WO2002072025A2 - Improving postsynaptic response by a combination of 4-minopyridine and agonist - Google Patents

Abstract

A method for improving postsynaptic neurotransmission comprises administering a 4-AP agent in combination with a postsynaptic agonist to prolong and potentiate agonist-induced postsynaptic elevation of intracellular Ca2+ levels as compared to the levels in the absence of the 4-AP agent.

Description

IMPROVING POSTSYNAPTIC RESPONSE BY A COMBINATION OF 4-AMINOPYRIDINE AND AGONIST

BACKGROUND The invention relates to methods, compounds and compositions for enhancing postsynaptic response to neurotransmitters by administering a 4-aminopyridine ("4-AP") or an analog in conjunction with a postsynaptic agonist in an amount effective to increase postsynaptic calcium mobilization, membrane voltage changes, contractions and/or secretions, h particular, the invention relates to use of combinations such as 4-AP and acetylcholinesterase (AChE) inhibitors or other postsynaptic agonists to treat traumatic brain injuries and neuromuscular and neurodegenerative disorders including myasthenia gravis and multiple sclerosis.

4-aminopyridine (4-AP) and related molecules have been widely recognized for their ability to block voltage-sensitive K⁺ channels (Aronson, 1992). 4-AP has also been used as a therapeutic agent for a number of neurological and neuromuscular disorders. A beneficial role for 4-AP and related molecules has been disclosed for multiple sclerosis (Schwid et al, 1997; Fujihara and Miyoshi, 1998), myasthenia gravis (Li and Zhang, 1994), and in a canine model of motoneuron disease (Pinter et al, 1997). Clinical applications of 4-AP have been extended to traumatic spinal cord injury (Segal et al, 1999) and to neurodegenerative disorders such as Alzheimer's disease (Andreani et al, 2000). The mechanism of action of 4-AP has been presumed to be the blockade of voltage-activated K⁺ channels, which, in turn, caused neuronal depolarization and potentiation of neurotransmission (Smith et al, 2000).

A number of reports address possible direct effects of 4-AP on calcium homeostatic mechanisms (Glover, 1981; Agoston et al, 1983; Furukawa et al, 1985; Fu et al, 1987; Gibson and Manger, 1988; Gobet et al, 1995; Fryer and Glover, 1997). However, attempts to enhance postsynaptic responses in the treatment of the above disorders have failed. Although known to block voltage-dependent K⁺ channels, 4-AP has not been used to target postsynaptic cells. There is a need for improved methods to treat neurological disorders including those caused by traumatic brain injuries as well as neuromuscular and neurodegenerative diseases such as myasthenia gravis and multiple sclerosis. SUMMARY OF THE INVENTION

The present inventors have discovered that 4-AP elevates intracellular Ca²⁺ concentration ("[Ca²⁺,]") in postsynaptic cells and cells targeted by neurons (such as astrocytes).

Thus, one embodiment of this invention is a combination therapy of 4-AP or an analogue thereof with (a) a neurotransmitter such as acetylcholine (ACh) or (b) a substance that prolongs the availability of a neurotransmitter, such as an acetylcholinesterase (AChE) inhibitor.

One aspect of the present invention relates to a pharmaceutical composition comprising a 4-AP agent, a postsynaptic agonist and a pharmaceutically acceptable carrier.

The postsynaptic agonist can be one or more of a neurotransmitter, a neurotransmitter receptor agonist, or a compound that prolongs the availability of a neurotransmitter to the postsynaptic cell. In one embodiment the neurotransmitter receptor agonist is a cholinergic agonist, more specifically, a muscarinic agonist. In another embodiment, the neurotransmitter can be acetylcholine. In yet another, the compound prolonging the availability of the neurotransmitter can be an acetylcholinesterase inhibitor. In one embodiment, the postsynaptic agonist can be an agonist of a ryanodine receptor (RyR) or of an inositol trisphosphate (IP3) receptor. Alternatively, the agonist component may comprise more than one of the foregoing types of compounds in combination.

In another embodiment of the invention, a combination of 4-AP and a "natural" neurotransmitter (e.g., ACh), a neurotransmitter receptor agonist (e.g., carbachol) or an agent that enhances neurotransmitter availability to the postsynaptic membrane (e.g. , an ACh inhibitor or a reuptake inhibitor) is used to alleviate symptoms of a condition in which neurotransmission is blocked or otherwise deficient. Thus another aspect of the invention relates to a method of treating a disorder of impaired neurotransmission in a subject, comprising administering to the subject an effective amount of a combination of an extrinsic postsynaptic agonist and a 4-AP agent effective for enhancing the neurotransmission.

Such conditions are caused by traumatic injury to the central nervous system, such as spinal cord injury or are neurological or neuromuscular disorders or dysfunctions including myasthenia gravis, multiple sclerosis, motor neuron disease and various neurodegenerative disorders including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). This therapeutic combination elevates postsynaptic [Ca ,] by any of a number of distinct mechanisms and thus improves synaptic function in a diseased individual. In particular, the present invention provides a method for improving or promoting synaptic transmission which comprises administration of a "4-AP agent," (either 4-AP or an analog or mimic), in combination with an extrinsic postsynaptic agonist, thereby increasing and prolonging the increased level of agonist-induced postsynaptic [Ca²⁺j] as compared to [Ca²⁺j] achieved in the absence of the 4-AP agent. As noted above, the postsynaptic agonist may be for example a small molecule neurotransmitter, such as acetylcholine, or ATP or a peptide such as bradykinin, or a cholinergic agonist such as carbachol, or a substance that inhibits breakdown or reuptake of a neurotransmitter, such as an AChE inhibitor, or another agonist of intracellular calcium mobilization. The 4-AP agent may be administered together with, prior to, or after the administration of the postsynaptic agonist, so long as both are present together at the synapse or target cell in effective amounts. The primary site of action of the 4-AP agent may be presynaptic or postsynaptic. This agent is delivered to a pre- or postsynaptic neuron, to the synapse or to another target cell on which a neuron acts, e.g., an astrocyte, a skeletal muscle cell, a cardiac muscle cell, or any peripheral target cell of an autonomic nerve or any combination of such cells.

As used herein, a synapse is intended to include the physical apposition or proximity between a neuron and its target cell which the neuron influences via release of a neurotransmitter, which acts on the target or postsynaptic cell. Postsynaptic cells according to this invention may be other neurons, glial cells such as astrocytes, muscle cells (striated, smooth or cardiac), or other effector cells that are target cells of neuro transmitters.

A suitable concentration of a 4AP agent is, for example, between about 1 mM and about 20 mM. The 4-AP agent and the postsynaptic agonist are administered in an amount effective to increase [Ca²⁺j] in the postsynaptic cell, thereby enhancing neurotransmission. The prolonged and potentiated agonist-induced postsynaptic [Ca²⁺j] elevations induced by the 4-AP agent are generally concentration dependent. These effects of the 4-AP agent are due in part to responses mediated by intracellular inositol trisphosphate (LP3) receptors and occur independently of its effect on voltage sensitive outer membrane K⁺ channels. However, the net effect of the 4AP agent may include the Ca²⁺ response to the inhibition of K⁺ channels.

The present invention also contemplates other modes of action of the 4AP agent and postsynaptic agonist combination that lead to elevated [Ca²⁺j], also referred to here as intracellular calcium mobilization. These included 4-AP-facilitated release of Ca²⁺ from intracellular calcium stores (ICS) by activation of phospholipase C (PLC), which leads to release

9-1- of IP3. In addition, the 4-AP agent may facilitate the modulation of IP3-linked Ca transients and potentiates intracellular Ca²⁺entry activated by depletion of ICS and increases ryanodine receptor (RyR) activity. A related mechanism is capacitative Ca²⁺ calcium entry (CCE) that occurs in astrocytes and muscle cells by triggering Ca²⁺ release-activated Ca²⁺ channels (CRAC) and/or store-operated Ca²⁺ channels (SOCC) to open, thereby allowing extracellular Ca²⁺ to enter the postsynaptic cell in response to depletion of ICS. The combined action of the 4-AP agent further blocks sarco/endo reticulum Ca -ATPase (SERCA) and may thereby prevent the uptake of free intracellular Ca²⁺ by the endoplasmic reticulum (ER), and lead to greater CCE. CCE contributes to the magnitude and duration of postsynaptic agonist-evoked intracellular Ca²⁺ transients, allowing intracellular stores to refill.

One benefit of prolonging and potentiating postsynaptic [Ca²⁺j] is to enhance excitation- contraction coupling between a motor neuron and a postsynaptic muscle cell, leading to improved neurotransmission and muscle contraction. In astrocytes or other glial cells, this effect on [Ca j] may activate these cells to secrete trophic factors that participate in repair or survival of surrounding neurons. These benefits are realized by administering the combination comprising the 4-AP agent to a subject in need of such treatment.

Enhanced Ca²⁺ mobilization follows administration to a postsynaptic cell of a 4-AP agent in combination with a postsynaptic agonist that increases CCE and is useful for treating and/or preventing a neurological or a neuromuscular disorder in a subject having, or at risk for, such a disorder.

In accordance with this invention, it is possible to screen potentiators of postsynaptic intracellular Ca²⁺ mobilization, both 4AP agents and postsynaptic agonists. In one embodiment a test substance can be screened for activity as a potentiator of postsynaptic response. This involves administering a known extrinsic postsynaptic agonist to a synapse model and administering a 4-AP test compound to the model in conjunction with the agonist. The postsynaptic response is measured. The test compound is selected if it produces a response, e.g., increased calcium mobilization, an elevated voltage change, contraction or secretion in comparison to results of administering a known 4-AP agent in combination with the known postsynaptic agonist.

In another embodiment of the invention, a test substance is screened for activity as an agonist of postsynaptic response. This involves administering a known 4-AP agent to a synapse model and administering a postsynaptic agonist test compound to the model in conjunction with the 4-AP agent. The postsynaptic response is measured and the test compound is selected if it produces elevated voltage change, contraction, or secretion in comparison to results of administering a known postsynaptic agonist in combination with the known 4-AP agent.

In yet another embodiment, it is possible to determine whether a candidate agent is a 4- AP agent. This is done first by (a) establishing a negative control by administering a known postsynaptic agonist to a synapse model and measuring postsynaptic intracellular Ca²⁺ levels or postsynaptic function, and (b) establishing a positive control by administering a combination of a known 4-AP agent and the known postsynaptic agonist at similar concentrations, as in the negative control, to a synapse model and measuring postsynaptic intracellular Ca²⁺ levels or postsynaptic function. This determines a differential effect of 4AP on said levels or function.

A third step is (c) administering a combination of the candidate agent with similar concentrations of the postsynaptic agonist as in (a) and (b) to the synapse model and measuring postsynaptic intracellular Ca²⁺ levels or postsynaptic function. This determines a differential effect of the candidate agent on said levels or function. If the differential effect of the candidate agent is at least about 20% of the differential effect of 4AP, the candidate agent is a 4AP agent.

In another embodiment, the screening comprises administering a candidate 4-AP agent to astrocytes in vitro; administering a known cholinergic agent to the cells; measuring the resulting postsynaptic [Ca²⁺,] and comparing that response to a response induced by the combination of 4AP itself with the cholinergic agonist. If the candidate agent has at least about 20% of the activity of 4AP in combination with the cholinergic agonist, it is considered to be a "4AP agent" that contributes to the synergistic elevated level of postsynaptic [Ca²⁺,].

The elements of the invention recited herein may be combined or eliminated among the particular embodiments described, as would be apparent to a person of ordinary skill.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB. Figure 1A shows the effect of 4-AP on [Ca²⁺],. Near-confluent cultured type I astrocytes were loaded with fura-2. Cells were perfused with increasing 4-AP concentrations applied at the times indicated by the top broken line arrows and washed at the times indicated by the solid line arrows below the trace. Increasing concentrations of 4-AP caused proportional [Ca²⁺]j elevation that reached an apparent plateau at 20 mM 4-AP, as shown in Figure IB. Removal of 4-AP caused a prompt return to baseline [Ca²⁺]j. The EC₅₀ of 4-AP was about 10 mM.

Figures 2A, 2B, 2C and 2D. Characterization of 4-AP-induced calcium response in type I astrocytes. A, Treatment with 10 mM 4-AP caused a slow-onset long-lasting [Ca²⁺]i elevation, which was promptly reversed by washout. B, When extracellular calcium was removed, 4-AP was still able to increase [Ca²⁺]j . However, the shape of the response was modified to a transient peak with a quick return to baseline. C, The effect of 4-AP was tested in the absence of extracellular calcium and after depletion of intracellular calcium stores with thapsigargin (Thap). D, A 10 mM concentration of 4-AP was applied to astrocytes in calcium-free KRB. Subsequently calcium was reintroduced, and [Ca²⁺]j was monitored. Perfusion of the testing substances is indicated by the horizontal bars.

Figures 3 A, 3B, 3C and 3D. Characterization of 4-AP-induced [Ca²⁺]i rise in cultured cortical neurons. A, A 10 mM concentration of 4-AP was applied to cortical neurons plated on glass coverslips. The compound caused a slow-onset long-lasting [Ca ]; elevation. The [Ca ]; elevation was promptly reversed by washing out the compound. B, When extracellular calcium was removed, 4-AP (10 mM) was still able to increase [Ca²⁺]j. However, the response was transient, and [Ca ]j returned promptly to baseline values. C, ICS were depleted in the absence of extracellular calcium with thapsigargin and CCE after reintroduction of calcium was measured. D, ICS were depleted with 4-AP in calcium-free medium. The neurons were subsequently exposed to thapsigargin, and 60 sec later calcium was reintroduced in the extracellular solution to elicit CCE. Perfusion of the testing substances is indicated by the horizontal bars.

Figures 4A-1, 4A-2 and 4B. Effect of 4-AP on InsP_t accumulation in astrocytes and neurons. A-l, 4-AP causes a concentration-dependent elevation of InsP_t accumulation in type I astrocytes. Compared with the stimulation obtained with a 10 μM ATP (EC₅₀, 30 μM), 10 mM

4-AP (EC₅₀) was 50% less powerful (Figure 4A-2). B, 4-AP increased InsP_t production in cortical neurons. Basal values are indicated by the open triangle and the dashed line. *p < 0.05 versus basal value.

Figures 5 A, 5B, 5C, 5D, 5E and 5F. Effect of 4-AP on neurotransmitter-evoked calcium transients in astrocytes. Astrocytes were challenged with 10 μM ATP (A) or 100 nM bradykinin (C). The calcium response to both agonists was characterized by a fast and sharp [Ca ], elevation and a rapid return to a much lower but prolonged [Ca²⁺], value. Astrocytes pre-exposed to 10 mM 4-AP and challenged with ATP (B) or bradykinin (D) showed a long-lasting large [Ca ], elevation. E and F show statistical analysis of the data extrapolated from the experiments in A and C 2 sec after agonist stimulation, and in B and D 2 sec before 4-AP washout. *p < 0.05 versus value in control cells. Perfusion of the testing substances is indicated by the horizontal bars.

Figures 6A, 6B, 6C, 6D, 6E and 6F. Effect of 4-AP on CCE triggered by agonist- induced ICS depletion in astrocytes. Intracellular calcium stores were depleted with ATP (A) or bradykinin in the absence of extracellular calcium. ICS emptying was controlled with a second ATP (A) or bradykinin (C) stimulation. When calcium was reintroduced, a weak [Ca²⁺], elevation was generated because of CCE activation. B, Intracellular calcium stores were depleted with 10 μM ATP in the presence of 4-AP. When calcium was reintroduced, [Ca²⁺], elevation was very high and lasted until 4-AP was removed from the cells. The same effect was recorded when stores were depleted with bradykinin in the presence of 4-AP (D). E and F show the statistical validation of the data presented A-D, respectively. Values were extrapolated from the experiments 2 sec before removal of 4-AP. *p value < 0.05 versus control cells. Perfusion of the testing substances is indicated by the horizontal bars.

Figures 7A, 7B and 7C. 4-AP potentiates thapsigargin-induced CCE in astrocytes. A, Calcium stores were depleted with a maximal concentration of thapsigargin (10 μM). B, Thapsigargin exposure in cells pretreated with 4-AP resulted in a large increase of CCE. C, Statistical validation of the data presented in A and B. Peak values were analyzed. *p value < 0.05 versus control cells. Perfusion of the testing substances is indicated by the horizontal bars. Figures 8A-1, 8A-2, 8B and 8C. 4-AP potentiates CCE in L6 cells. A-l, Application of 10 μM thapsigargin in calcium-free KRB caused an elevation of [Ca²⁺]j, (8A-2). Reintroduction of calcium in the extracellular buffer was followed by CCE. The effect of thapsigargin alone is highlighted in the 8A-2. B, In the presence of 10 mM 4-AP, CCE was increased approximately ninefold. C, Statistical validation of the data extracted from control at peak and from 4-AP- exposed cells 2 sec before 4-AP washout. *p value < 0.05 versus control cells. Perfusion of the testing substances is indicated by the horizontal bars.

Figures 9A, 9B, 9C and 9D. 4-AP inhibits voltage-gated K⁺ currents in astrocytes. A, Astrocytes were voltage-clamped at a membrane voltage of about 60 mV. Fifty-millisecond-long pulses were delivered at increasing voltages from about 80 mV up to +50 mV, at 10 mV intervals. The 7-Fprofile was generated with a positive current that was recorded up to about

2 nA amplitude. The steady-state current was measured 40 msec after the start of the pulse. B,

Example traces recorded in astrocytes before, after exposure to 10 mM 4-AP and after washout. C, 4-AP application reversibly blocked the late component (40 msec), whereas the early, fast inactivating component did not recover from blockage after 10 min washout (open bars are the normalized control current; black bars represent the currents after 4-AP application; hatched bars represent the amplitude of the current after 10 min washout). The fast component was calculated as the difference between the current at 5 and 40 msec after the beginning of the pulse. D displays the inhibitory effect of 20 mM TEA on potassium current in type I astrocytes.

Control current was about 1.38 + 0.63 pA and used as 100%> in the open bar. D, The effect of

20 mM TEA is represented by the black bar. Cells were washed out, and the recovery of K⁺ current is summarized in the hatched bar.

Figures 10A, 10B, 10C and 10D. Effect of DTx on CCE in astrocytes. We tested whether voltage-gated K⁺ channel blocker α-dendrotoxin ("DTx) potentiated CCE evoked by ATP-induced ICS depletion. A, Response to 10 μM ATP in control cells. B, Cells were challenged with 10 μM ATP after a 3 min exposure to 100 nM DTx. C and D were the same experiments as shown in A and B, respectively, and they were performed in the absence of extracellular calcium. Calcium was reintroduced in the perfusion buffer, and CCE was monitored. Perfusion of the testing substances is indicated by the horizontal bar. DETAILED DESCRIPTION

The present inventors have discovered that certain combinations of agents that

7+ contribute independently and synergistically to the elevation of intracellular Ca levels in "postsynaptic" cells targeted by neurons can be harnessed to facilitate central and peripheral neurotransmission in conditions in which this neurotransmission is blocked or suboptimal. This invention relates to methods, compounds and compositions for prolonging and potentiating

7+ agonist-induced postsynaptic intracellular Ca levels. The term "4-AP agent" or "postsynaptic response facilitator" as used herein encompasses compounds such as 4-aminopyridine (Fampridine), and prodrugs, metabolites, derivatives, structural analogs and addition salts thereof. Nonlimiting examples include 3,4- diaminopyridine; 4-amino-3-(phenethylamino)pyridine dihydrochloride; 4-aminopyridine- 1- oxide; N-(3-chlorophenyl)-N-4-pyridinyl-4-morpholinepropanamine bis(2-hydroxybenzoate); N-4-pyridinyl-N-(3 -(trifluoromethyl)phenyl)-4-moφholinepropanamine bis(2-hydro xybenzoate) ; N-(3-chlorophenyl)-beta-methyl-N-4-pyridinyl-4-morpholinepropanamine (Z)-2-butenedioate (1:2); 4-aminopyridine hydrochloride; 3-Methyl-4-aminopyridine; 3-((Dimethylamino)carbonyl) amino-4-aminopyridine (LF 14); 3-Methoxy-4-aminopyridine; 4-Aminopyridine methiodide; and 4-Nitrosomethylaminopyridine. Other 4-AP agents include potassium channel blockers having the inventive facilitating effect in combination with the postsynaptic agonist.

The term "postsynaptic agonist" as used herein means a compound added exogenously that acts ultimately on a postsynaptic target cell to raise the intracellular concentration in that cell and contribute to the stimulation of a postsynaptic response measurable as an electrophysiological, electromechanical or secretory response. The "extrinsic" or "exogenous" postsynaptic agonists of the invention are added to biological systems or administered to subjects as opposed to an "intrinsic" postsynaptic agonist, i.e. one that is produced naturally in vivo. The extrinsic postsynaptic agonist may be the same chemical entity as an intrinsic molecule such as a neurotransmitter, or other small organic molecule such as ATP and bradykinin, which may not be classical neurotransmitters but act similarly, or may be a synthetic compound such as a recombinant polypeptide. A "postsynaptic agonist" as defined herein may be a substance that prolongs the availability of a neurotransmitter at the postsynaptic membrane, for example an inhibitor of metabolic breakdown such as an AChE inhibitor, or inhibitors of reuptake of the neurotransmitter by the presynaptic neuron, for example, tricyclic or newer- generation antidepressants that inhibit reuptake of catecholamines, indoleamines or combinations of these amines.

Inositol trisphosphate (IP3) receptor agonists and their derivatives may be used in the present invention. Nonlimiting examples include: bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F_2α and vasopressin.

Cholinesterase (e.g., acetylcholinesterase) inhibitors and their derivatives may be used in the present invention. Nonlimiting examples include: 1,10-phenanthroline; 1,3,2- dioxaphosphorinane-2-oxide; 1 ,5-bis(4-trimethylammoniumphenyl)pentan-3-one; 1 ,7-N- heptylene-bis-9,9'-amino- 1 ,2,3,4-tetrahydroacridine; 1 -(2-methyl-6-benzothiazolyl)-3-(N- benzyl-4-piperidinyl )propan-l-one; l-(3,4-(methylenedioxy)benzoyl)-3-(2-(l-benzyl-4- piperidinyl )ethyl)thiourea; 1 -benzyl-4-(2-(N-(4'-(benzylsulfonyl)benzoyl)-N-methylamino )ethyl)piperidine; l-benzyl-4-(N-methyl-N-((phenylmethylsulfonyl)phenylcarbonyl)aminoethyl) piperidine; 1-bromopinacolone; l-methyl-l,2,3,3a,4,9b-hexahydrochromeno(4,3-b)pyrrol-6-yl N -methylcarbamate; l-methyl-S-(3-methylthiophosphoryl) imidazolium; 10,10- dimethylhuperzine A; 10,11-methylene dioxycamptothecin; 14-fluorohuperzine A; 2'- heptylcarbamoyloxy-2-methyl-6,7-benzomorphan; 2,5-hexanedione; 2-((((4,6-dimethoxy-(2- pyrimidinyl)amino)carbonyl)amino)sulfonyl)-N,N-dimethyl-3- pyridinecarboxamide; 2- (methylsulfonyl)ethanol; 2-(trimethylsilyl)ethanol; 2-(trimethylsilyl)ethyl acetate; 2- (trimethylsilyl)methyl acetate; 2-chloro-12-(2-piperidinoethyl)dibenzo(d,g)-l,3,6 -dioxazocine; 2-dimethylaminoethyl-(dimethylamido)fluorophosphate; 2-heptyl-2- nitrophenylmethylphosphonate; 2-hydroxymethyl-N,N-dimethylpiperidinium; 2-hydroxytacrine; 2-isopropyl-S-(2-diisopropylaminoethyl)methylthiophosphonate; 2-N,N-dimethylaminomethyl- 5-methylfuran; 2-tert-butyl-9-amino-l,2,3,4-tetrahydroacridine; 20-glycinate-10,l 1- methylenedioxycamptothecin; 20-glycinate-7-chloromethyl- 10, 11 -methylenedioxycamptothecin; 217AO; 3 alpha, 17 beta-dibutyryloxy-2 beta, 16 beta-dipiperidino-5 alpha- androstane dimethobromide; 3'-chloro-4-stilbazole; 3,3-dimethyl-2-butyl methylphosphonofluoridate; 3- (2,3-dihydro-2,2-dimethylbenzofuran-7-yl)-5-methoxy-l,3,4 -oxadiazol-2(3H)-one; 3-(2-(l- benzylpiperidin-4-yl)ethylamino)-6-phenylpyridazine; 3-(2-methoxyphenyl)-5-methoxy-l,3,4- oxadiazol-2(3H)-one; 3-(N,N-dimethylamino)trifluoroacetophenone; 3-(tert- butyl)trifluoroacetophenone; 3-carbamyl-N-allylquinuclidinium; 3-deoxyvasicine; 3- diethylaminophenyl-N-methylcarbamate methiodide; 3-hydroxy-N,N-dimethylpiperidinium; 3- hydroxymethyl-N,N-dimethylpiperidinium; 3-hydroxytacrine; 3-MPAM-ES; 33 SN; 4- azidobretylium tosylate; 4-hydroxy-N,N-dimethylpiperidinium; 4-hydroxytacrine; 4-nitrophenyl methyl(4-trifluoromethylphenyl)phosphinate; 4-phenylazophenyltrimethylammonium; 4- stilbazole; 5,7-dihydro-3-(2-(l-(phenylmethyl)-4-piperidinyl)ethyl)-6H -pyrrolo(3,2-f)-l,2- benzisoxazol-6-one; 5-(l,3,3-trimethylindolinyl)-N-(l-phenylethyl)carbamate; 5-(7-nitrobenz-2- oxa-l,3-diazol-4-yl) aminoethylmethylphosphonofluoridate; 5-(7-nitrobenz-2-oxa-l,3-diazol-4- yl)pentyl methylphosphonofluoridate; 5-aza-3-(((3-((methylaminocarbonyl)oxy)phenylmethyl )methylamino)propyl)-9H-xanthen-9-one; 51 C; 6,7,8,9-tetrahydro-3-methyl-lH-pyrano-(4,3- b)quinolin-l-one; 6-methoxy-3-(2-(l-(phenylmethyl)-4-piperidinyl)ethyl)-l,2 -benzisoxazole; 7-((methylethoxyphosphinyl)oxy)- 1 -methylquinolinium; 7-bromotacrine; 9-amino-8-fluoro- 1 ,2,3,4-tetrahydro-2,4-methanoacridine; 9-dehydro- 10-N-demethyl-- 10-N-( 10'-phthalimidodecyl )galanthaminium; 9-dehydro- 10-N-demethyl- 10-N-(8'-phthalimidooctyl )galanthaminium; AA 31; Alternariol; Ambenonium Chloride; Amiridin; Ammophos; Arisugacin; Armin; Azamethiphos; Azinphosmethyl; B 156; bis(isopropyl methyl)phosphonate; bis(trichloromethyl)sulfone; bromophos; butonate; Carbaryl; Carbofuran; CHF 2819; Chinotilin; Chlorfenvinphos; Chlorpyrifos; Coroxon; Coumaphos; Crotylsarin; cui xing an; cui xing ning; cyanofenphos oxon; cycloguanide phenylsulfone; cyclohexyl methylphosphonofluoridate; cyclophostin; dehydroevodiamine; demecarium bromide; DEP-2PAM; DEP-4PAM; diamminediaqua platinum(II); Diazinon; Dichlorvos; dicyclopropyloketoxime diethylphosphoric acid ester; diethyl mesoxalate; diethyl S-n-propyl phosphorothiolate; diisopropylamine dichloroacetate; diisopropylphosphorylthiocholine iodide; Dimethoate; dimethylcarbamyl fluoride; dimethylcarbamylcholine; dimethylthionocarbamylcholine; distigmine; E 2020; Echothiophate Iodide; Edifenphos; Edrophonium; EPN oxon; ethyl 4-nitrophenyl methylphosphonate; ethylparaoxon; faleoconitine; fasciculin; Fenitrothion; Fenthion; Fonofos; Fordine; Galanthamine; GD 7; GT 161; GT-165; Hexafluorenium; hexamethylenebis(dimethyl- (3-phthalimidopropyl)ammonium bromide); hexyl 2,5-dichlorophenylphosphoroamidate; huperzine A; huperzine B; huperzinine; huprine X; huprine Y; indolinyl-N,N- dimethylcarbamate; Isoflurophate; isopropyl S-2-trimethylammoniumethylmethyl phosphonothioate; itopride; KW 5092; m-(N,N,N-trimethylammonio)trifluoroacetophenone; Malathion; Methacyne; methanesulfonyl fluoride; methiocarb sulfoxide; Methomyl; Methyl Parathion; Methylphosphonfluoridate; Methylphosphonothiolate; Methylsulfomethylate; Metrifonate; Mevinphos; MF 268; MHP 133; Monocrotophos; N(1),N(3),6- trimethylcycloheptadiimidazole-2,8-diamine; N(l),N(8)-bisnoφhenserine; N(1),N(8)- bisnoφhysostigmine; N(8)-noφhenserine; N(8)-noφhysostigmine; N,N'-diisopropyl phosphorodiamidic anhydride; N,N,N',N^'-tetrakis(2-pyridylmethyl)ethylenediamine; N,N- dimethylcarbamic acid 2,3-dihydro-l,3,3-trimethylindol-5 -yl ester; N,N- methylethylphenylcarbamate; N-(4-(2-(dimethylamino)ethoxy)benzyl)-3,4-dimethoxybenzmide; N-(epsilon-aminocaproyl)-p-aminophenyltrimethylammonium; N-butyl-3-butylpyridinium; N- demethylhuperzinine; N-methylhuperzine B; N-methylpiperidine; N-octyl-l,2,3,4-tetrahydro-9- aminoacridine; Naled; Neostigmine; Nibufm; Normeperidine; Norneostigmine; Noφyridostigmine; O,O,S-trimethylphosphorodithionate; O-(3-(trimethylammonium)phenyl)- 1,3,2-dioxaphosphorinane 2 -oxide; O-ethyl N,N-dimethylamino-S-(2-diethylaminoethyl )thiophosphate; O-ethyl O-4-nitrophenyl phosphoramidate; O-ethyl-S-hexyl- methylthiophosphonate; O-hexyl-S-hexylmethyl thiophosphonate; O-methyl-S-n- hexylmethylthiophosphonate; octamethyl pyrophosphoramide; onchidal; P 10358; Panpal; Paraoxon; Parathion; parazoanthoxanthin A; PD 142676; Phenserine; Phenylphosphonothioic Acid, 2-Ethyl 2-(4-Nitrophenyl) Ester; phlegmariurine C; Phorate; Phosalone; Phosphamidon; Phoxim; Physostigmine; physostigmine heptyl; physostigmine methiodide; pinacolyl S-(2- dimethylaminoethyl)methylphosphonothioate; pinacolyl S-(2-trimethylaminoethyl) methylphosphonothioate; PK-154; poly- APS; prothiophos; pseudozoanthoxanthin; Pyridostigmine Bromide; Quilostigmine; RH 218; Rivastigmine; Ro 20683; Ro 46-5934; RX 67668; RX 72601; S 27; S 9977; S-(2-(diethylamino)ethyl) 4-methylbenzothiohydroximate; S- (2-(diethylamino)ethyl) alpha-keto-4 -methylbenzothiohydroximate; S-methyl-methylparathion; Salioxon; Sanguiritrine; Sarin; Silatrane; Soman; Stephaglabrine; Sulfotepp; Tabun; Tacrine; TAK 147; Teration; territrem B; Tetrachlorvinphos; tetraethyl pyrophosphate; tetrahydropyridostigmine; Tetraisopropylpyrophosphamide; THB 013; Thiofanox; Tolserine; Trichlorfon; trichloronate oxon; tripropylammonium; TV3326; Velnacrine; Visoltricin; VX; Zifrosilone;

Muscarinic agonists and their derivatives may be used in the present invention. Nonlimiting examples include: cis 2-methyl-spiro[l-azabicyclo[2.2.2]octane 3,5'- [l,3]oxothiolane (AF-102B, cevimeline); l-methyl-l,2,5,6-tetrahydropyridine-3-carboxaldehyde methoximine (CI-979/RU35926); l-azabicyclo[2.2.2]octane, 3-(6-chloroρyrazinyl)-(2)- butendioate (L-689,660); alvameline (Lu25-109); N,N-diethyl-4-[3-(2,3,6,7-tetrahydro- 1,3,7- trimethyl-2,6-dioxo-lHpurin-8-yl)propyl-l-piperazinecarboxamide hydrochloride (S-9977-2); sabcomeline (SB 202,026); 3-[(N-(2-diethylamino-2-methylpropyl)-6-phenyl-5- propyljpyridazinamine sesquifumarate (SR46559); Thiopilocaφine (SDZ ENS 163); (R)-3-(2- propanyloxy)-l-azabicyclo[2.2.2]octane, (E)-2-butendioate (WAL 2014); xanomeline (-)-S-2,8-dimethyl-3-methylene-l-oxa-8-azaspiro (4,5) decane, L-tartrate monohydrate (YM 796); 2-ethyl-8-methyl-2,8-diazaspiro(4,5)-decane-l,3-dione hydrobromide (RS-86); arecoline; pilocaφine; muscarones; spirodioxolanes; oxotremorine; milameline; and talsaclidine. The term "synapse" as used herein includes a site of contact and interaction between two neurons or between neuron or muscle cell (also termed a "neuromuscular junction). Also included in this definition is a site in which a neuron and other target cells such as a glial cell or other tissue or glandular effector cell meet and across which a signal can pass. The presynaptic neuron may convert an electrical impulse to a chemical signal near the synapse and transmit the chemical signal, in the form of secreted neurotransmitter material, to the target cell. This transfer is referred to as neurotransmission.

A "postsynaptic cell" is any target cell that is located sufficiently close to a neuron so it can receive a chemical signal from the neuron. Typically, at a synapse, the neuron and postsynaptic cell (be it a neuron, muscle cell or other type) are separated by a gap often called the "synaptic cleft". The site of action of the compositions and methods of the invention may be at the synapse or elsewhere on the target cell(s).

The neuronal cell membrane from which the neurotransmitter is released is referred to as the "presynaptic membrane"; the target cell membrane at which it is received is referred to as the "postsynaptic membrane". The results produced by the action of 4-AP or a 4AP agent as described herein are primarily viewed as facilitating events that result in propagation of a neural signal. The term "facilitate" as used herein means to prolong and/or increase the level of mobilized intracellular

7+

Ca (calcium transients) in a postsynaptic cell. Thus, 4-AP will facilitate propagation of a nerve impulse across a synapse that results in enhanced postsynaptic function such as electrophysiological activity (e.g. , postsynaptic neuronal firing), electromechanical activity (e.g. , muscle contraction) or neuronal or non-neuronal target cell secretory activity The term "treatment" as used herein is intended to encompass prophylactic administration to prevent or suppress the development of an undesired condition, and/or therapeutic administration to eliminate or reduce the extent or symptoms of an existing condition. Treatment according to the invention is given to a human or other animal having a disease or condition creating a need for such treatment. The present methods also include providing the present compositions to a "synapse model" which can be cells, tissues or organs in vitro.

A preferred composition according to the invention comprises an effective amount of a combination of a postsynaptic agonist and a 4-AP agent and is preferably administered to a subject to treat a "disorder of impaired neurotransmission." The disorder is caused by a failure in the above referenced signaling pathways or activities that are manifest as deficiencies in the postsynaptic cell or the delivery of the "message" to that cell. The novel combination treatment is directed to the normalization of such deficiencies. The neurological or neuromuscular disorders are generally characterized by impaired or dysregulated neurotransmission and may be caused by traumatic injury to the central nervous system, such as spinal cord injury. Other disorders may have autoimmune or inflammatory pathophysiologies, for example, myasthenia gravis or multiple sclerosis. Yet others are neurodegenerative diseases whose causes are more complex or obscure at this time, e.g., Alzheimer's disease, amyotrophic lateral sclerosis or other CNS disorders with compromised neurotransmission. The chemical compositions useful in the present invention are "converted" into pharmaceutical compositions by dissolution in, and/or addition of, appropriate, pharmaceutically acceptable carriers or diluents. Thus, the compositions may be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injectables, inhalants, and aerosols, using conventional means. Known methods are used to prevent release or absoφtion of the active ingredient or agent until it reaches the target cells or organ or to ensure time-release of the agent. A pharmaceutically acceptable form is one that does not inactivate or denature the active agent. In pharmaceutical dosage forms useful herein, the present compositions may be used alone or in appropriate association or combination with other pharmaceutically active compounds. Accordingly, the pharmaceutical compositions of the present invention can be administered to any of a number of sites in a subject and thereby delivered via any of a number of routes to achieve the desired effect. Local or systemic delivery is accomplished by administering the pharmaceutical composition via injection, infusion or instillation into a body part or body cavity, or by ingestion, inhalation, or insufflation of an aerosol. Preferred routes of administration are parenteral, which include intramuscular, intracranial, intravenous, intraperitoneal, subcutaneous intradermal or topical routes.

The present compositions can be provided in unit dosage form, wherein each dosage unit, e.g., a teaspoon, a tablet, a fixed volume of injectable solution, or a suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other pharmaceutically active agents. The term "unit dosage form" refers to physically discrete units suitable for a human or animal subject, each unit containing, as stated above, a predetermined quantity of the present pharmaceutical composition or combination in an amount sufficient to produce the desired effect. Any pharmaceutically acceptable diluent or carrier may be used in a dosage unit, e.g., a liquid carrier or vehicle such as a saline solution, a buffer solution, or other physiologically acceptable aqueous solution). The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamic properties of the pharmaceutical composition in the particular host.

An "effective amount" of a composition is an amount that produces the desired effect in a host, which effect can be monitored, using any end-point known to those skilled in the art. Furthermore, the amounts of each of the active agents exemplified herein are intended to provide general guidance as to the dose range of each component which may be utilized by the practitioner upon optimizing these methods for practice either in vitro or in vivo. Exemplified dose ranges do not preclude use of higher or lower doses, as might be warranted in a particular application. For example, the actual dose and schedule may vary depending on (a) whether a composition is administered in combination with other pharmaceutical compositions, or (b) inter-individual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts may vary for in vitro applications. One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, parenteral and topical administration. Other contemplated formulations include nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically based formulations. The formulations may be prepared by any method known or hereafter developed. In general, preparation includes bringing the active ingredients into association with a carrier or one or more other additional components, and if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. As used herein, "additional components" include, but are 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; sweetening agents; flavoring agents; coloring 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; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; pharmaceutically acceptable polymeric or hydrophobic materials as well as other components.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood that such compositions are generally suitable for admimstration to any mammal or other animal and a skilled veterinary practitioner will know how to design and perform such modifications using only routine experimentation.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose as described above, or as a plurality of single unit doses. The amount of the active ingredient in each unit dose may be the total amount of active ingredient to be administered, or a convenient fraction of that total such as, for example, one-half or one-third.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may in the form of a discrete solid dosage unit. Solid dosage units include, for example, a tablet, a caplet, a hard or soft capsule, a cachet, a troche, or a lozenge. Each solid dosage unit contains a predetermined amount of the active ingredient, for example a unit dose or fraction thereof. Other formulations suitable for administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an "oily" liquid is one that comprises a carbon or silicon based liquid that is less polar than water. Liquid formulations of a pharmaceutical composition of the invention which are suitable for administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with a suitable solution vehicle prior to use.

Liquid suspensions, in which the active ingredient is dispersed in an aqueous or oily vehicle, and liquid solutions, in which the active ingredient is dissolved in an aqueous or oily vehicle, may be prepared using conventional methods. Liquid suspension of the active ingredient may be in an aqueous or oily vehicle and may further include one or more additional components such as, for example, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Liquid solutions of the active ingredient may be in an aqueous or oily vehicle and may further include one or more additional components such as, for example, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.

Powdered and granular formulations according to the invention may be prepared using known methods or methods to be developed. Such formulations may be administered directly to a subject, or used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Powdered or granular formulations may further comprise one or more of a dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion. Such compositions may further comprise one or more emulsifying agents. These emulsions may also contain additional components including, for example, sweetening or flavoring agents. The terms "in conjunction with" and "in combination with" as used herein refer to the use of both active ingredients to produce the desired effects of the invention. The 4-AP agent may be used or administered together with the postsynaptic agonist or administered separately either prior to or after the administration of the postsynaptic agonist, and by the same or different routes, so long as the 4-AP agent and the postsynaptic agonist are present at the synapse together and/or the desired effects of the invention are achieved. This may be achieved by administration and delivery via various routes and to various sites as discussed elsewhere. The combination of 4-AP agent and postsynaptic agonist may be achieved in several different ways. For example, both compounds may be administered simultaneously either separately or physically combined. Alternatively, one may be administered before the other so long as both compounds are present together at the synapse or the site where a neuron interacts with a target cell.

The active ingredients may be simultaneously co-administered in different dosage forms, or the two compounds may be combined in a pharmaceutical composition and delivered in a single dosage form, with each compound in a pharmaceutically effective amount in combination with the other. The term "active ingredient" means any substance, chemical, compound, composition, or formulation that achieves the desire inventive effect. The classes of compounds useful in the invention include groups of biologically active compounds related by their mode of biological activity. For example, acetylcholinesterase inhibitors are compounds that reduce activity of acetylcholinesterase. Individual compounds within a class of compounds may or may not be structurally related. For example, the class of acetylcholinesterase inhibitors includes several structurally related phosphate, phosphonate and phosphinate esters. However, the class of compounds designated acetylcholinesterase inhibitors also includes other structurally unrelated compounds, for example, 1-bromopinacolone.

Administration of the above referenced combinations of compounds has advantages over the administration of either members of the combination alone. First, the combinations are at least additive and preferably synergistic. Second, the administration of a combination may make possible a reduction in total dosage thus reducing the risk of individual side effects. Thus, where a desired dose of one of the active ingredients is toxic, the other agent may be used to enhance the prolonged administrability of the toxic agent permitting administration of a lesser non-toxic amount of the toxic agent while maintaining a therapeutic effect. Third, the active ingredients may be administered by the same or different routes.

An effective dose, amount, level or concentration of a combination of a 4AP agent and a postsynaptic agonist require that the amount of each substance be such that, together, the desired response is achieved. One can speak in terms of the effective dose, amount, level or concentration of each component separately. However, it should be kept in mind that it is the effective dose of the combination that is paramount to achieving the described biological 7+ responses: a prolonged increase in intracellular Ca in the postsynaptic target cell that will enhance neurotransmission and postsynaptic function.

The action of the 4-AP agent of primary relevance to this invention is its ability to facilitate a variety of events resulting in the propagation of a signal. The term "facilitate" and "potentiate" are used interchangeably herein to indicate the promotion or hastening of natural process. This is the reverse of inhibition or suppression. For example, in the case of neurotransmission between neurons, facilitation or potentiation relates to the effect of a nerve impulse moving across a synapse, resulting in continued or enhanced generation of action potentials in the postsynaptic cell. The postsynaptic agonist of this invention is one that stimulates mobilization of intracellular Ca²⁺ thereby creating a variety of effects known to be associated with such Ca ⁺ elevation. The combination of the 4AP agent prolongs and/or potentiates the agonist-induced elevation of intracellular Ca²⁺ levels compared to the levels achieved in the absence of the 4-AP agent. Despite the known actions of 4-AP on cells, the effects of the 4-AP agent noted above occur by mechanisms not previously known to be associated with 4AP and separate from the known effects on voltage sensitive K⁺ channels. The 4-AP agent will act to facilitate the release of Ca²⁺ from intracellular Ca²⁺ stores (ICS). The 4-AP agent may also facilitate activation of phospholipase C (PLC) leading to release of inositol trisphosphate (IP3) which is a known inducer of Ca²⁺ release from ICS and may facilitate the modulation of IP3 -linked Ca²⁺ transients.

The combined action of the 4-AP agent and postsynaptic agonist can increase CCE and thus elevate the Ca²⁺ mobilization induced by the neurotransmitter ACh. Thus, in one embodiment, this combination of substances is provided to neurons that are situated in close proximity to other neurons, astrocytes, muscle cells, etc. This may be done in vitro or in vivo in a subject, for example, one in need of enhanced neurotransmission due to the presence of a neurological or neuromuscular disorder.

In yet another embodiment, the 4-AP agent and the postsynaptic agonist may be employed in a test to screen an unknown substance for its potential activity as a potentiator of postsynaptic intracellular Ca²⁺ mobilization. In this approach, a 4-AP agent alone (control) is contacted with CNS tissue or other agglomeration of neurons and target cells in vivo or in vitro, e.g. or a synapse model in vitro, such as in the Example below. In the test group, the tissue or cell preparation is contacted with a test substance in combination with the 4-AP agent. Resulting postsynaptic intracellular Ca²⁺ levels are measured. If the combination produces an increase relative to the 4-AP agent alone, this is indicative that the test substance is a potentiator of postsynaptic intracellular Ca²⁺ mobilization. Again, in the literature, the ability of 4-AP to enhance neuromuscular and central neurotransmission has been explained by its activity as a K channel blocker. Here, the present inventors describe novel effects of 4-AP, occurring via different mechanisms: the stimulation of IP₃-receptor mediated mobilization of intracellular Ca²⁺ from intracellular Ca²⁺ stores (ICS), PLC activation, and the potentiation of agonist responses through a large potentiation of CCE. Ca²⁺ plays various roles in promoting the signaling effect of neurotransmitters. When a neuron is in a resting state, Ca²⁺ concentrations are much lower in the cytosol than in the endoplasmic reticulum and extracellular matrix. Calmodulin, a Ca -binding protein, regulates Ca²⁺ concentrations by activating Ca²⁺ pumps, which transport the Ca²⁺ out of the cytosol when concentrations are high. This creates a concentration gradient, and when the membrane is

7+ 7+ excited (depolarized), Ca voltage-channels open, leading to an influx of extracellular Ca . Ca²⁺ influx into a neuron is initiated by (a) depolarization of the cell membrane, thereby opening Ca²⁺ voltage-channels, or (b) the binding of a neurotransmitter (e.g., ACh to a G- protein-coupled receptor, triggering a signaling cascade that leads to Ca²⁺ influx. The latter

7+ pathway includes activation of phospholipase C, which activates IP3, which opens Ca voltage channels in the ER (or via ryanodine receptors (RyR), in sarcoplasmic reticulum in a contracting muscle fiber.

Once a cell is stimulated and Ca²⁺ is released, the Ca²⁺ binds to Ca²⁺ Kinase π, which activates neurotransmitter (e.g., ACh) secretion and re-synthesis. The neurotransmitter propagates the signal by activating G-proteins (or other types of receptors in the case of other neurotransmitters) on postsynaptic cells. Ca²⁺ also acts on Na⁺ channels, depolarizing the membrane and propagating the signal. The following series of events is involved:

1. Ca²⁺ activates PKC.

2. PKC and CaM inhibit K⁺ channels and other ion channels.

3. Ca²⁺ activates calexcitin (CE), which also inhibits K⁺ channels, making the membrane more excitable. 4. CE elicits Ca²⁺ release from RyR on the endoplasmic reticulum membrane, amplifying Ca²⁺ signals

5. CE also activates Ca²⁺-ATPase in the endoplasmic reticulum membrane to remove excess Ca²⁺. 6. CE or Ca²⁺ acts on transcription factors resulting in protein synthesis.

A number of neurologic and neuromuscular disorders (e.g., myasthenia gravis, multiple sclerosis, motor neuron diseases, and Alzheimer's disease) are associated with compromised presynaptic release of neurotransmitters. The result is reduced post-synaptic activity, in

7+ 7+ particular increased intracellular Ca concentration ([Ca ,]).

7+ • The present inventors have discovered that 4-AP increased [Ca ], in cortical astrocytes, neurons and skeletal muscle cells in a concentration-dependent manner. The elevation consisted

7+ • • of two phases. The first phase was dependent on intracellular Ca mobilization; this was

7+ • • confirmed by the observation that depletion of intracellular Ca with the agent thapsigargin in the absence of extracellular Ca²⁺ prevented 4-AP-induced [Ca²⁺], elevations. The second phase required the presence of Ca²⁺ in the extracellular environment.

The effect of 4-AP on astrocytes provides a suitable model that predicts potentiation of ACh post-synaptic stimulation. ATP and bradykinin mimic the effect of ACh at the neuromuscular junction, thereby acting as "postsynaptic agonists" as the term is defined herein.

7+

In astrocytes, 4-AP potently prolonged and increased [Ca ], elevations induced by phospholipase C activating agents (i.e., ATP and bradykinin). Both in astrocytes and neurons, 4-AP also increased the production of IP in a concentration dependent manner. 4-AP enhanced Ca²⁺ entry induced by depletion of ICS (also termed CE). 4-AP significantly and reversibly enhanced CCE. CCE is known to contribute to the magnitude and duration of agonist evoked [Ca²⁺], transients to Ca²⁺ oscillation and is the principal mechanism for refilling ICSs. The previously unknown modification of the Ca²⁺ transients induced by 4-AP were not correlated with the previously known effects on K⁺ channels:

(1) The potentiation of CCE caused by 4-AP was not mimicked by blocking the fast inactivating K⁺ current with α-dendrotoxin or low 4-AP concentrations,

(2) Blockade of delayed rectifier K⁺ current with TEA or blockade of both currents with low concentrations of 4-AP in the presence of TEA did not increase CCE. Thus, effects of 4-AP on intracellular Ca²⁺ homeostasis described herein and elsewhere do not depend on its action on K⁺ homeostasis despite its previously known activity as a K⁺ channel blocker. That is, these effects of 4-AP and the mechanisms by which they occur would not have been predictable prior to the making of this invention. Modulation of [Ca²⁺], opens the path for new therapeutic uses of 4-AP. The newly discovered way in which 4-AP modulates [Ca ⁺], homeostasis is based on a new mechanism of action of this compound particularly as it is manifest postsynaptically in both brain and neuromuscular junctions. Understanding this mechanism provides a new approach for enhancing neurotransmission by intervening at the postsynaptic level. In particular, the use of 4-AP and its analogs provides a pharmacologically effective way to increase the activity of store-operated Ca²⁺ channels. 4-AP activates PLC, enhancing intracellular release of _P , which facilitates release of Ca²⁺ from ICSs. Thus, exposing cells to 4-AP modifies their response to a neurotransmitter or other postsynaptic agonist.

To achieve the effects described herein, 4-AP (or an equivalent or analog) and [Ca ]_ex must be present. 4-AP powerfully potentiates activated Ca²⁺ entry from the extracellular

7+ • environment that is caused by store-depletion. As a result of 4-AP application, [Ca ], mcreases above baseline in a steady-state manner.

Changes in CCE induced by 4-AP also affect responses to neurotransmitters by

7+ • prolonging and increasing [Ca ], elevation. Based on work focused on activity in presynaptic cells, it was thought that 4-AP acts on smooth endoplasmic reticulum Ca²⁺ ATPase to prevent pumping of Ca²⁺ back into the stores. However, in neurons 4-AP does not potentiate CCE, although it increases resting [Ca²⁺],. Therefore, another mechanism must be invoked to explain the large potentiation of CCE. The present inventors conceived that CCE is related to the opening of (a) Ca²⁺ release-activated Ca²⁺ channels or (b) stored-operated Ca²⁺ channels (CRAC-SOCC).

When intracellular stores are depleted and IP3 is increased, a robust signal triggers the opening of CRAC/SOC channels. This could be due to a soluble factor or a physical interaction of CRAC/SOC with an IP3 receptor. The CRAC/SOC channels maybe the target for 4-AP that causes a large influx of extracellular Ca²⁺. The 4-AP agent may lock these channels in an open position resulting in increased Ca ⁺ influx at a time that the endoplasmic reticulum is unable to sequester Ca²⁺ because 4-AP is blocking SERCA. This combination of effects may explain the high potentiation of CCE.

EXAMPLE 1 In the following example, the inventors analyzed how 4-AP may affect calcium homeostasis. In particular, the effect of 4-AP was analyzed on intracellular calcium homeostasis in cortical type I astrocytes, primary cortical neurons, and skeletal muscle cells using fura-2 ratiometric calcium imaging. The studies showed that there are at least three novel actions of 4-AP, unrelated to its ability to block voltage-sensitive K⁺ channels. 4-AP regulates calcium homeostasis by elevating inositol trisphosphate levels and therefore causing calcium release from intracellular calcium stores (ICS), by potentiating capacitative calcium entry (CCE) and therefore agonist-evoked calcium transients.

In particular, the inventors analyzed the effect of 4-aminopyridine (4-AP) on free cytosolic calcium concentration ([Ca²⁺],) in basal conditions, after stimulation with neurotransmitters, and during capacitative calcium entry. Using fura-2 ratiometric calcium imaging, they found that 4-AP increased [Ca²⁺], in type I astrocytes, neurons, and in skeletal

7+ muscle cells. The [Ca ], elevation induced by 4-AP was concentration-dependent and consisted of two phases: the first was dependent on intracellular calcium mobilization, and the second was dependent on extracellular calcium influx. 4-AP also increased the second messenger inositol trisphosphate in both neurons and astrocytes. In astrocytes, 4-AP treatment potentiated the sustained phase of the [Ca²⁺], elevation induced by ATP and bradykinin. In addition, capacitative calcium entry was potentiated several fold by 4-AP, in astrocytes and muscle cells but not in neurons. These effects of 4-AP were completely and promptly reversible. 4-AP blocked voltage-sensitive K⁺ currents in astrocytes. However, voltage-sensitive K⁺ channel blockers inhibiting these currents did not affect agonist-induced calcium transients or capacitative calcium entry, indicating that 4-AP effects on [Ca²⁺], were not caused by the blockade of voltage-gated K⁺ channels. The inventors conclude that 4-AP is able to affect calcium homeostasis at multiple levels, from increasing basal [Ca²⁺], to potentiating capacitative calcium entry. The potentiation of capacitative calcium entry in astrocytes or muscle cells may explain some of the therapeutic activities of 4-AP as a neurotransmission enhancer. MATERIALS AND METHODS

Preparation of primary cultures of cortical type f astrocytes.

Embryonic type I astrocyte cultures were obtained from embryonic day 17 rat fetuses, according to a published protocol, with slight modifications (Grimaldi et al, 1994). Briefly, fetuses were obtained by means of C-section from a 17 d pregnant Wistar rat and quickly decapitated. The heads were placed in PBS (Life Technologies, Gaithersburg, MD) containing 4.5 gm/1 of glucose. Cerebral cortices were dissected, minced, and enzymatically digested with papain (Worthington, Freehold, NJ). The tissue fragments were then mechanically dissociated. The cells in suspension were counted and plated in 25 cm² flasks (10⁶ cells per flask). Medium was changed after 6-8 hr, to wash away unattached cells. Subsequently, the medium was changed every 2 d. This yielded cultures consisting of 95% or more type I astrocytes, as characterized by glial fibrillary acidic protein (GFAP) immunoreactivity (Grimaldi et al, 1999).

Primary cultures of cortical neurons. Neuronal cultures were prepared as described above, with some modifications (Grimaldi and Cavallaro, 1999). Briefly, after obtaining the cell suspension, the inventors counted and tested the cells for viability using the trypan blue exclusion test (viability was >97%). Cells were then plated on poly-L-lysine and collagen-coated glass coverslips in 12 multi-well plates. After 96 hr, 10 μM of fluorodeoxyuridine was added to the medium to inhibit glial cell division, while the medium was supplemented with 10 μM uridine to allow mRNA synthesis.

L6 rat skeletal muscle cell line.

L6 cells were purchased from the American Tissue Culture Collection (Rockville, MD). On arrival, cells were cultured, expanded, and frozen. Cell aliquots were thawed and used between passage 1 and 5. Cells were maintained in DMEM with 10% fetal bovine seram (HyClone, Logan, UT) and Pen/Strep (Life Technologies).

Total inositol phosphate accumulation assay.

Inositol phosphate (InsP_t) accumulation was assayed in astrocytes and neurons as previously described (Grimaldi and Cavallaro, 1999; Grimaldi et al, 1999). Briefly, near-confluent astrocyte cultures were switched to serum-free, myo-inositol-free DMEM containing 2 Ci/ml of myo-[2-³H]-inositol (30 Ci/mmol) (American Radiolabeled Chemicals, St. Louis, MO). After 36 hr, cells were rinsed twice in a saline solution (KRB) containing (in mM): NaCl 125, KC1 5, Na₂HPO₄ 1, MgSO₄ 1, CaCl₂ 1, glucose 5.5, and HEPES 20, pH 7.2. Cells were incubated with KRB containing 20 mM LiCl for 20 min to block InsPl degradation. Cells were then exposed to testing substances for 90 min, at which time the reaction was stopped with 6% ice- cold perchloric acid. Supernatants were transferred to test tubes, and acidity was neutralized with a solution containing 9 mM sodium tetraborate and 0.5 M potassium hydroxide. Five hundred microliters of the neutralized solution were transferred to a new test tube, to which 750 μ\ of anionic exchange resin (Dowex AG 1X-8; 100-200 mesh; mixed 1:3 in water) (Bio-Rad, Hercules, CA) was added. The resin was centrifuged and washed with 1 ml of water. InsP_t were eluted with 500 μ\ of a solution containing 1.2 M ammonium formate and 0.1 M formic acid solution.

Finally, 200 μ\ of the eluant was transferred to scintillation vials, mixed with 4 ml of scintillation liquid (Biosafe II; RPI, IL) and counted for 4 min. Results were expressed as DPM/well. Each experiment was repeated at least three times, and each data point was run in quadruplicate.

Single cell [Ca ⁺], measurements.

Astrocytes, L6, or neurons were seeded on glass coverslips (Assistent, Germany). Before each experiment, the cells were washed once in KRB and loaded with 2 μM fura-2 AM (Molecular Probes, Eugene, OR) for 22 min at room temperature, to minimize probe compartmentalization (Roe et al, 1990), under continuous gentle shaking. After loading, the cells were washed once with KRB and then incubated for 22 min in fura-2 AM-free KRB at room temperature (to minimize the compartmentalization of the probe; Roe et al, 1990), to allow washout of the unesterified probe (Grimaldi et al, 1999). Finally, the coverslips were mounted in a low-volume, self-built 150 μ\ perfusion chamber. Preparations were perfused with calcium or calcium-free KRB saline solution at a speed of 1 ml/min. Experiments were imaged using an inverted microscope equipped with an intensified CCD camera (Videoscope, VA) and a 403 lens (Zeiss fluar series). Calcium-free KRB contained no added calcium and 100 μM EGTA. Image pairs obtained every 2 sec by exciting the preparations at 340 and 380 nm were used to obtain ratio images. Excitation wavelengths were changed using a filter wheel (Metaltek), and the emission wavelength was set to 510 nm. Captured images were processed with a Matrox-LC acquisition board and analyzed by using the software MetaFluor (Universal Imaging, West Chester, PA). Regions of interest were obtained by delimiting the profile of the cells and averaging the fluorescence intensity within the delimited area. Intensity values were converted to [Ca ], using different methods for neurons, muscle cells, and astrocytes. Ratio values were calibrated to [Ca²⁺], for neurons and muscle cells obtaining E_max and R_max and F_mιn and R_mιn by exposing the cells to lOμM ionomycin in presence of 10 mM calcium. After the maximal signal was obtained, cells were perfused with calcium-free KRB containing 10 mM

74-

EGTA. [Ca ], in neurons and muscle cells was then calculated using the equation developed by Grynkiewicz et al. (1985). Ratio values were calibrated to [Ca²⁺], in astrocytes with a titration method. The titration calibration curve was obtained in living cells exposed to known extracellular calcium concentration in the presence of 5 μM ionomycin (containing 5.2% calcium) as previously published (the following are the ratio values measured in astrocytes: 760 nM Caext R340/380 5 18; 1260 μM Caext R340/380 5 32; R340/380 at 0 Caext 5 0.29; R340/380 at 10 mM Caext 5 60) (Grimaldi et al, 1999). Compartmentalization of fura-2 after loading of the cells in the above-specified conditions was assessed to exclude any significant trapping of the probe in organelles. The inventors perfused fura 2-loaded cells with digitonin at 12.5 g/ml and monitored residual fluorescence in the cells with the excitation set at 360 and emission at 510 nm, the isobestic point for fura 2. As a result of cell membrane permeabilization, fura-2 freely diffused from the cytosol to the extracellular space. No significant residual fluorescence was detected inside the cells, indicating negligible probe compartmentalization (data not shown).

Patch-clamp recording.

Astrocytes seeded on glass coverslips were washed in KRB, placed in a perfusion chamber, and then perfused continuously. Glass pipettes were pulled (2-3 MΩ) and filled with a solution containing (in mM): 130 K⁺gluconate, 10 HEPES, 5 BAPTA, 2 ATP, 0.3 GTP, and 1.0 MgCl₂. Cells were voltage-clamped in the whole-cell configuration using an Axopatch-ID amplifier driven by a personal computer running the pClamp acquisition software (Axon Instruments, Foster City, CA). Data were analyzed off-line using the same software.

Fast and transient currents were measured at the peak, whereas steady-state currents were measured at the end of the voltage pulse, at a delay >150 msec. In a second set of experiments, voltage changes before and after drug application were measured in the current- clamp configuration, after waiting several minutes for membrane voltage stabilization and after adjusting the resting voltage to -75 mV with a steady current injection. A fixed current pulse inducing a voltage step of ~5 mV was delivered every 8 sec to continuously monitor the cell input resistance. Only cells with stable holding currents before drug application were considered in the analysis.

Materials.

All materials were purchased from Sigma (St. Louis, MO), unless otherwise specified in the text.

Use of laboratory animals.

Adequate measures were taken to minimize unnecessary pain and discomfort to the animal and to minimize animal use, according to National Institutes of Health regulations on animal handling and care (Guide for the Care and use of Laboratory Animals; National Academy Press, 1996). Pregnant animals were killed by exposure to CO₂.

Statistical analyses.

Experiments were performed at least three times using different cell preparations. For [Ca²⁺]j measurements, digital images were converted to analog data and imported to a spreadsheet. Numeric values, representing the [Ca²⁺]j determined every 2 sec, were averaged, and the SE was calculated. Data are displayed as averages ± SE. When statistical validation was required, data were analyzed by ANOVA followed by a t test and shown as a bar inset in the corresponding figure. Differences were considered statistically significant when the/ 0.05. RESULTS

Effect of 4-AP on [Ca²⁺J,

4-AP moderately increased [Ca²⁺], in cortical type I astrocytes (Figs. 1 A, 2A), cortical neurons (Fig. 3 A), and L6 cells (see Fig. 7B). In astrocytes, increasing concentrations of 4-AP between 1 and 20 mM caused a linear elevation of [Ca²⁺], , with an apparent plateau at 20 mM (Fig. 1A-2). The calculated EC₅₀ was between 5 and 10 mM of the drug (Fig. 1A-2). The elevation of [Ca²⁺], induced by 4-AP had a slow onset and reached a steady-state level that was maintained as long as the drug was applied (Figs. 1 A, 2A). After washing, calcium concentration returned promptly to baseline values (Figs. 1 A, 2A). The 4-AP-induced [Ca²⁺], elevation did not show desensitization (Fig. 1 A). Similar results were found in neurons (Fig. 3A) and in muscle cells (see Fig. 8B-2).

The role of ICS and extracellular calcium in 4-AP-induced [Ca²⁺], elevation in astrocytes and in neurons was analyzed. In the absence of extracellular calcium, 4-AP was still able to elevate [Ca²⁺], in both astrocytes and neurons (Figs. 2B, 3B). However, the prolonged sustained phase of [Ca²⁺], elevation was absent (Figs. 2B , 3B). Muscle cells displayed a similar behavior (see Fig. SB-2). These data suggest that this property of 4-AP is general rather than cell type- specific. In the absence of extracellular calcium, treatment with thapsigargin prevented 4-AP- induced calcium mobilization in astrocytes and neurons (Fig. 2C). The latter evidence suggests that 4-AP-induced calcium elevation is caused by release of calcium from thapsigargin-sensitive ICS.

4-AP increases InsP_t production

To test whether the effect of 4-AP on [Ca²⁺], was caused by the activation of the phospholipase C (PLC)/InsP pathway, the inventors measured InsP_t production, an index of PLC activation, in astrocytes (Fig. 4A) and neurons (Fig. 4B) after exposure to 4-AP. In both cell types, after 4-AP treatment a concentration-dependent increase of InsP production was detected (Fig. 4). Sensitivity of neurons and astrocytes to 4-AP was almost identical. Stimulation of the InsP_t production caused by 4-AP (10 mM, the EC₅₀ for 4-AP) was not very large, compared with the effectiveness of a less than half-maximal concentration of ATP (Grimaldi et al, 1999) (Fig. 4A-2). 4-AP potentiates CCE in astrocytes and muscle cells

Astrocytes exposed to 10 mM 4-AP in calcium-free buffer released calcium from ICS. The subsequent reintroduction of calcium in the perfusion buffer was followed by elevation of [Ca ]„ indicating the activation of CCE (Fig. 2D). If the latter finding is compared with CCE evoked by an agonist such as ATP or bradykinin, it is clear that CCE in the presence of 4-AP is potentiated (see Figs. 6A, C). This finding suggested that 4-AP interfered with CCE. To test whether CCE potentiation by 4-AP could modulate agonist-evoked calcium responses, the inventors analyzed calcium transients after exposure to InsP -linked agonists known to mobilize calcium from ICS. Astrocytes pre-exposed to 4-AP and then challenged with ATP or bradykinin displayed markedly changed calcium transient dynamics. In particular, the amplitude of the sustained phase of the calcium transient, an indicator of CCE, became larger and was significantly prolonged.

In the case of 10 μM ATP, the pattern of [Ca²⁺], elevation in control cells was

9+ characterized by a spike followed by a lower but prolonged phase of [Ca ], elevation (Fig. 5A). When astrocytes were pre-exposed to 10 mM 4-AP and then challenged with 10 μM ATP, the calcium transient induced by ATP lacked the initial spike but had a very high and prolonged plateau phase, which was promptly reversed by removing 4-AP from the perfusion medium (Fig.

5R; statistical validation is displayed in Fig. 5E, values were taken 2 sec before 4-AP removal).

Bradykinin responses were similarly affected by 4-AP. The typical spike-plateau response to bradykinin in control astrocytes (Fig. 5C) was, after treatment with 10 mM 4-AP,

7+ modified to a persistent high [Ca ], elevation, which was promptly reversed after washing out the 4-AP (Fig. 5_9; statistical validation in Fig. 5E, values extracted 2 sec before 4-AP washout).

To further prove that 4-AP potentiates CCE, the inventors designed a group of experiments in which CCE was measured. Control experiments were conducted to determine [Ca²⁺], elevation in response to ICS depletion induced by three different paradigms. First, when ICS were depleted by ATP stimulation in the calcium- free KRB, the reintroduction of calcium induced a very small [Ca²⁺], elevation (Fig. 6 A). ICS were completely emptied by the ATP pulse, as shown by the lack of response to a second stimulation with ATP (Fig. 6A) or bradykinin (Fig. 6C). When the same experiments were performed in the presence of 10 M 4-AP, CCE was greatly potentiated, when compared to ATP or 4-AP alone (Fig. 2D). In 4-AP- pretreated astrocytes, ATP-induced emptying of ICS triggered a large CCE after reintroduction of calcium in the extracellular medium. [Ca²⁺], rapidly rose to an extremely high plateau, which was maintained as long as 4-AP was present and promptly decreased to baseline, once the compound was washed out (Fig. 6R; see statistical validation in Fig. 6E, values extracted 2 sec before 4-AP washout). A similar CCΕ potentiation by 4-AP was seen after ICS depletion with bradykinin (Fig. 6D; statistical validation displayed in Fig. 6F, values extracted 2 sec before 4- AP washout). The effect of 4-AP on CCΕ was concentration-dependent. A 5 mM concentration of 4-AP caused a smaller potentiation than 10 mM 4-AP of CCΕ induced by ICS depletion with either ATP (control, 173 ± 5 nM; 5 mM 4-AP, 765 ± 10 nM; 10 mM 4-AP, 1489 ± 75 r M) or bradykinin (control, 182 + 8 nM; 5 mM 4-AP, 488 ± 16 nM; 10 mM 4-AP, 1084 ± 68 nM). In a second set of experiments, the inventors analyzed CCΕ after depletion of ICS with thapsigargin, an irreversible blocker of the smooth endoplasmic reticulum calcium ATPase (SΕRC A), to exclude that the 4-AP potentiation of CCΕ observed after ATP-and bradykinin- induced ICS emptying was not caused by interaction with secondary signal-transducing mechanisms activated by the two agonists. Exposure to a maximal concentration of thapsigargin (10 μM) would also allow us to assess the role of SERCA blockade in CCE potentiation by 4- AP (Fig. 7 A). Thapsigargin, at 10 μM, applied with calcium- free KRB, completely discharged ICS. When calcium was reintroduced in the extracellular environment, a transient [Ca²⁺], elevation was triggered, which decreased to a lower steady-state [Ca²⁺], (Fig. 7 A). When this experiment was conducted in the presence of 4-AP, CCE triggered by thapsigargin was powerfully potentiated (Fig. 7B; statistical validation presented in Fig. 7C, values were extrapolated at the peak of the response). The inventors tested whether a similar phenomenon occurred in muscle cells and neurons. A strong potentiation of CCE induced by 4-AP was also observed in muscle cells (Fig. 8A, B; statistical validation is displayed in Fig. 8C, values were extracted 2 sec before 4-AP washout). On the contrary, potentiation of CCE by 4-AP was not observed in neurons (Fig. 3, compare D, C).

4-AP inhibits voltage-dependent K⁺ currents in astrocytes

Astrocytes were voltage-clamped at a membrane voltage of -60 mV. Fifty millisecond current pulses were delivered at 10 mV intervals from -80 mV up to +50 mV. Positive currents were recorded, and the resultant f-V profile is shown in Fig. 9 A. Currents measured 40 msec after the start of each pulse were reversibly blocked by bath-applied 4-AP (10 mM) (Fig. 9 A; sample traces displayed in Fig. 9_5). The early, fast inactivating component, measured as the difference between the current 5 and 40 msec after the beginning of the pulse, did not recover from 4-AP blockade even after a 10 min washout (Fig. 9 . In addition, application of 4-AP did not significantly affect resting membrane potential of astrocytes held in current clamp (data not shown). Moreover, the contribution of other conductances at resting potential was negligible, because input resistance, measured with 100 pA current injections, did not change after application of 4-AP (Jin = 32 MΩ + 12 before and after 4-AP application). The voltage- dependent K⁺ channels blocker TEA (20 mM) reduced K⁺ currents in a similar manner (Fig. 9D).

Voltage-sensitive fX channel blockers have no effect on CCE

To assess whether the effects of 4-AP on CCE resulted from blockade of voltage- sensitive K⁺ channels, the inventors analyzed the effect of specific blockers on CCE. They studied ATP-evoked [Ca²⁺]j transients in astrocytes pretreated with α-dendrotoxin (DTx), an inhibitor of the fast inactivating K⁺ cunent (Ransom and Sontheimer, 1995; Rowan and Harvey, 1996; Frizzo and Barbeito, 1997). DTx did not potentiate CCE, although peak responses to ATP were affected (Fig. 10). The voltage-gated K⁺ channel blocker TEA up to 120 mM, did not affect CCE (data not shown). Simultaneous treatment of astrocytes with low concentrations of 4-AP (500 μM) in combination with 50 mM TEA were not able to mimic the effect of 10 mM 4-AP on CCE (data not shown).

Because the inventors found that 10 mM 4-AP caused a slight alkalization of KRB (pH 7.8), the inventors conducted experiments to determine whether changes in extracellular pH might have been involved in CCE potentiation. Increasing the pH of KRB up to 8.4 was not able to reproduce the effect of 4-AP on CCE (data not shown).

DISCUSSION OF THE EXPERIMENTAL RESULTS

4-AP and its analogs have numerous clinical applications, including treatment of neuromuscular and neurodegenerative disorders and traumatic injuries of the CNS (Li and Zhang, 1994; Pinter et al, 1997; Fujihara and Miyoshi, 1998; Gruner and Yee, 1999; Segal et al, 1999; Andreani et al, 2000). All of the therapeutic activities of 4-AP are c rently explained by blockade of voltage-activated K⁺ channels (Vislobokoe et al, 1983; Davies et al, 1991; Choquet and Kom, 1992; Kirsch and Drewe, 1993; Castle et al, 1994). However, effects of 4- AP on calcium homeostasis have also been reported (Agoston et al, 1983; Pant et al, 1983; Tapia et al, 1985; Gibson and Manger, 1988; Campbell et al, 1993). Here, the inventors show that 4-AP causes a complex change of calcium homeostasis, which includes mobilization of calcium from ICS, likely caused by InsP₃ elevation, modulation of InsP -linked calcium transients, and potentiation of CCE activated by ICS depletion. 4-AP modulation of calcium homeostasis may represent an additional molecular mechanism for the therapeutic actions of 4- AP and could be useful in the study of store-operated calcium channels.

The data the inventors present show a weak and concentration-dependent increase of total InsP_t production in astrocytes and neurons after exposure to 4-AP. The inventors have discovered that this effect of 4-AP might be ascribed to an increase of PLC activity. Activation of PLC and subsequent production of InsP₃ may be responsible for 4-AP-evoked calcium mobilization from ICS.

In addition, 4-AP appears to play a significant role in the regulation of calcium entering the cells after ICS depletion, a phenomenon known as CCE. CCE contributes to the magnitude and duration of agonist-evoked [Ca²⁺]j transients, to calcium oscillation, and is the principal mechanism through which ICS are refilled (Putney, 1986). CCE is caused by the opening of the calcium release-activated calcium channel (CRAC) or store-operated calcium channel (SOCC).

This channel has been identified as homologous to the transient receptor potential channels in Drosophila (Petersen et al, 1995). Here, the inventors report that 4-AP potentiates CCE in astrocytes and muscle cells, but not in neurons. Other have already shown that 4-AP can inhibit SERCA (Ishida and Honda, 1993). The fact that in neurons 4-AP does not potentiate CCE, although it increases resting [Ca²⁺]i, strongly suggests that the target of 4-AP is not present in neurons. Because SERCA is present in astrocytes as well as in muscle cells and in neurons, this strongly suggests that 4-AP potentiation of CCE does not involve SERCA. Furthermore, the inventors show that blockade of SERCA with the irreversible inhibitor thapsigargin used at maximal concentrations (Thastrup et al, 1990) evoked CCE to a lesser degree than in the presence of 4-AP. When thapsigargin and 4-AP were added together, the resultant effect on CCE was synergistic. This would not be possible if the target of 4-AP action was only the SERCA. Therefore, the inventors have discovered that 4-AP may be acting on targets different than SERCA. The inventors also have demonstrated that 4-AP can potently prolong and increase [Ca²⁺]j elevations caused by neurotransmitters such as ATP and bradykinin, which are linked to the intracellular messenger InsP . This latter evidence suggests that the physiology of the response to neurotransmitters can be modified when cells are exposed to 4-AP. This indicates that an interaction in vivo is likely to happen and will result in a longer duration of calcium transients. Because the effect of 4-AP alone on CCE is not so large as when it is triggered by a large calcium mobilization, the inventors believe that other mechanisms must be activated to uncover the potentiation of CCE that the inventors observed. When ICS are depleted either using an agonist able to cause a large production of fnsP , such as ATP or bradykinin, or an agent able to completely discharge ICS, such as thapsigargin, a robust signal is generated that triggers the opening of CRAC. Such a signal has not been definitively identified and characterized. However, in cortical type I astrocytes a soluble factor, probably belonging to the family of the eicosanoid derivatives (Rzigalinski et al, 1999), may be responsible for the opening of CRAC channel. Alternatively, a physical association between SOC/CRAC and the InsP receptor (LP3), may be involved in the opening of the CRAC channel after the emptying of ICS (Boulay et al, 1999). Regardless of the signal used to trigger the opening of the CRAC channels, the presence of 4-AP causes a considerably larger influx of calcium from the extracellular space than in control cells. CRAC and voltage-sensitive K⁺ channels have some similarity in the amino acid sequence (Harteneck et al, 2000), therefore, it is conceivable that 4-AP interacts with the open CRAC channels, in a similar manner to K⁺ channels, and thereby increases CCE. The increased calcium influx coupled with the inability of the endoplasmic reticulum to sequestrate it, because 4-AP is blocking SERCA, would ultimately result in the great potentiation of CCE demonstrated in our experiments.

That 4-AP may interact with other targets cannot be excluded. In particular, ligand-gated calcium channels may participate in the calcium transient evoked by ATP (for review, see

Bumashev, 1998). However, the fact that 4-AP potentiates CCE when the ICS are depleted by thapsigargin suggests that these alternative mechanisms may participate but are not required.

Regardless of the mechanisms underlying CCE potentiation, the prolongation and potentiation of agonist-induced [Ca²⁺]i elevation may enhance excitation-contraction coupling of the muscle cells with a consequent improvement of neuromuscular function. Moreover, in the CNS such a potentiation of calcium responses may cause astrocytes to change their state of activation and to secrete trophic factors, which could play an important role in repairing mechanisms and in survival of surrounding neurons.

The inventors have also shown that the effects of 4-AP are not attributable to blockade of voltage-sensitive K⁺ channels. All experiments with different types of compounds affecting voltage-sensitive K⁺ channels were not able to reproduce the effect of 4-AP on CCE.

In conclusion, the inventors report novel effects of 4-AP, namely mobilization of calcium from ICS, PLC activation, and the potentiation of agonist responses through a large potentiation of CCE. These actions may explain some of the therapeutic effects of 4-AP in disorders in which impairment of neurotransmission is involved. Moreover, changes in calcium homeostasis induced by 4-AP in astrocytes might cause the release of trophic factors that would likely support regrowth of neuronal extensions. Finally, the inventors have discovered that 4-AP potentiates CCE by interfering with SOC/ CRAC channels and may thus be a useful tool to study this channel for which specific agonists and antagonists are not yet developed.

In describing preferred embodiments of the present invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. It is to be understood that each specific element includes all technical equivalents, which operate in a similar manner to accomplish a similar pmpose. The above-described embodiments of the invention may be modified or varied, and elements added or omitted, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. Each reference cited here is incoφorated by reference as if each were individually incoφorated by reference.

References

U.S. provisional patent application 60/275,463, filed March 14, 2001, and the following publications are incoφorated herein by reference.

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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a 4-AP agent, an agonist of postsynaptic intracellular Ca²⁺mobilization, and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the 4-AP agent is 4-AP.

3. The composition of claim 1, wherein the 4-AP agent is selected from the group consisting of 3,4-diaminopyridine; 4-amino-3-(phenethylamino)pyridine dihydrochloride; 4- aminopyridine-1 -oxide; N-(3-ch_orophenyl)-N-4-pyridinyl-4-moφholinepropanamine bis(2- hydroxybenzoate); N-4-pyridinyl-N-(3-(trifluoromethyl)phenyl)-4-moφholinepropanamine bis(2 -hydroxybenzoate); N-(3-chlorophenyl)-beta-methyl-N-4-pyridinyl-4- moφholinepropanamine (Z)-2-butenedioate (1 :2); 4-aminopyridine hydrochloride; 3-Methyl-4- aminopyridine; 3-((Dimethylamino)carbonyl)amino-4-aminopyridine (LF 14); 3-Methoxy-4- aminopyridine; 4-Aminopyridine methiodide; and 4-Nitrosomethylaminopyridine.

4. The composition of claim 1-3 wherein the postsynaptic agonist is an agonist of a ryanodine receptor (RyR) or inositol trisphosphate (IP3) receptor.

5. The composition of any of claims 1-3, wherein the postsynaptic agonist is one or more of

(a) a neurotransmitter;

(b) a neurotransmitter receptor agonist;

(c) a compound that prolongs the availability of a neurotransmitter to the postsynaptic cell; (d) a ryanodine receptor (RyR) agonist; or

(e) an inositol trisphosphate (IP3) receptor agonist.

6. The composition of claim 5, wherein the agonist is a cholinergic agonist.

7. The composition of claim 6, wherein the cholinergic agonist is a muscarinic agonist.

8. The composition of claim 1, wherein the agonist is acetylcholine.

9. The composition of claim 1, wherein the agonist is an acetylcholinesterase inhibitor.

10. A method of treating a disorder of impaired neurotransmission in a subject, comprising administering to the subject an effective amount of a combination of an extrinsic postsynaptic agonist and a 4-AP agent effective for enhancing the neurotransmission.

11. The method of claim 10, wherein the disorder is one caused by a traumatic injury to the central nervous system.

12. The method of claim 11, wherein the disorder is a neurologic or neuromuscular disease or dysfunction.

13. The method of claim 12, wherein the disease or dysfunction is myasthenia gravis, multiple sclerosis, Alzheimer's disease, or amyotrophic lateral sclerosis.

14. The method of claim 10, wherein the 4-AP agent is administered together with the postsynaptic agonist.

15. The method of claim 10, wherein the 4-AP agent is administered prior to or after the administration of the postsynaptic agonist, the 4-AP agent and the postsynaptic agonist being present at the synapse together in effective amounts.

16. The method of claim 10, wherein the effective amount of the combination is such that the 4-AP agent attains a concentration between about 1 mM and about 20 mM in the vicinity of target cells of said neurotransmission.

17. The method of claim 16, wherein the target cells are neurons, astrocytes, and/or muscle cells.

18. The method of claim 16, wherein the vicinity is a synaptic space between neurons or a neuromuscular junction.

19. The method of claim 10, wherein the effective amount is one that results in a prolonged increase in the concentration of intracellular Ca²⁺ in the target cells in response to the agonist.

20. A method for promoting neurotransmission, comprising contacting neurons and their target cells with a combination of a 4-AP agent and an extrinsic postsynaptic agonist of

94- intracellular Ca mobilization, thus prolonging and/or potentiating agonist-induced elevation

9+ of intracellular Ca levels as compared to the levels achieved in the absence of the 4-AP agent, thereby promoting neurotransmission.

21. The method of claim 20, wherein the 4-AP causes the prolonged and/or potentiated elevation of intracellular Ca²⁺ levels independently of inhibition of outer membrane voltage sensitive potassium (K⁺) channels.

22. The method of claim 20, wherein the prolonged and/or potentiated elevation depends on the concentration of the 4AP agent.

23. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores (ICS).

24. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by facilitating activation of phospholipase C (PLC) leading to release of inositol trisphosphate (L?3) and, thereby, release of Ca²⁺ from ICS.

25. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by facilitating the modulation of _P3-_inked Ca²⁺ transients.

26. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by potentiating store-depletion-activated elevation of intracellular Ca²⁺ entry.

27. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by facilitating capacitative Ca²⁺ entry (CCE).

28. The method of claim 27, wherein the CCE occurs in muscle cells and/or astrocytes.

29. The method of claim 20, wherein the 4-AP agent, the postsynaptic agonist, or both, acts by facilitating the opening of Ca²⁺ release-activated Ca²⁺ channel (CRAC) and/or store-operated Ca²⁺ channel (SOCC), allowing extracellular Ca²⁺ to enter the target cell, thereby leading to capacitative Ca²⁺ entry.

30. The method of claim 29, wherein the 4-AP agent, the postsynaptic agonist, or both, acts to prevent uptake of intracellular Ca²⁺ by endoplasmic reticulum through inhibition of sarco/endo reticulum Ca²⁺- ATPase.

31. The method of claim 20, wherein the target cells are muscle cells and said increased potentiated intracellular Ca²⁺ levels enhance excitation-contraction coupling leading to improved neuromuscular function.

32. The method of claim 20, wherein the target cells are astrocytes and the elevated Ca²⁺ levels activate the astrocytes to secrete trophic factors active in tissue repair and neuronal survival.

33. The method of claim 20, wherein the contacting is in vivo.

34. A method of potentiating Ca²⁺ mobilization induced by an intrinsic neurotransmitter in a target cell, comprising administering to the target cell a combination of a 4-AP agent and a postsynaptic agonist of Ca²⁺ mobilization such that capacitative Ca²⁺ entry (CCE) is increased.

35. The method of claim 34, wherein the administering is to a subject in vivo.

36. The method of claim 35, wherein the subject has a neurological and/or neuromuscular disorder.

37. A method for screening a test substance for activity as a potentiator of postsynaptic response, comprising: administering a known extrinsic postsynaptic agonist to a synapse model; administering a 4-AP test compound to the model in conjunction with the agonist; measuring postsynaptic response; and selecting the test compound if it produces an elevated intracellular calcium mobilization, voltage change, contraction and/or secretion in comparison to administering a known 4-AP agent in combination with the known postsynaptic agonist.

38. A method for screening a test substance for activity as an agonist of postsynaptic response, comprising: administering a known 4-AP agent to a synapse model; administering a postsynaptic agonist test compound to the model in conjunction with the 4-AP agent; measuring postsynaptic response; and selecting the test compound if it produces an elevated intracellular calcium mobilization, voltage change, contraction and/or secretion in comparison to administering a known postsynaptic agonist in combination with the known 4-AP agent.

39. A method to determine whether a candidate agent is a 4-AP agent, comprising (a) as a negative control, administering a known postsynaptic agonist to a synapse

9+ model and measuring postsynaptic intracellular Ca levels or postsynaptic function; (b) as a positive control, administering a combination of a known 4-AP agent and the known postsynaptic agonist at similar concentrations as in (a) to a synapse

7+ model and measunng postsynaptic intracellular Ca levels or postsynaptic function; thereby determining a differential effect of 4AP on said levels or function; (c) administering a combination of the candidate agent with similar concentrations of the postsynaptic agonist as in (a) and (b) to the synapse model and measuring postsynaptic intracellular Ca²⁺ levels or postsynaptic function, thereby determining a differential effect of the candidate agent on said levels or function; wherein, if the differential effect of the candidate agent is at least about 20% of the differential effect of 4AP, then the candidate agent is a 4AP agent.