΟ + PAP-1. Measurements were made in the conditioned medium and normalized to the amount of total cellular protein in each culture. n=4-6, _* p < 0.001 compared with control, _** p < 0.001 compared with the "ΑβΟ" group. ΑβΟ, 20 nM, doxycycline, 20 μΜ, and PAP-1, 1 μΜ. NO released by ΑβΟ-treated microglia is the major soluble mediator of ΑβΟ-induced microglial neurotoxicity. This toxicity was blocked by co- treating ΑβΟ-stimulated cultured microglia with PAP-1.

[0045] Applicants also performed in situ experiments using hippocampal slices, which better reflect the conditions in the brain in terms of microglial density and their interaction with astroglia and neurons, showed that PAP-1 treatment substantially reduced ΑβΟ-induced microglia activation and blocked ΑβΟ-induced neuronal damage (indicated by propidium iodide uptake and Fluoro-Jade C staining). Three consecutive hippocampal slices were obtained from mouse brain and from mice received the same indicated treatment. One slice was used for CD1 lb staining (green) for activated microglia (slices outlined by Hoechst stain), one for propidium iodide (PI) uptake, and one for Fluoro-Jade C stain for neuronal damage. DG: dentate gyrus. ΑβΟ, 20 nM; doxycycline, 20 μΜ; and PAP-1, 1 μΜ. Because of the restricted microglial expression of Kvl .3 in the brain, these observations support a conclusion that the PAP-1 effect was through inhibiting microglial Kvl.3.

PAP-1 did not impair the ability of microglia to phagocytose Αβ

[0046] Using an Αβ uptake assay, Applicants pretreated microglia with either control (vehicle only), anti-SRA (scavenger receptor A) antibody, doxycycline or PAP-1. Figure 2 is a bar graph showing cell-associated fluorescence as measured by flow cytometry after 2 hrs of such pre-treatment. («=3, _* p < 0.01 and _** p < 0.001 compared with control). The anti-SRA antibody and doxycycline pretreatments caused significant decreases in APcell-associated fluorescence but PAP-1 pretreatment did not. Thus, PAP-1 did not impair the ability of microglia to phagocytose Αβ but doxyxyxline did. These data suggest that using PAP-1 for treatment of amyloid neurodegenerative diseases (such as AD) may have an advantage over doxycycline treatment in that PAP-1 does not hamper Αβ clearance by microglia.

Kyl.3 is expressed and functional in mouse microglia

[0047] In order to determine if Kvl.3 is indeed functionally expressed in mouse microglia Applicants performed electrophysiological experiments on cultured mouse microglia in the whole-cell mode of the patch-clamp technique. Kv currents were elicited with 500 millisecond pulses from a holding-potential of -100 mV to +40 mV applied every 45 sec. Under these conditions a Kv current exhibiting the characteristic use-dependence and inactivation of Kvl .3 was observed in a majority of cells. Figure 3 is a graph comparing the response of the cultured microglia to a 500 millisecond pulse following treatment with control (saline only) and Ι μΜ PAP-1. These data demonstrate that Kvl .3 is expressed in mouse microglia.

Stimulation with AbO and LPS Increases Kyl.3 Expression in Cultured Microglia

[0048] Microglia cultured from newborn C57B1/6 mice were treated with lipopolysaccharides (LPS) or AbO for 24 or 48 hrs. Figure 4A is a graph showing that AbO and LPS significantly increase Kv peak current density (** p<0.01) determined by whole-cell voltage-clamp recordings. Kv currents were elicited by 500 ms voltage steps from -80 to 40 mV (representative traces on right).

[0049] Figure 4B shows the patch clamp response of the cultured microglia to a

500 millisecond pulse following a 48 hour pre-treatment with control (saline).

[0050] Figure 4C shows the patch clamp response of the cultured microglia to a

500 millisecond pulse following a 48 hour pre-treatment with ΑβΟ.

[0051] Figure 4D shows the patch clamp response of the cultured microglia to a

500 millisecond pulse following a 48 hour pre-treatment with LPS demonstrating that microglial activation increases Kvl .3 expression.

[0052] Additionally, cultured microglia were immuno-fluorescently stained for Kvl .3 and the microglial marker CD l ib, and counterstained with DAPI in accordance with the techniques described generally above. Both LPS and AbO stimulated the activated morphology of microglia and enhanced Kvl.3 immunoreactivity.

[0053] Figure 4E shows qRT-PCR data indicating that 24-hr treatment of microglia with AbO and LPS significantly increased the transcript level of Kvl .3 (n = 3,/? < 0.05).

The Kv Channel in AbO-Stimulated Microglia Exhibits the Biophysical Properties of

[0054] Figures 5 A through 5C show Applicants conducted biophysical characterization of cultured microglia by whole-cell voltage-clamp recordings of currents elicited by voltage steps from -80 to 100 mV in 20 mV increments with Boltzmann fit of normalized peak currents: AbO (Vl/2 - 25.6 mV) Use-dependent inactivation elicited by repetitive depolarization from -80 to +40 mV (1 pulse/sec) for 10 pulses. Figures 5 A through 5F show a pharmacological characterization of currents from cultured microglia stimulated for 48 hours with AbO or LPS (IC ₅₀ s for the two conditions): ShK-186 (68.5 pM and 79.2 pM), MgTX (79.7 pM and 78.9 pM), PAP-1 (6.8 nM and 9.5 nM), respectively. ShK-186 is a novel analog of Shk, a natural peptide isolated from the sea anemone, Stichodactyla heliantus. ShK-186 has been shown to be a selective and potent blocker of the Kvl.3 potassium channel.

Microglia in 5xFAD Mice Express More Functional Kvl.3 Than WT Microglia

[0055] Figure 6 shows Peak K+ current densities from microglia isolated from WT and 5xFAD mice, determined by whole-cell voltage-clamp recordings, elicited by 500 ms voltage step -80 to 40 mV.

[0056] Additionally, cerebral sections from 4 month-old WT and 5xFAD mice were immunostained with anti-Kvl .3 (red) and the amyloid dye FSB (blue) and examined in accordance with the general methods described above. Representative images of an amyloid plaque were also costained for Kvl .3 (red) and CD l ib (green) and counterstained with DAPI (blue). Enhanced Kvl.3 immunoreactivity was observed in microglia around the FSB-positive amyloid plaques.

[0057] These data indicate that 5xFAD Mice Express More Functional Kvl .3 Than WT Microglia. PAP-1 Prevents the Inhibitory Action of ΑβΟ on the Induction of CAl LTP in Rat Hippocampal Slices

[0058] Figure 7 summarizes an experiment wherein, under control conditions, CAl LTP was induced by high frequency stimulation (HFS), which consists of 4 trans of 100Hz basal intensity stimulation lasting for Is per train. In control (vehicle only) group, following the HFS, the amplitude or slope of fEPSP was increased to 209 ± 28% of baseline at 45 min after HFS. The bath application of AbO (50 nM) for 10 min blocked the induction of LTP (126±6.7% of control). Pretreatment with PAP-1 (10 μΜ) for 30 min prior to AbO application prevented the inhibition of AbO on the induction of LTP. The mean of five consecutive measurements at the end of LTP induction (45 min) was normalized to the baseline (100%) which was the mean of five consecutive measurements just before the HFS.

[0059] It is to be appreciated that, although the invention has been described hereabove with reference to certain examples or embodiments of the invention, various additions, deletions, alterations and modifications may be made to those described examples and embodiments without departing from the intended spirit and scope of the invention. For example, any elements, steps, members, components, compositions, reactants, parts or portions of one embodiment or example may be incorporated into or used with another embodiment or example, unless otherwise specified or unless doing so would render that embodiment or example unsuitable for its intended use. Also, where the steps of a method or process have been described or listed in a particular order, the order of such steps may be changed unless otherwise specified or unless doing so would render the method or process unsuitable for its intended purpose. Additionally, the elements, steps, members, components, compositions, reactants, parts or portions of any invention or example described herein may optionally exist or be utilized in the substantial absence of other elements, steps, members, components, compositions, reactants, parts or portions unless otherwise noted. All reasonable additions, deletions, modifications and alterations are to be considered equivalents of the described examples and embodiments and are to be included within the scope of the following claims.