WO2009099563A2

WO2009099563A2 - Combination of a nmda receptor channel blocker and a pkc activator for treatment of alzheimer's disease

Info

Publication number: WO2009099563A2
Application number: PCT/US2009/000610
Authority: WO
Inventors: Daniel L. Alkon; Changhai Cui
Original assignee: Blanchette Rockefeller Neurosciences Institute
Priority date: 2008-02-05
Filing date: 2009-01-30
Publication date: 2009-08-13
Also published as: WO2009099563A3

Abstract

Background: Excitotoxicity, which is associated with the excessive activation of NMDA receptors, has been implicated as playing an important role in the progressive neuronal damage in.Alzheimer's disease (AD). Memantine, an open-channel blocker of NMDA receptors, is being used clinically to prevent the excitotoxicity. However, excitotoxicity is not the only process in which NMDA receptors can be dysregulated in AD. Recent studies have demonstrated that Aβ targets excitatory synapses, down regulates the surface expression of NMDA receptors, and induces reversible synaptic loss through NMDA receptor dependent pathways. To effectively treat Alzheimer's disease, a new therapeutic approach, which not only reduces the exitotoxicity but also potentiates normal NMDA receptor functions, needs to be explored. Objective: Given the fact that PKC activation potentiates functions of NMDA receptors, we aim to determine effects of the combination of a PKC activator, bryostatin-1, and memantine on the excitotoxicity and synaptic function. Methods: We used a biotinylation method and cell viability assays to determine the surface expression of NMDA receptors and the excitotoxicity of hippocampal neurons with or without the presence of bryostatin-1 and/or memantine. Results: We demonstrated that bryostatin-1 significantly enhanced the surface expression of NMDA receptors in hippocampal neurons. Treatment with the combination of bryostatin-1 together with memantine did not increase the excitotoxicity. On the contrary, it was more effective than either bryostatine-1 or memantine alone in the prevention of neuronal death caused by Aβ, regardless of the synaptic or extrasynaptic origin of the excitotoxicity. Conclusion: This study demonstrates that the combination of bryostatin-1 with memantine has potential as a new therapeutic approach for the effective treatment of Alzheimer's disease.

Description

Combination of a NMDA receptor channel blocker and a PKC activator for treatment of Alzheimer's Disease

FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods to alter cellular modulation of ion channels for the treatment of Alzheimer's disease. The present invention further relates to use the combination of an NMDA receptor channel blocker and a PKC activator to synergistically treat Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive decline of memory and cognitive functions. Although there are many hypotheses for the possible mechanisms of AD, the central theory is that the excessive accumulation of beta amyloid (AB) peptides either directly or indirectly affects a variety of cellular events and leads to neuronal damage and cell death (Selkoe, 1991 , 2002). Among many targets of Aβ accumulation, alternations of NMDA receptor functions have been considered to be one factor responsible for the synaptic failure in AD.

[0003] As a major subfamily of glutamate receptors, NMDA receptors perform essential roles in excitatory synaptic transmission and plasticity via both postsynaptic and presynaptic terminals (Dingledine et al., 1999). These receptors have distinct channel properties that distinguish them from other glutamate receptors. These receptors are highly permeable to Ca2+ and voltage-dependently blocked by Mg2+ (Cull-Candy and Leszkiewicz, 2004). Under normal physiological conditions, NMDA receptors are activated by the transient synaptic release of glutamate. However, under a variety of acute and chronic disease conditions, such as ischemia, seizures, traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, NMDA receptors activated by the transient synaptic release of glutamate. However, under a vaiety of acute and chronic disease conditions, such as ischemia, seizures, traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotropic lateral seclerosis, NMDA receptors are activated in a sustained fashion. This leads to excessive influx of Ca2+ that, in turn, causes neuronal injury and death (Palmer, 2001 ). This process, termed excitotoxicity, has been hypothesized to play a critical role in the progressive neuronal damage in AD (Rogawski and Wenk, 2003). Many research discoveries have provided supporting evidence for this hypothesis. Studies revealed the increased tonic glutamate concentration in AD brain (Jimenez, 1998), which could be attributed to the down-regulated functions of glutamate transporters associated with AD (Topper et al., 1995; Harkany et al., 2000). Furthermore, AD peptides could directly activate or potentiate NMDA receptor responses thus cause the excitotoxicity (Koh et al., 1990; Mattson et al., 1992; Snyder et al., 2005). Moreover, the oxidative stress and the increased intracellular Ca2+ concentration generated by AP further enhance the glutamate-mediated neuronal toxicity. In addition, the membrane depolarization caused by the neuronal injury could relieve the normal Mg2+ block and lead to the over activation of NMDA receptors (Zeevalk and Nicklas, 1992). Thus, many factors that occur during AD pathophysiology can contribute to NMDA receptor mediated excitotoxicity.

[0004) The excitotoxicity theory of AD has led to the emergence of memantine as a therapeutic strategy for AD (Parsons et al., 1999; Rogawski and Wenk, 2003) (Lipton, S. A., US patent 5,614,560). Memantine, l-amino-3,5- dimethyladamantane, is a member of the aminoadamantane class of organic molecules. It blocks NMDA receptors in a voltage-dependent manner with IC50 of 0.5-3uM at -60 to -7OmV membrane potentials (Rogawski and Wenk, 2003). Thus, memantine prevents excitotoxicity by blocking the sustained activation of NMDA receptors in AD. Clinical trials have demonstrated that memantine improves cognitive functions such as attention and memory, and reduces symptoms of insomnia, lack of drive and fatigue (Palmer, 2001 ).

|0005| However, excitotoxicity is not the only way that NMDA receptors are dysregulated in AD. Recent studies from several laboratories have demonstrated that Aβ targets excitatory synapses, down regulates the surface expression of NMDA receptors, and induces reversible synaptic loss through NMDA receptor dependent pathways (Lacor et al., 2004; Snyder et al., 2005; Shankar et al., 2007). Therefore, AB accumulation will significantly reduce the efficacy of the excitatory synaptic transmission and the synaptic plasticity mediated by NMDA receptors (Snyder et al., 2005; Hsieh et al., 2006). Thus, the functions of NMDA receptor can be altered in two opposite directions in AD. First, these receptors are subjected to sustained activation, which leads to the excitotoxicity. Second, the surface expression of these receptors is down regulated, which causes the suppression of NMDA receptor mediated synaptic functions and the neurogenesis. Given the essential roles of NMDA receptors in a variety of neuronal functions, such as synaptic plasticity, dendritic remodeling, neuronal growth and survival (Constantine-Paton and Cline, 1998; Bennett, 2000; Luscher et al., 2000; Hardingham et al., 2003), suppressing functions of NMDA receptors will jeopardize normal brain functions.

[0006] Although the administration of memantine to Alzheimer disease patients blocks the exicitotoxicity, it does not correct the reduced efficacy of the excitatory synaptic transmission and synaptic plasticity. Thus, a new approach that can prevent the excitotoxicity and concurrently recover the normal functions of NMDA receptors are presented in this invention for the effective treatment of AD.

(0007] Protein kinase C (PKC) is one of the largest gene families of protein kinase (Liu and Heckman, 1998). Among various PKC isozymes, 131 1 , 8, y, e, were found in the brain. PKC is primarily a cytosolic protein, but with stimulation it translocates to the membrane. Studies have demonstrated that the translocated PKC can phosphorylate glutamate receptors, including NMDA receptors, as well as other proteins that are located in the postsynaptic density (Suzuki et al., 1993). PKC has several impacts on NMDA receptors (MacDonald et al., 2001). Specifically, PKC enhances the surface expression of NMDA receptors (Xiong et al., 1998; Lan et al., 2001 ), potentiates the peak current amplitude via a tyrosine kinase cascade, and decreases the steady state current vs. peak amplitude ratio by a tyrosine kinase-independent mechanism (MacDonald et al., 2001). Furthermore, PKC has been repeatedly implicated to play important roles in synaptic plasticity and learning memory (Akers et al., 1986; Bank et al., 1989; Malenka et al., 1989; Olds et al., 1989; Olds et al., 1990; Ben-Ari et al., 1992; Alkon et al., 2005; Sossin, 2007). Moreover, studies have demonstrated that one of PKC activators, such as bryostatin-1 , reduces the levels of soluble beta amyloid and enhances recent memory (Etcheberrigaray et al., 2004) (Etcheberrigaray and Alkon, US Patent 6825229).

[0008] Although PKC activation potentiates functions of NMDA receptors, it does not prevent the excitotoxicity associated with the sustained activation of NMDA receptors in AD. Conversely, NMDA receptor channel blockers, such as memantine, prevent the excitotoxicity, they do not recover the down regulated functions of NMDA receptors in AD. In the present invention, we use the combination of an NMDA receptor channel blocker and a PKC activator as a new therapeutic approach to effectively treat AD. Memantine, as a NMDA receptor channel blocker, prevents the excitotoxicity in AD; while bryostatin- 1 , or its analogues, as a PKC activator, restores the physiological functions of NMDA receptors.

SUMMARY OF THE INVENTION

|0009] The invention provides a pharmaceutical composition comprising a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for altering cellular modulation of ion channels and a pharmaceutically acceptable carrier. In one embodiment, the modulation of the pharmaceutical composition is in vivo or in vitro modulation. In another embodiment, ion channel of the invention is a Ca++ channel.

(00010) The invention also provides a pharmaceutical composition that the combined effect of the amounts of the NMDA receptor and the PKC activator administered is greater than the sum of the therapeutic effects of the amounts of the individual therapeutic agents separately administered. In one embodiment, the NMDA channel blocker and the PKC activator are administered simultaneously. In another embodiment, the NMDA channel blocker and the PKC activator are administered sequentially in any order.

[00011] In another aspect, the NMDA receptor channel blocker of the The pharmaceutical composition inhibits NMDA receptor mediated excitotoxicity or prevents sustained activation of NMDA receptor. [00012] In one embodiment, the NMDA receptor channel blocker of the invention is an antagonist for the NMDA receptor or a substance that blocks a major intracellular consequence of NMDA receptor activation.

(00013] In another embodiment, the NMDA receptor channel blocker of the invention is selected from the group consisting of memantine and derivatives thereof that have similar pharmacodynamic effects in respect to alter cellular modulation of ion channels.

|00014] In a preferred embodiment, the NMDA receptor channel blocker of the invention is a memantine, an amantadine, a dextromethorphan, a dextrorphan, an obogaine, a ketamine, a nitrous oxide, a phencyclidine, a tramadol, magnesium, dizocilpine, phencyclidine, tiletamine, budipine, flupirtine, l -[l -(2- thienyl)cyclohexyllpiperidine, (+)-(3S,4S)-7-hydroxy-delta6-tetrahydrocannabinol-l ,l - dimethylheptyl or a combination thereof. Of particular interest is a memantine.

[00015] In another aspect, the PKC activator of the invention enhances surface expression of NMDA receptor or restores the down-regulated function of NMDA receptor.

[00016] In one embodiment, the PKC activator of the invention is a macrocyclic lactone, benzolactam, or pyrrolidinone.

[00017] In preferred embodiment, the PKC activator of the invention is bryostatin that is selected from the group consisting of bryostatin- 1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, - 17, and -18. Of particular interest is bryostatin-1.

[00018] In another embodiment, the PKC activator of the invention is neristatin. Of particular interest is neristatin-1.

[00019] Further, the invention provides a method for altering cellular modulation of ion channels comprising administering a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for altering cellular modulation of ion channels and a pharmaceutically acceptable carrier.

|00020) The invention also provides a method of inhibiting NMDA receptor mediated excitoxicity in a neuronal cell, which method comprises contacting the neuronal cell with an effective amount of a NMDA receptor channel blocker in combination with a PKC activator.

[00021] Provided is also a method of treating a disorder associated with cellular modulation of ion channels, which method comprising administering a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for treating disorder and a pharmaceutically acceptable carrier.

[00022] In one embodiment, the disorder the invention relates to is Alzheimer's

Disease, multi-infarct dementia, the Lewy-body variant of Alzheimer's Disease with or without association with Parkinson's disease, Creutzfeld-Jakob disease, Korsakow's disorder, or attention deficit hyperactivity disorder. Or particular interest is Alzheimer's Disease.

BRIEF DESCRIPTION OF THE DRAWINGS Figure IA-B. Fig. 1 depicts AB reduced the surface expression of NMDA receptors. AB decreased the surface expression of NMDA receptors in hippocampal neurons. A, surface expression of NMDA receptors were detected by biotinylation and Western Blot with anti-NRl antibody. C, control; AB, neurons were treated with l μM AB for three days. B, l μM AB decrease the surface expression of NMDA receptors by %.

Figure 2. Fig. 2 depicts PKC activators, bryostatin-1 and TPA, enhanced surface expression of NMDA receptors. PKC activators, TPA and bryostatin-1 , increased the surface expression of NMDA receptors. Neurons were treated with either 10OnM of TPA or 2nM of bryostatin-1 for 20minuts. NMDARl was detected as described in Figure 1 .

DCI :#8I 5823I 6A Figure 3. Fig. 3 shows results that Memantine did not interfere with the effect of bryostatin- 1 on the surface expression of NMDA receptors. Memantine did not prevent the effect of bryostatin-1 of the surface expression of NMDA receptors. A, 1.5μM memantine had a small enhancement effect on the NMDA receptor expression, 2nM bryostain- 1 further increased NMDA receptor surface expression. B, Percentages of NMDA receptor surface expression changes with 1 μM Aβ, (I μM AB + l .5μM memantin), and (l μM Aβ + l .5μM memantine + 2nM bryostatin-1 ).

Figure 4. Fig. 4 shows Aβ induced neuronal death without excitotoxic stimulation. AB induced neuronal death under various conditions. LDH levels were determined for hippocampal neuros ( 12 day in culture) treated with l μM or AB, (I μM AB + 2.5μM Memantine), (I μM AB + 2nM Bryostatine-1 ), or (l μM AB + 2nM Bryostatin-1 + 2.5μM Memantine).

Figure 5. Fig. 5 depicts excitotoxicity assay on bryostatin- 1 and memantine treated neurons. The combination of bryostatin- 1 with memantine is more effective for the prevention of excitotoxicity in hippocambal neurons. A, effects of memantine and bryostatine-1 on the excitotoxicity mediated by synaptic glutamate receptors. B, effects of memantine and bryostatine-1 on the excitotoxicity mediated by NMDA recetors.

DETAILED DESCRIPTION

[00023] The present invention is related to the combination use of a NMDA receptor channel blocker and a PKC activator to synergistically treat Alzheimer's disease. Specifically, memantine, a NMDA receptor channel blocker, is used to prevent the excitotoxicity, while bryostatin-1 , or its analogues, as a PKC activator is used to promote the surface expression and physiological functions of NMDA receptors.

6B [00024] As used herein, "synergistic" means that the combined effect of the NMDA receptor and the PKC activator is greater than the sum of the therapeutic effects of the amounts of the individual therapeutic agents separately administered.

|0002S) As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition, compound, or solvent with which an active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject. As used herein, "pharmaceutically acceptable carrier" includes, but is not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; preservatives; ^' physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; antioxidants; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials and other ingredients known in the art and described, for example in Genaro, ed. (1985) Remington's Pharmaceutical Sciences Mack Publishing Co., Easton, Pa., which is incorporated herein by reference. In one embodiment the pharmaceutical compositions of the present invention are sterile.

(00026| The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[00027) To treat Alzheimer's disease, bryostatin- 1 and memantine may be administered in various ratios, routes, forms (solid or liquid), and in amounts sufficient to prevent excitotoxicity and potentiate NMDA receptor functions. These two compounds could be administrated in the same or separate form, either simultaneously or sequentially.

[00028] For memantine, the dose could be 20mg per day, as recommended for the

Alzheimer's disease treatment in the form of Namenda. Bryostatine- 1 , which has undergone toxicity and safety studies in animals and humans for the cancer treatment, has a safety limit of 40m/m2/week. A significant reduction in the Ap 40 accumulation was detected when administering bryostatin-1 to transgenic mice APPrV717I)/PSl [A246E] at 4Oug/kg for 5.5 month (Etcheberrigaray et al., 2004). In addition, a dramatic increase of sAPP secretion was detected with 0.1 nM of bryostatin-1.

(00029] In addition to memantine and bryostatin-1, other pharmacologically carriers, diluent, and inactive ingredients, such as preservatives, buffer, antioxidants, could be formulated with memantine and bryostatin-1.

(00030] The combination of memantine and bryostatin-1 can also be used to treat other acute neurological insults and neurological disorders, such as stroke, traumatic brain injury, Huntington's disease, Parkinson's disease, and other neurodegenerative disorders, where both the prevention of the exitotoxicity and normal physiological functions of NMDA receptors are required for the effective treatment.

(000311 Macrocyclic lactones, and particularly bryostatin-1 is described in U.S. Patent

4,560,774 (incorporated herein by reference in its entirety). Macrocyclic lactones and their derivatives are described elsewhere in the art for instance in U.S. Patent 6,187,568, U.S. Patent 6,043,270, U.S. Patent 5,393,897, U.S. Patent 5,072,004, U.S. Patent 5,196,447, U.S. Patent 4,833,257, and U.S. Patent 4,611,066 (each of which are incorporated herein by reference in their entireties). The above patents describe various compounds and various uses for macrocyclic lactones including their use as an anti-inflammatory or anti-tumor agent. Other discussions regarding bryostatin class compounds can be found in: Szallasi et al. (1994) Differential Regulation of Protein Kinase C Isozymes by Bryostatin 1 and Phorbol 12-Myristate 13-Acetate in NIH 3T3 Fibroblasts, Journal of Biological Chemistry 269(3): 21 18-24; Zhang et al. (1996) Preclinical Pharmacology of the Natural Product Anticancer Agent Bryostatin 1, an Activator of Protein Kinase C, Cancer Research 56: 802-808; Hennings et al. (1987) Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin, Carcinogenesis 8(9): 1343-46; Varterasian et al. (2000) Phase II Trial of Bryostatin 1 in Patients with Relapse Low-Grade Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia, Clinical Cancer Research 6: 825-28; and Mutter et al. (2000) Review Article: Chemistry and Clinical Biology of the Bryostatins, Bioorganic & Medicinal Chemistry 8: 1841 - 1860 (each of which is incorporated herein by reference in its entirety).

(00032) Myalgia is the primary side effect that limits the tolerable dose of a PKC activator. For example, in phase II clinical trials using bryostatin-1, myalgia was reported in 10 to 87% of all treated patients. (Clamp et al. (2002) Anti-Cancer Drugs 13: 673-683). Doses of 20 μg/m² once per week for 3 weeks were well tolerated and were not associated with myalgia or other side effects. (Weitman et al. (1999) Clinical Cancer Research 5: 2344-2348). In another clinical study, 25 μg/m² of bryostatin-1 administered once per week for 8 weeks was the maximum tolerated dose. (Jayson et al. (1995) British J. of Cancer 72(2): 461-468). Another study reported that 50 μg/m² (a 1 hour i.v. infusion administered once every 2 weeks for a period of 6 weeks) was the maximum-tolerated dose. (Prendville et al. (1993) British J. of Cancer 68(2): 418-424). The reported myalgia was cumulative with repeated treatments of bryostatin-1 and developed several days after initial infusion. Id. The deleterious effect of myalgia on a patient's quality of life was a contributory reason for the discontinuation of bryostatin-1 treatment. Id. The etiology of bryostatin-induced myalgia is uncertain. Id.

(00033) The National Cancer Institute has established common toxicity criteria for grading myalgia. Specifically, the criteria are divided into five categories or grades. Grade 0 is no myalgia. Grade 1 myalgia is characterized by mild, brief pain that does not require analgesic drugs. In Grade 1 myalgia, the patient is fully ambulatory. Grade 2 myalgia is characterized by moderate pain, wherein the pain or required analgesics interfere with some functions, but do not interfere with the activities of daily living. Grade 3 myalgia is associated with severe pain, wherein the pain or necessary analgesics severely interfere with the activities of daily living. Grade 4 myalgia is disabling.

(00034) The compositions of the present invention increase the tolerable dose of the PKC activator administered to a patient and/or ameliorate the side effects associated with PKC activation by attenuating the activation of PKC in peripheral tissues. Specifically, PKC inhibitors inhibit PKC in peripheral tissues or preferentially inhibit PKC in peripheral tissues. Vitamin E, for example, has been shown to normalize diacylglycerol-protein kinase C activation in the aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. (Kunisaki el al. (1994) Diabetes 43(1 1): 1372-1377). In a double-blind trial of vitamin E (2000 IU/day) treatment in patients suffering from moderately advanced Alzheimer's Disease, it was found that vitamin E treatment reduced mortality and morbidity, but did not enhance cognitive abilities. (Burke el al. (1999) Post Graduate Medicine 106(5): 85-96).

[0003S) Macrocyclic lactones, including the bryostatin class, represent known compounds, originally derived from Bigula neritina L. While multiple uses for macrocyclic lactones, particularly the bryostatin class are known, the relationship between macrocyclic lactones and cognition enhancement was previously unknown.

|00036] The examples of the compounds that may be used in the present invention include macrocyclic lactones (i.e. bryostatin class and neristatin class compounds). While specific embodiments of these compounds are described in the examples and detailed description, it should be understood that the compounds disclosed in the references and derivatives thereof could also be used for the present compositions and methods.

(00037) As will also be appreciated by one of ordinary skill in the art, macrocyclic lactone compounds and their derivatives, particularly the bryostatin class, are amenable to combinatorial synthetic techniques and thus libraries of the compounds can be generated to optimize pharmacological parameters, including, but not limited to efficacy and safety of the compositions. Additionally, these libraries can be assayed to determine those members that preferably modulate α-secretase and/or PKC.

[00038] Several classes of PKC activators have been identified. Phorbol esters, however, are not suitable compounds for eventual drug development because of their tumor promotion activity, (Ibarreta et al. (1999) Neuro Report 10(5&6): 1035-40). Of particular interest are macrocyclic lactones (i.e. bryostatin class and neristatin class) that act to stimulate PKC. Of the bryostatin class compounds, bryostatin- 1 has been shown to activate PKC and proven to be devoid of tumor promotion activity. Bryostatin- 1, as a PKC activator, is also particularly useful since the dose response curve of bryostatin- 1 is biphasic. Additionally, bryostatin-1 demonstrates differential regulation of PKC isozymes, including PKCD, PKCD and PKCD. Bryostatin- 1 has undergone toxicity and safety studies in animals and humans and is actively

10 investigated as an anti-cancer agent. Bryostatin-1 's use in the studies has determined that the main adverse reaction in humans is myalgia. One example of an effective dose is 20 or 30 μg/kg per dose by intraperitoneal injection.

[00039] Macrocyclic lactones, and particularly bryostatin-1 , are described in U.S. Patent

4,560,774 (incorporated herein by reference in its entirety). Macrocyclic lactones and their derivatives are described elsewhere in U.S. Patent 6,187,568, U.S. Patent 6,043,270, U.S. Patent 5,393,897, U.S. Patent 5,072,004, U.S. Patent 5,196,447, U.S. Patent 4,833,257, and U.S. Patent 4,61 1 ,066 (each incorporated herein by reference in its entirety). The above patents describe various compounds and various uses for macrocyclic lactones including their use as an antiinflammatory or anti-tumor agent. (Szallasi et al. (1994) Journal of Biological Chemistry 269(3): 2118-24; Zhang et al. (1996) Carter Research 56: 802-808; Hennings et al. (1987) Carcinogenesis 8(9): 1343-1346; Varterasian et al. (2000) Clinical Cancer Research 6: 825-828; Mutter et al. (2000) Bioorganic & Medicinal Chemistry 8: 1841-1860)(each incorporated herein by reference in its entirety).

[00040] As will also be appreciated by one of ordinary skill in the art, macrocyclic lactone compounds and their derivatives, particularly the bryostatin class, are amenable to combinatorial synthetic techniques and thus libraries of the compounds can be generated to optimize pharmacological parameters, including, but not limited to efficacy and safety of the compositions. Additionally, these libraries can be assayed to determine those members that preferably modulate α-secretase and/or PKC.

[00041] Combinatorial libraries high throughput screening of natural products and fermentation broths has resulted in the discovery of several new drugs. At present, generation and screening of chemical diversity is being utilized extensively as a major technique for the discovery of lead compounds, and this is certainly a major fundamental advance in the area of drug discovery. Additionally, even after a "lead" compound has been identified, combinatorial techniques provide for a valuable tool for the optimization of desired biological activity. As will be appreciated, the subject reaction readily lend themselves to the creation of combinatorial libraries of compounds for the screening of pharmaceutical, or other biological or medically- related activity or material-related qualities. A combinatorial library for the purposes of the

1 1 present invention is a mixture of chemically related compounds, which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support. The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes that need to be carried out. Screening for the appropriate biological property may be done by conventional methods. Thus, the present invention also provides methods for determining the ability of one or more inventive compounds to bind to effectively modulate α-secretase and/or PKC.

[00042] A variety of techniques are available in the art for generating combinatorial libraries described below, but it will be understood that the present invention is not intended to be limited by the foregoing examples and descriptions. (See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14: 83; U.S. Patents 5,359,1 15; 5,362,899; U.S. 5,288,514: PCT publication WO 94/08051; Chen et al. (1994) JACCS 1 6:266 1 : Kerr et al. (1993) JACCS 1 1 5:252; PCT publications W092/10092, W093/09668; W091/07087; and W093/20242; each of which is incorporated herein by reference). Accordingly, a variety of libraries on the order of about 16 to 1 ,000,000 or more diversomers can be synthesized and screened for a particular activity or property.

|00043) Analogs of bryostatin, commonly referred to as bryologs, are one particular class of PKC activators that are suitable for use in the methods of the present invention. The following Table summarizes structural characteristics of several bryologs, demonstrating that bryologs vary greatly in their affinity for PKC (from 0.25 nM to 10 μM). Structurally, they are all similar. While bryostatin- 1 has two pyran rings and one 6-membered cyclic acetal, in most bryologs one of the pyrans of bryostatin- 1 is replaced with a second 6-membered acetal ring. This modification reduces the stability of bryologs, relative to bryostatin-1, for example, in both strong acid or base, but has little significance at physiological pH. Bryologs also have a lower molecular weight (ranging from about 600 to 755), as compared to bryostatin-1 (988), a property which facilitates transport across the blood-brain barrier.

12

[00044] Analog 1 (Wender et al. (2004) Curr Drug Discov Technol. 1 : 1 ; Wender et al.

(1998) Proc Natl Acad Set U S A 95: 6624; Wender et al. (2002) Am Chem Soc. 124: 13648 (each incorporated herein by reference in their entireties)) possesses the highest affinity for PKC. This bryolog is aboutlOO times more potent than bryostatin-1. Only Analog 1 exhibits a higher affinity for PKC than bryostatin. Analog 2, which lacks the A ring of bryostatin-1 is the simplest analog that maintains high affinity for PKC. In addition to the active bryologs, Analog 7d, which is acetylated at position 26, hasvirtually no affinity for PKC.

Analog 2; K₁ = 8.0 nM 3 R = t-Bu

4 R = Ph

5 R = (CH₂J₃P-Br-Ph

Bryostatin 1 ; Kj = 1.35 nM Analog 1 ; /<i = 0.25 nM

|00045) B-ring bryologs are also suitable for use in the methods of the present invention.

These synthetic bryologs have affinities in the low nanomolar range (Wender et al. (2006) Org

13 Lett. 8: 5299 (incorporated herein by reference in its entirety)). The B-ring bryologs have the advantage of being completely synthetic, and do not require purification from a natural source.

3. PKC Ki = 1.2 ± 0.6 nM 4: PKC Kj = 0.67 ± 0.5 πM

5: PKC Kt = 2.0 ± 0.5 nM β: PKC K, = 2.6 ± 0.5 nM

PKC binding affinities for B-ring bryologs

[00046] A third class of suitable bryostatin analogs is the A-ring bryologs. These bryologs have slightly lower affinity for PKC than bryostatin I (6.5, 2.3, and 1.9 nM for bryologs 3, 4, and 5, respectively) but have a lower molecular weight.

(00047) A number of derivatives of diacylglycerol (DAG) bind to and activate protein kinase C (Niedel et al. (1983) Proc. Natl. Acad. Sci. USA 80: 36; Mori et al. (1982) J. Biochem (Tokyo) 91 : 427; Kaibuchi et al. (1983) J. Biol. Chem. 258: 6701). However, DAG and DAG derivatives are of limited value as drugs. Activation of PKC by diacylglycerols is transient, because they are rapidly metabolized by diacylglycerol kinase and lipase (Bishop et al. (1986) J. Biol. Chem. 261 : 6993; Chung et al. (1993) Am. J. Physiol. 265: C927; incorporated herein by reference in their entireties). The fatty acid substitution determines the strength of activation. Diacylglycerols having an unsaturated fatty acid are most active. The stereoisomeric

14 configuration is also critical. Fatty acids with a 1,2-sn configuration are active, while 2,3-sn- diacylglycerols and 1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids are synergistic with diacylglycerols. In one embodiment of the present invention, the term "PKC activator" expressly excludes DAG or DAG derivatives, such as phorbol esters.

[00048) Isoprenoids are PKC activators suitable for use in the methods of the present invention. Famesyl thiotriazole, for example, is a synthetic isoprenoid that activates PKC with a Kd of 2.5 μM. Farnesyl thiotriazole, for example, is equipotent with dioleoylglycerol (Gilbert et al. (1995) Biochemistry 34: 3916; incorporated herein by reference in its entirety), but does not possess hydrolyzable esters of fatty acids. Farnesyl thiotriazole and related compounds represent a stable, persistent PKC activator. Because of its low MW (305.5) and absence of charged groups, farnesyl thiotriazole would readily cross the blood-brain barrier.

[00049] Octylindolactam V is a non-phorbol protein kinase C activator related to teleocidin. The advantages of octylindolactam V, specifically the (-)-enantiomer, include greater metabolic stability, high potency (Fujiki et al. (1987) Adv. Cancer Res. 49: 223; Collins et al. (1982) Biochem. Biophys. Res. Commun. 104: 1 159; each incorporated herein by reference in its entirety)(ECso = 29nM) and low molecular weight that facilitates transport across the blood brain barrier.

15 |00050] Gnidimacrin is a daphnane-type diterpene that displays potent antitumor activity at concentrations of 0.1 - I nM against murine leukemias and solid tumors. It acts as a PKC activator at a concentration of ~3 nM in K562 cells, and regulates cell cycle progression at the Gl/S phase through the suppression of Cdc25A and subsequent inhibition of cyclin dependent kinase 2 (Cdk2) (100% inhibition achieved at 5 ng/ml). Gnidimacrin is a heterocyclic natural product similar to bryostatin, but somewhat smaller (MW = 774.9).

(000511 I ri pallidal is a bicyclic triterpenoid isolated from Iris pallida. Iripallidal displays anti-proliferative activity in a NCI 60 cell line screen with GISO (concentration required to inhibit growth by 50%) values from micromolar to nanomolar range. It binds to PKCa with high affinity (Ki = 75.6 nM). It induces phosphorylation of ERK1/2 in a RasGRP3 -dependent manner. M. W. 486.7. Iripallidal is only about half the size of bryostatin and lacks charged groups.

[00052] Ingenol is a diterpenoid related to phorbol but possesses much less toxicity; It is derived from the milkweed plant Euphorbia peplus. Ingenol 3,20-dibenzoate, for example, competes with [3H]phorbol dibutyrate for binding to PKC (Ki for binding=240 nM) (Winkler et

16 al. (1995) J.Org.Chem. 60: 1381 ; incorporated herein by reference). Ingenol-3-angelate possesses antitumor activity against squamous cell carcinoma and melanoma when used topically (Ogbourne et al. (2007) Anticancer Drugs. 18: 357; incorporated herein by reference).

[00053J Napthalenesulfonamides, including N-(n-heptyl)-5-chloro-l- naphthalenesulfonamide (SC-10) and N-(6-Phenylhexyl)-5-chloro-l-naphthalenesulfonamide, are members of another class of PKC activators. SC-10 activates PKC in a calcium-dependent manner, using a mechanism similar to that of phosphatidylserine (Ho et al. (1986) Biochemistry 25: 4179; incorporated herein by reference). Naphthalenesulfonamides act by a different mechanism from bryostatin and would be expected to show a synergistic effect with bryostatin or a member of another class of PKC activators. Structurally, naphthalenesulfonamides are similar to the calmodulin (CaM) antagonist W-7, but are reported to have no effect on CaM kinase.

17

(00054] The linoleic acid derivative DCP-LA (2-[(2-pentylcyclopropyl)methyl] cyclopropaneoctanoic acid) is one of the few known isoform-specific activators of PKC known. DCP- LA selectively activates PKCε with a maximal effect at 100 nM. (Kanno el al. (2006) J. Lipid Res. 47: 1 146). Like SC-10, DCP-LA interacts with the phosphatidylserine binding site of PKC, instead of the diacylglycerol binding site.

|00055] An alternative approach to activating PKC directly is to increase the levels of the endogenous activator, diacylglycerol. Diacylglycerol kinase inhibitors such as 6-(2-(4-[(4- fluorophenyl)phehylmethylene]-l-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-l-yl)ethyl]-2,3-dihydro-2-thioxo- 4(lH)-quinazolinone (R59949) enhance the levels of the endogenous ligand diacylglycerol, thereby producing activation of PKC (Meinhardt el al. (2002) Anti-Cancer Drugs 13: 725).

[00056] A variety of growth factors, such as fibroblast growth factor 18 (FGF- 18) and insulin growth factor, function through the PKC pathway. FGF- 18 expression is upregulated in learning and receptors for insulin growth factor have been implicated in learning. Activation of the PKC signaling pathway by these or other growth factors offers an additional potential means of activating protein kinase C.

(00057] Growth factor activators, such as the 4-methyl catechol derivatives, such as 4- methylcatechol acetic acid (MCBA), that stimulate the synthesis and/or activation of growth factors such as NGF and BDNF, also activate PKC as well as convergent pathways responsible for synaptogenesis and/or neuritic branching.

(00058] The present compounds can be administered by a variety of routes and in a variety of dosage forms including those for oral, rectal, parenteral (such as subcutaneous, intramuscular and intravenous), epidural, intrathecal, intra-articular, topical and buccal administration. The dose range

18 for adult human beings will depend on a number of factors including the age, weight and condition of the patient and the administration route.

(00059) For oral administration, fine powders or granules containing diluting, dispersing and/or surface-active agents may be presented in a draught, in water or a syrup, in capsules or sachets in the dry state, in a non-aqueous suspension wherein suspending agents may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening or emulsifying agents can be included.

[00060] Other compounds which may be included by admixture are, for example, medically inert ingredients, e.g. solid and liquid diluent, such as lactose, dextrose, saccharose, cellulose, starch or calcium phosphate for tablets or capsules, olive oil or ethyl oleate for soft capsules and water or vegetable oil for suspensions or emulsions; lubricating agents such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate, binding agents such as starches, Arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuff; sweeteners; wetting agents such as lecithin, polysorbates or laurylsuphates; and other therapeutically acceptable accessory ingredients, such as humectants, preservatives, buffers and antioxidants, which are known additives for such formulations.

[00061] Liquid dispersions for oral administration may be syrups, emulsions or suspensions.

The syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol. In particular a syrup for diabetic patient can contain as carriers only products, for example sorbitol, which do not metabolize to glucose or which metabolize only a very small amount to glucose. The suspensions and the emulsions may contain a carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

[00062] Suspension or solutions for intramuscular injection may contain, together with the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol and, if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water that is generally Water for Injection. Preferably, however, they may take the form of a sterile, aqueous, isotonic saline

19 solution. Alternatively, the present compounds may be encapsulated within liposomes. The present compounds may also utilize other known active agent delivery systems.

|00063) The present compounds may also be administered in pure form unassociated with other additives, in which case a capsule, sachet or tablet is the preferred dosage form.

(00064) Tablets and other forms of presentation provided in discrete units conveniently contain a daily dose, or an appropriate fraction thereof, of one of the present compounds. For example, units may contain from 5 mg to 500 mg, but more usually from 10 mg to 250 mg, of one of the present compounds.

|00065] It will be appreciated that the pharmacological activity of the compositions of the invention can be demonstrated using standard pharmacological models that are known in the art. Furthermore, it will be appreciated that the inventive compositions can be incorporated or encapsulated in a suitable polymer matrix or membrane for site-specific delivery, or can be functional i zed with specific targeting agents, capable of effecting site specific delivery. These techniques, as well as other drug delivery techniques are well known in the art.

(00066| All books, articles, or patents references herein are incorporated by reference to the extent not inconsistent with the present disclosure. The present invention will now be described by way of examples, which are meant to illustrate, but not limit, the scope of the invention.

EXAMPLES Example 1

[00067] To mimic the Alzheimer's Disease condition, primary cultured hippocampal or cortical neurons were treated with amyloid beta (AB) peptides for three days. Subsequently, the surface expression of NMDA receptors was determined with a biotinylation assay and Western blot analysis. As shown in- Figure 1, AP reduced the surface expression of NMDA receptors comparing to the control. This indicated that, with the presence of AP peptides, NMDA receptors were not sufficiently expressed on the neuronal membrane. Thus, in addition to the excitotoxicity, AP would suppress the physiological functions of NMDA receptors. To effectively treat Alzheimer's disease, a new therapeutic approach which not only reduces the excitotoxicity but also potentiates NMDA receptor functions is explored in this invention.

20 Example 2

|00068] Memantine, as a NMDA receptor channel blocker, is currently being used as an

Alzheimer's Disease treatment to prevent the excitotoxicity associated with the dementia. To determine whether memantine has an effect on the surface expression of NMDA receptors, experiments were performed using memantine over a concentration range that corresponds to that used in the clinical practice. For Alzheimer's disease treatment, memantine is typically administrated orally in a daily dose of 20mg, and it can be rapidly and completely absorbed to reach a maximum plasma concentration following 4 to 8 hours of the administration. The concentration of memantine in CSF fluid is about 0.2uM - 0.5uM with a daily dose of 20mg for 14 days (Kornhuber and Quack, 1995). The effect of memantine on the surface expression of NMDA receptors was tested at 0.2uM, 0.5uM, and 1.5uM concentrations. As demonstrated in Figure 3, 1.5 uM memantine slightly up regulated the surface expression of NMDA receptors, while 0.2uM and 0.5 uM memantine had no effect (data not shown). This shows that, at 20mg/day dosage, memantine will reduce the excitotoxicity by blocking NMDA receptors, but it will not recover the reduced surface expression of NMDA receptors.

Example 3

(00069] To effectively treat Alzheimer's disease, a new therapeutic approach, which not only reduces the exitotoxicity but also potentiats NMDA receptor in response to synaptic stimulation is explored in this invention. It has been well established that PKC activation enhances the surface expression and modulates functions of NMDA receptors (MacDonald et al., 2001). Bryostatin- 1 , as a partial agonist for PKC, can selectively activate PKC isozymes, such as PKCα, PKCγ, and PKCε, at nM or sub-nM concentrations (Sun and Alkon, 2005). Therefore, whether bryostatin- 1 would affect the surface expression of NMDA receptors was examined. As illustrated in Figure 2, 2nM bryostatin- 1, as well as 10 nM TPA, could enhance the surface expression of NMDA receptors in neurons which were treated with Aβ.

Example 4

[00070| To determine whether bryostatin- 1 can enhance the surface expression of NMDA receptors with the presence of memantine, the surface expression of NMDA receptors under the condition that neurons are treated with Aβ and exposed memantine was examined. As shown in Figure 3, bryostatin-1 could increase the surface expression of NMDA receptors in the presence of memantine.

21 Example S

[00071) Given the fact that PKC activation increases the surface expression of NMDA receptors, one of the concerns of using a PKC activator for the Alzheimer's disease treatment is that it may worsen the excitotoxicity. To validate our strategy, we performed a neuronal viability assay to determine the excitotoxicity in the presence of bryostatin-1 and memantine. We used LDH measurement and fluorescent microscopy methods to evaluate the excitotoxic cell death. As shown in Figure 4, AB treatment, even without the excitotoxic stimulation, caused higher LDH release compared to that of the control. This increased LDH release could be partially prevented by 2ruM bryostatine-1 , but not by memantine, and completely prevented by the combination of bryostatine-1 and memantine. We next determined effects of memantine and bryostatin-1 on the exitotoxic cell death mediated by synaptic vs. extrasynaptic NMDA receptors. The results shown in Figure 5A suggest that with the presence of bicuculline, which causes Ca²⁺ influx through synaptic NMDA receptors, bryostatin-1 plus memantine significantly reduced LDH release comparing to the effect of memantine or bryostatine-1 alone. Thus, the combination of bryostatin-1 with memantine could effectively prevent the excitotoxic cell death mediated by synaptic NMDA receptors. To evaluate the excitotoxic neuronal death mediated by extrasynaptic NMDA receptors, 2004 or 30μM NMDA was added to the culture media to evoke excitotoxicity (Figure 5B). Similar to that mediated by the synaptic NMDA receptors, the excitotoxicity could be more effectively prevented by the combination of memantine and bryostatine-1. These results suggest that treatment with the combination of bryostatin- 1 with memantine does not increase the excitotoxicity. On the contrary, it is more effective than either bryostatine-1 or memantine alone in the prevention of neuronal death caused by A(3 treatment, regardless of the nature of excitotoxicity.

[00072] The results shown in Figure 1-5 demonstrate that using the combination of memantine and bryostatin-1 to synergistically prevent NMDA receptor mediated excitotoxicity and potentiate NMDA receptor functions.

Example 6

[00073J Primary hippocampal or cortical cultures were prepared using E 17-18 embryonic rats.

Briefly, hippocampi or cortices were dissected out from the embryonic rat brain, dissociated; and plated on poly-D-lysine coated 35mm dishes. The neuronal culture was maintained in Neurobasal medium supplemented with B27, 0.5mM Lglutamine, and 25uM glutamate. Three days after plating, the medium was change to the one without glutamate added to reduce the excitotoxicity.

22 Example 7

|00074) 4 days after plating, neurons were treated with IuM of Af3 for three days. To determine the effect of memantine on NMDA receptor expression, 0.2uM, 0.5uM, orl .5uM of memantine were tested. The PKC activator, bryostatine-1, was added to neurons that had been treated with AD or Ar3 + memantine. Biotinylation and Western blot analysis were performed immediately after 25 minute treatment with bryostatin- 1.

Example 8

(00075) Surface expression of NMDA receptors was determined by biotinylation assay followed by Western blot analysis. For the biotinylation experiment, neurons were incubated in sulfo-NHS-LC- biotin (Pierce) for 30 mM. The surface biotinylation was stopped by removal and replacing the above solution with 10 mM D-lysine. Cells were then washed three times with cold PBS and lysed with RJPA buffer containing protease inhibitors. Biotinylated proteins were precipitated with 100 Ul of ImmunoPure Immobilized Streptavidin (Pierce) and separated on a 10% Trisglycine gel. Subsequently, the proteins were transferred to a PVD membrane (Invitrogen) and Western blot analysis was perform using a anti NMDA receptor subunit, NRlA, antibody (Chemicon).

Example 9

(00076) To determine the excitotoxicity, neuron viability assay was performed using the primary cultured hippocampal neurons which were platted in a 96 well plate. Following the treatment with IuM Af3 for three days, neurons were washed once with standard solution and challenged with 20 and 3OuM NMDA either with or without 2.5uM memantine. After incubation at 37oC for 1 hours, neurons were washed and then returned to the original medium. The viability of neurons was determined using CytoTox-One Homogeneous membrane Integrity Assay (Promega).

23

Claims

WHAT IS CLAIMED:

1. A pharmaceutical composition comprising a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for altering cellular modulation of ion channels and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1 , wherein said modulation is in vivo or in vitro modulation.

3. The pharmaceutical composition of claim 1, wherein said ion channel is a Ca++ channel.

4. The pharmaceutical composition of claim 1 , wherein the NMDA receptor channel blocker is present in an amount effective to inhibit NMDA receptor mediated excitotoxicity.

5. The pharmaceutical composition of claim 1, wherein the NMDA receptor channel is present in an amount effective to prevent sustained activation of NMDA receptor.

6. The pharmaceutical composition of claim 1, wherein the NMDA receptor channel blocker is selected from the group consisting of memantine and derivatives thereof.

7. The pharmaceutical composition of claim 1, wherein the NMDA receptor channel blocker is a memantine, an amantadine, a dextromethrophan, a dextrorphan, an obogaine, a ketamine, a nitrous oxide, a phencyclidine, a tramadol, magnesium, dizocilpine, phencyclidine, tiletamiπe, budipine, flupirtine, l-[l-(2-thienyl)cyclohexyllpiperidine, (+)-(3S,4S)-7-hydroxy-delta6- tetrahydrocannabinol-l,l-dimethylheptyl or a combination thereof.

8. The pharmaceutical composition of claim 7, wherein the NMDA receptor channel blocker is memantine.

9. The pharmaceutical composition of claim 1, wherein the PKC activator enhances surface expression of NMDA receptor.

10. The pharmaceutical composition of claim 1, wherein the PKC activator restores the down-regulated function of NMDA receptor.

1 1. The pharmaceutical composition of claim 1, wherein the PKC activator is a macrocyclic lactone.

24

12. The pharmaceutical composition of claim 1, wherein the PKC activator is a benzolactam.

13. The pharmaceutical composition of claim 1, wherein the PKC activator is a pyrrolidinone.

14. The pharmaceutical composition of claim 1 1, wherein the macrocyclic lactone is a bryostatin.

15. The pharmaceutical composition of claim 14, wherein the bryostatin is selected from the group consisting of bryostatin- 1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1, -12, -13, -14, -15, -16, -17, and - 18.

16. The pharmaceutical composition of claim 15, wherein the bryostatin is bryostatin- 1.

17. The pharmaceutical composition of claim 14, wherein the macrocyclic lactone is a neristatin.

18. The pharmaceutical composition of claim 17, wherein the neristatin is neristatin- 1.

19. A method for altering cellular modulation of ion channels comprising administering a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for altering cellular modulation of ion channels and a pharmaceutically acceptable carrier.

20. The method of claim 19, wherein said modulation is in vivo or in vitro modulation.

21. The method of claim 19, wherein said ion channel is a Ca++ channel.

22. The method of claim 19, wherein the combined effect of the amounts of the NMDA receptor and the PKC activator administered is greater than the sum of the therapeutic effects of the amounts of the individual therapeutic agents separately administered.

23. The method of claim 19, wherein the NMDA channel blocker and the PKC activator are administered simultaneously.

24. The method of claim 19, wherein the NMDA channel blocker and the PKC activator are administered sequentially in any order.

25

25. The method of claim 19, wherein the NMDA receptor channel blocker inhibits NMDA receptor mediated excitotoxicity.

26. The method of claim 19, wherein the NMDA receptor channel blocker blocks sustained activation of NMDA receptor.

27. The method of claim 19, wherein the NMDA receptor channel blocker is an antagonist for the NMDA receptor or a substance that blocks a major intracellular consequence of NMDA receptor activation.

28. The method of claim 19, wherein the NMDA receptor channel blocker is selected from the group consisting of memantine and derivatives thereof that have similar pharmacodynamic effects in respect to alter cellular modulation of ion channels.

29. The method of claim 28, wherein the NMDA receptor channel blocker is a memantine, an amantadine, a dextromethrophan, a dextrorphan, an obogaine, a ketamine, a nitrous oxide, a phencyclidine, a tramadol, magnesium, dizocilpine, phencyclidine, tiletamine, budipine, flupirtine, 1 - [ 1 -(2-thienyl)cyclohexyllpiperidine, (+)-(3S,4S)-7-hydroxy-delta6-tetrahydrocannabinol- 1 ,1 - dimethylheptyl or a combination thereof.

30. The method of claim 29, wherein the NMDA receptor channel blocker is a memantine.

31. The method of claim 19, wherein the PKC activator enhances surface expression of NMDA receptor.

32. The method of claim 23, wherein the PKC activator restores the down-regulated function of NMDA receptor.

33. The method of claim 19, wherein the PKC activator is a macrocyclic lactone.

34. The method of claim 19, wherein the PKC activator is a benzolactam.

35. The method of claim 19, wherein the PKC activator is a pyrrol idinone.

36. The method of claim 33, wherein the macrocyclic lactone is a bryostatin.

37. The method of claim 36, wherein the bryostatin is selected from the group consisting of

26 bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, and -18.

38. The method of claim 37, wherein the bryostatin is bryostatin-1.

39. The method of claim 33, wherein the macrocyclic lactone is a neristatin.

40. The method of claim 39, wherein the neristatin is neristatin- 1.

41. A method of inhibiting NMDA receptor mediated excitoxicity in a neuronal cell, comprising the step of contacting the neuronal cell with an effective amount of a NMDA. receptor channel blocker in combination with a PKC activator.

42. The method of claim 41, wherein the combined effect of the amounts of the NMDA receptor and the PKC activator administered is greater than the sum of the therapeutic effects of the amounts of the individual therapeutic agents separately administered.

43. The method of claim 41 , wherein the NMDA channel blocker and the PKC activator are administered simultaneously.

44. The method of claim 41 , wherein the NMDA channel blocker and the PKC activator are administered sequentially in any order.

45. The method of claim 41, wherein the NMDA receptor channel blocker is an antagonist for the NMDA receptor or a substance that blocks a major intracellular consequence of NMDA receptor activation.

46. The method of claim 41, wherein the NMDA receptor channel blocker is selected from the group consisting of memantine and derivatives thereof that have similar pharmacodynamic effects in respect to alter cellular modulation of ion channels.

47. The method of claim 46, wherein the NMDA receptor channel blocker is a memantine, an amantadine, a dextromethrophan, a dextrorphan, an obogaine, a ketamine, a nitrous oxide, a phencyclidine, a tramadol, magnesium, dizocilpine, phencyclidine, tiletamine, budipine, flupirtine, 1- [ 1 -(2-thieny l)cyclohexy 1 lpiperidine, (+)-(3 S,4S)-7-hydroxy-delta6-tetrahydrocannabinol- 1,1- dimethylheptyl or a combination thereof.

48. The method of claim 47, wherein the NMDA receptor channel blocker is a memantine.

27

49. The method of claim 41, wherein the PKC activator enhances surface expression of NMDA receptor.

50. The method of claim 41, wherein the PKC activator restores the down-regulated function of NMDA receptor.

51. The method of claim 41, wherein the PKC activator is a macrocyclic lactone.

52. The method of claim 41 , wherein the PKC activator is a benzolactam.

53. The method of claim 41 , wherein the PKC activator is a pyrrolidinone.

54. The method of claim 51, wherein the macrocyclic lactone is a bryostatin.

55. The method of claim 54, wherein the bryostatin is selected from the group consisting of bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, and -18.

56. The method of claim 55, wherein the bryostatin is bryostatin-1.

57. The method of claim 51, wherein the macrocyclic lactone is a neristatin.

58. The method of claim 57, wherein the neristatin is neristatin- 1.

59. A method of making the pharmaceutical composition of claim 1 , comprising combining the NMDA channel blocker and the PKC activator simultaneously.

60. A method of treating a disorder associated with cellular modulation of ion channels, which method comprising administering a NMDA receptor channel blocker in combination with a PKC activator in an amount effective for treating disorder and a pharmaceutically acceptable carrier.

61. The method of claim 60, wherein the disorder is Alzheimer's Disease, multi-infarct dementia, the Lewy-body variant of Alzheimer's Disease with or without association with Parkinson's disease, Creutzfeld-Jakob disease, Korsakow's disorder, or attention deficit hyperactivity disorder.

62. The method of claim 61 , wherein the disorder is Alzheimer's Disease.

63. The method of claim 60, wherein said modulation is in vivo or in vitro modulation.

64. The method of claim 60, wherein said ion channel is a Ca-H- channel.

28

65. The method of claim 60, wherein the combined effect of the amounts of the NMDA receptor and the PKC activator administered is greater than the sum of the therapeutic effects of the amounts of the individual therapeutic agents separately administered.

66. The method of claim 60, wherein the NMDA channel blocker and the PKC activator are administered simultaneously.

67. The method of claim 60, wherein the NMDA channel blocker and the PKC activator are administered sequentially in any order.

68. The method of claim 60, wherein the NMDA receptor channel blocker inhibits NMDA receptor mediated excitotoxicity.

69. The method of claim 60, wherein the NMDA receptor channel blocker blocks sustained activation of NMDA receptor.

70. The method of claim 60, wherein the NMDA receptor channel blocker is an antagonist for the NMDA receptor or a substance that blocks a major intracellular consequence of NMDA receptor activation.

71. The method of claim 60, wherein the NMDA receptor channel blocker is selected from the group consisting of memantine and derivatives thereof that have similar pharmacodynamic effects in respect to alter cellular modulation of ion channels.

72. The method of claim 71, wherein the NMDA receptor channel blocker is a memantine, an amantadine, a dextromethrophan, a dextrorphan, an obogaine, a ketamine, a nitrous oxide, a phencyclidine, a tramadol, magnesium, dizocilpine, phencyclidine, tiletamine, budipine, flupirtine, 1- [l-(2-thienyl)cyclohexyllpiperidine, (+)-(3S,4S)-7-hydroxy-delta6-tetrahydrocannabinol-l ,l- dimethylheptyl or a combination thereof.

73. The method of claim 72, wherein the NMDA receptor channel blocker is a memantine.

74. The method of claim 60, wherein the PKC activator enhances surface expression of NMDA receptor.

75. The method of claim 60, wherein the PKC activator restores the down-regulated

29 function of NMDA receptor.

76. The method of claim 60, wherein the PKC activator is a macrocyclic lactone.

77. The method of claim 60, wherein the PKC activator is a benzolactam.

78. The method of claim 60, wherein the PKC activator is a pyrrolidinone.

79. The method of claim 76, wherein the macrocyclic lactone is a bryostatin.

80. The method of claim 79, wherein the bryostatin is selected from the group consisting of bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, and -18.

81. The method of claim 80, wherein the bryostatin is bryostatin- 1.

82. The method of claim 76, wherein the macrocyclic lactone is a neristatin.

83. The method of claim 82, wherein the neristatin is neristatin- 1.

30