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WO1995023132A1 - Agents bloquants des canaux ioniques et leur procedes d'utilisation - Google Patents

Agents bloquants des canaux ioniques et leur procedes d'utilisation Download PDF

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WO1995023132A1
WO1995023132A1 PCT/US1995/002301 US9502301W WO9523132A1 WO 1995023132 A1 WO1995023132 A1 WO 1995023132A1 US 9502301 W US9502301 W US 9502301W WO 9523132 A1 WO9523132 A1 WO 9523132A1
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channels
compounds
activated
voltage
ion
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Inventor
Stanley M. Goldin
Robert N. Mcburney
Lee D. Margolin
N. Laxma Reddy
Subbarao Katragadda
Andrew G. Knapp
Lain-Yen Hu
Graham J. Durant
James B. Fischer
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Cenes Pharmaceuticals Inc
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Cambridge Neuroscience Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/02—Guanidine; Salts, complexes or addition compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/16—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/18—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/20—Acenaphthenes; Hydrogenated acenaphthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
    • C07C2603/24—Anthracenes; Hydrogenated anthracenes

Definitions

  • the present invention pertains to ion channel blockers and methods of treatment and pharmaceutical compositions.
  • Voltage-activated sodium (“Na”) and calcium (“Ca”) channels of electrically excitable cells are currently understood as being protein molecules which are embedded in the lipid bilayer membranes of cells [Hille, B (1992) Ionic Channels of Excitable Membranes. 2nd Edition, Sunderland, MA., Sinauer Assoc, pp. 59-1 14; Goldin, S.M. (1986) Molecular Level Characterization of Ion channels, in The Heart &
  • Said ion channels play a central role in regulation of a wide range of cellular functions in mammals.
  • the opening and closing of a pathway comprising a "pore" within the ion channel molecule regulates the movement of Na and Ca across the cell membrane; this in turn alters the electrical potential across the cell membrane.
  • the movement of said ions through the channels may alter the ion concentrations within or outside the cell.
  • said ion channels As a result of the ability of said ion channels to produce voltage-activated transmembrane movement of Ca and Na, they are responsible for the electrical signals which underlie the rapid flow of information within the brain, and from the brain to the spinal cord [Catterall, W.A. (1992) Physiol. Rev. 72, S15-S48]; Bean, B.P. (1989) Ann. Rev. Physiol. 51: 367-384; Hess, P. (1990) Ann. Rev. Neurosci. 13:337-56]. They produce and propagate the electrical signals in nerve cells which initiate and regulate muscle contraction [Catterall (1992) ibid. ; Bean (1989), ibid.].
  • voltage-activated Ca channels regulate processes which occur on a longer time frame of minutes to hours. These processes include but are not limited to secretion of peptide hormones such as growth hormone from the pituitary gland [Tse et al. (1993) Science 260, 82-84; Chang, J.P. et al. (1988) Endocrinology 123, 87-94; Tan, K.N., and Tashjian, A.H. (1984) J. Biol. Chem. 259: 418-426 ] and insulin from pancreatic beta cells [Larner, J. (1985) in The Pharmacological Basis of Therapeutics, 7th Ed. , Goodman, L.S. and Gilman, A.G. eds., New York, MacMillan, pp. 1490-1503];
  • an ion channel In response to depolarization of the cell membrane to, typically, -10 to +20 mV, an ion channel is more likely to be found in the open state or in the closed, inactivated state than in the closed, activatable state most favored for a metabolically "healthy” cell which is most frequently found at the hyperpolarized, "resting" membrane potential of, typically -60 to - 100 mV.
  • Certain pathophysiological circumstances produce sustained and/or repetitive depolarization of the cell membrane, as occurs in acute disorders including but not limited to brain ischemia resulting from stroke, cardiac arrest [Choi, D.W. (1990) Cerebrovascular and Brain Metab. Rev. 2, 105-147; Meldrum, B.S.(1990) Cerebrovasc. Brain Metab. Rev. 2, 27-57] and in chronic disorders, among them epilepsy [Porter, R. J. (1989) Epilepsia 30, Suppl. 1, S29-S34; Rogawski, M.A., and Porter, R.J. (1990) Pharmacol. Rev. 42, 224-270]; head trauma [Marshall, L.F. (1990) Curr. Opin. in Neurol. and Neurosurg. 3, 4-9]; amyotrophic lateral sclerosis (LAS) [Appel, S.H.
  • Parkinson's Disease [Miller, W.C., and DeLong, M. (1987) in Carpenter, M.B. and Jayaraman, A, eds. The Basal Ganglia, vol. II, New York, Plenum Press, pp. 415-427; Mitchell, I.J. et al., (1989) Neurosci. 32, 213-226] , and hyperkalemic periodic paralysis [Cannon et al. (1991) Neuron 6, 619-626], excessive depolarization-induced activity of said voltage-activated ion channels produces adverse physiological consequences in the excitable cells of, respectively, cardiac, nerve, or muscle tissue. In the case of cardiac arrhythmias, the result is inappropriately timed contractions of cardiac muscle with potentially lethal consequences.
  • the molecular targets of a large subset of compounds of the invention are the voltage-activated ion channels which govern the release of glutamate and other neurotransmitters.
  • voltage-activated ion channels which govern the release of glutamate and other neurotransmitters.
  • two classes of voltage-sensitive ion channels play a central role in governing presvnaptic glutamate release:
  • Antagonists of the neuron-specific type II subclass of voltage-gated Na channels are neuroprotective [Stys, P. K., S. G. Waxman, and B. R. Ransom (1992) J. Neurosci. 12, 430-439; Graham, S.H., J. Chen, F.H. Sharp, and R.P. Simon (1993) J. Cereb. Blood Flow and Metab.
  • BW1003C87 reportedly possesses potent antifolate activity, unrelated to its ability to block Na channels, which causes anemia at neuroprotective dose levels and may limit its clinical utility [Graham, S.H., J. Chen, F.H. Sharp, and R.P. Simon (1993) J. Cereb. Blood Flow and Metab. 13, 88-97].
  • This is one of several examples demonstrating that, although the therapeutic efficacy of glutamate release blockers in animal models has been clearly established, there is clearly a need for novel neuroprotective glutamate release blockers with an acceptable safety profile.
  • L-type Ca channels are particularly sensitive to dihydropyridine Ca antagonists such as nifedipine and nimodipine, and N-type Ca channels are specifically blocked by the cone snail peptide, ⁇ -conotoxin-GVIA [Bean, B.P. (1989) Ann. Rev. Physiol. 51, 367-384].
  • Q-type a new subclass of presynaptic Ca channels controlling neurotransmitter release
  • P- and Q-type Ca channels are closely related to P-type Ca channels: P- and Q-type Ca channels are insensitive to dihydropyridine Ca antagonists or w-conotoxin-GVIA. However, both P- and Q-type channels are specifically blocked by the spider venom peptide ⁇ -aga-IVA, the former subclass being more sensitive to this toxin than the latter.
  • SNX 11 1 is currently in human clinical trials for prevention of ischemic brain damage (ref). SNX 11 1 , which is hypothesized to interact with the ion-selectivity filter [Boland, L., Morrill, J., and Bean, B. (1994) J.
  • NMDA antagonists are highly effective in animal models of focal cerebral ischemia [Albers, G.W., M.P.
  • Figure 2 illustrates the generally accepted key steps in the cascade of events that lead to neuronal cell death in ischemia.
  • NMDA antagonists prevent nerve cell death resulting from the events depicted in the lefthand limb of the diagram.
  • Antagonists of non-NMDA receptors block cell death resulting from the events shown on the righthand limb of the diagram.
  • a blocker of glutamate release by acting at an earlier stage of the process, prevents excessive activation of both NMDA and non-NMDA receptor subclasses, and thus combines the advantages of both NMDA and non-NMDA receptor antagonists. This has recently been demonstrated in in vivo studies of the glutamate release blocker BW1003C87 [Graham, S.H., J. Chen, F.H.
  • BW1003C87 is as effective in models of global ischemia as non-NMDA antagonists, and is as effective in models of focal ischemia as noncompetitive NMDA antagonists such as MK 801 and CNS 1102.
  • a compound that is therapeutically in both focal and global ischemia should be ideally suited to acute treatment of brain damage resulting from high risk cardiovascular surgery.
  • cardiovascular surgery For example, in coronary artery bypass surgery, subsequent neurological deficits have been attributed not only to interruption of the brain's blood supply from the heart, which constitutes a global ischemic insult, but also to small clots
  • Excitable Membranes 2nd Edition, Sunderland, MA., Sinauer Assoc, pp. 59-67] do not block said ion channels in a use-dependent manner as operationally defined herein.
  • the insufficiency of use-dependence in blockade of ion channels limits their therapeutic utility, because said toxins indiscriminately block said ion channels whether or not they are in the open, activatable, or closed conformations.
  • This indiscriminate blockade of said channels whether or not they are in cells functioning in a normal manner not requiring channel blockade, results in untoward toxicity, which in the cases of tetrodotoxin and saxitoxin includes respiratory paralysis [Narahashi, T.
  • the present application describes and claims novel, therapeutically useful compounds and methods for identifying such compounds which exhibit the property of "use-dependent" block of ion channels which conduct cations across the cell membrane. These compounds are particularly well suited to treatment of disorders involving repetitive, persistent or inappropriate depolarization of the cellular membrane potential.
  • the present invention relates to methods of treating mammalian diseases which are associated with increased frequency or duration of depolarization of cells, comprising the administration of a therapeutically effective amount of a compound which inhibits said cells' voltage-activated ion channels preferentially when said channels are activated by repetitive or sustained depolarization by interacting with the SS1/SS2 selectivity filter of said ion channels.
  • the present invention further relates to compounds and methods of identifying compounds which 1) preferentially inhibit cellular ion channels activated by repetitive or sustained depolarization and 2) interact with the SS1/SS2 selectivity filter of said ion channels.
  • These compounds can preferentially inhibit the excessive influx of cations through voltage-activated sodium and calcium channels of cells that experience increased frequency or duration of depolarization while exerting minimal affects on said sodium and calcium ion-channel activity in normal cells, which are relatively more likely to be found in the hyperpolarized state. Specifically, they block the excessive and/or inappropriate entry of cations into cells affected by pathophysiological circumstances which produce sustained and/or repetitive depolarization of the cell membrane while not affecting significantly the function of normal cells. As such, these compounds are particularly advantageous in treating diseases and/or disorders caused by cells which experience increased frequency or duration of depolarization.
  • a preferred embodiment of the present invention offers a significant advantage over compounds currently available for the treatment of the diseases and/or disorders mentioned above.
  • This advantage relates to the ability of a subset of the invention's compounds to block both voltage-activated calcium and sodium channels due to the specific interaction of the compounds with a site contained within both classes of channels. This site, known as the SS1/SS2 ion-selectivity filter, shares a high degree of structural and functional homology in calcium and sodium channels, allowing interaction and blockage of both channel types by the same compounds.
  • Figure 1 illustrates key features of the pathophysiology of focal ischemic damage in stroke.
  • Figure 2 illustrates a cascade of biological events initiated by brain ischemia and leading to nerve cell death.
  • Figure 3 is a diagram of the arrangement in the cell membrane of the ⁇ subunits of a voltage-activated Ca channel and Na channel, illustrating the structural homology between said channels.
  • Figure 4a and 4b illustrate a structural model of voltage-activated Na and Ca channels.
  • Figure 4c displays mutations in the SS2 regions of a voltage-activated Ca channel which affect the ion-conducting behavior of said channels.
  • Figure 5 illustrates the ability of compounds of the invention to competitively inhibit the binding of radiolabeled [ 3 H]-saxitoxin to the SS1/SS2 ion-selectivity filter region of voltage activated Na channels of rat brain nerve terminals (synaptosomes).
  • [ 3 H]-saxitoxin to the SS1/SS2 ion-selectivity filter region of voltage activated Na channels of rat brain nerve terminals (synaptosomes).
  • Figure 6a provides protein sequence data of the SS1/SS2 regions of the internal repeat I of a variety of voltage-activated Ca channel and Na channel clones.
  • Figure 6b depicts the amino acid sequence of the SS2 region of voltage-activated Na channels, illustrating the differences in the sequences between tetrodotoxin-sensitive and tetrodotoxin-insensitive forms of the channel.
  • Figure 7 illustrates structural similarities between tetrodotoxin and two compounds of the invention, CNS 1145 and CNS 1237.
  • Figures 8a and 8b illustrate the use dependent actions of compounds of the invention on nerve terminal Ca channels which results in the ability of said compounds to accelerate the decay of glutamate release from brain nerve terminals as measured by rapid superfusion. It further contrasts this property with the behavior of cone snail and spider venom blockers of said channels, demonstrating that glutamate release blockade is relieved by sustained depolarization.
  • Example 4 Example 4.
  • Figure 9 illustrates the ability of a compound of the invention to block voltage- activated Na channels expressed in CHO cells, in a use-dependent manner enhanced by repetitive depolarization of the cell membrane. It contrasts this property with the lack of use dependence block of said channels by of a compound of the prior art, tetrodotoxin.
  • Figure 10a and 10b illustrates another manifestation of use-dependent block of said voltage-activated Na channels by two compounds of the invention: the ability of depolarization to enhance the rate of block of Na channels by said compounds.
  • Figure 10c illustrates the lack of depolarization-dependent enhancement of the rate of block of said voltage-activated Na channels by a compound of the prior art,
  • Figure 11 demonstrates block by the cone snail venom peptide co-conotoxin MVIIC of glutamate release from brain nerve terminals as measured by rapid superfusion.
  • Figure 12 illustrates the ability of a compound of the invention, CNS 1237, to provide neuroprotection in an animal model of stroke, the rat middle cerebral artery (MCAO) occlusion model.
  • MCAO rat middle cerebral artery
  • Figure 13 displays the ability of compounds of the invention to reduce the size of kainate-induced lesions of rat brain (refer to Example 10).
  • Figure 14 provides the amino acid sequences of the SS1/SS2 ion selectivity filter domain for a representative group of voltage-activated Na and Ca channels.
  • administration delivery of a substance to the individual in need of treatment.
  • a substance in particular a compound of the invention
  • compound a homogenous substance of defined or definable chemical structure.
  • Examples of compounds of relevance are the substituted guanidine compounds of the invention such as CNS 1237, substituted guanidine toxins outside the scope of the current invention such as saxitoxin and tetrodotoxin, and peptides outside the scope of the current invention such as the ⁇ -conotoxins GVIA and MVIIA.
  • depolarization reduction of the membrane potential of a cell from values found in a cell in the "resting, hyperpolarized" state. Said values are normally about -70 mV to -90 mV (negative on the inside surface of the cell membrane).
  • the depolarization When a cell fires action potentials due to the opening of voltage-activated Na or Ca channels, the depolarization is usually robust, reaching values of, typically, about -10 to +20 mV. In ischemic and/or hypoxic situations, the depolarization is typically more gradual and smaller in magnitude, declining to values of, not atypically, about -60 to -30 mV. When said values are reached, a pathophysiological condition of abnormal excitability and concomitant dysfunction of the cell may occur, adversely affecting the surrounding tissue and the health of the individual. depolarization, increased frequency of: a state of an excitable cell in which more frequent depolarization occurs. The use of this term that is of greatest relevance to the current invention involves the more frequent reduction of the membrane potential.
  • Each depolarizing event occurs on the subsecond timescale and may result in activation of the opening of voltage-activated Na or Ca channels.
  • the opening of said channels is often the event which initiates, and/or exacerbates, said increased frequency of depolarization.
  • said increased frequency of depolarization is more likely to occur at inappropriate times and may have
  • depolarization increased duration of: a state of an excitable cell in which the length of time said cell is in the depolarized state is increased. Again, in ischemic and/or hypoxic situations, said increased duration of depolarization is more likely to occur at inappropriate times and may have pathophysiological consequences (e.g., excessive release of glutamate to levels causing neuronal injury and death).
  • depolarization repetitive: depolarizing events that are discrete and recur over a period of time. Repetitive depolarization is a normal and beneficial phenomenon resulting, for example, in appropriately timed release of neurotransmitters from nerve terminals or rhythmic contractions of the heart.
  • Pathophysiological situations may cause and/or be exacerbated by repetitive depolarizations that are inappropriately timed and/or are excessive in frequency, resulting, for example, in cardiac arrhythmias or excessive release of neurotrans-mitters.
  • single cell electrophysiological methods are used to demonstrate the ability of repetitive depolarization to potentiate and/or accelerate block of ion channels by said compounds.
  • depolarization, sustained depolarizing events that endure over time. As used herein, this term usually implies events that last for at least several seconds and may have pathophysiological consequences (e.g., excessive release of glutamate to levels causing neuronal injury and death).
  • hypoxic hypoxia: levels of oxygen below those normally occurring in air, blood, or tissues. Sustained hypoxia may have pathophysiological consequences.
  • inhibition reduction of the ability of a process to occur in the normal manner. For example, sustained depolarization or exposure to an ion-channel blocker may result in inhibition of the ability of a voltage-activated ion channel to conduct ions.
  • inhibition, preferential circumstances favoring reduction of the ability of a process to occur in the normal manner.
  • compounds of the invention preferentially inhibit the ion-conducting ability of voltage-activated ion channels under circumstances of sustained or repetitive depolarization. This is due to the fact that said channels are more susceptible to block by compounds of the invention when said channels are in the conformational states that are more frequently generated by depolarization.
  • ion channels protein molecules, embedded within and spanning the cell membrane, that are normally capable of conducting ions across the cell membrane through a transmembrane "pore" formed within said protein molecule.
  • ion channels, activated an ion channel in a functional/conformational state that allows conduction of ions through its pore, across the cell membrane.
  • ion channels, activatable an ion channel in a functional/conformational state that enables the channel to be activated by a particular initiating event.
  • said initiating event is usually the depolarization of the cell membrane.
  • ion channels, inactivated an ion channel in a functional/conformational state that prevents the conduction of ions through its pore, unless some event occurs that shifts it to the activatable functional/conformational state.
  • ischemia local lack of blood supply in a tissue due to mechanical obstruction of blood vessels delivering blood to the tissue. For example, narrowing of the arteries (arterial stenosis) caused by cholesterol-generated formations (plaques) in the coronary arteries is a frequent cause of ischemia in the heart (myocardial ischemia).
  • MCAO rat middle cerebral artery occlusion
  • normally hyperpolarized cell excitable cells in the "resting" state in healthy tissue normally exhibit membrane potential values, typically, of about -70 to -90 mV as measured electrophysiologically. This is termed a normally hyperpolarized cell.
  • depolarization is a process that initiates normal physiological processes such as neurotransmitter release and myocardial contractility.
  • What is abnormal in the pathophysiological sense are cells undergoing excessive or inappropriate frequency or duration of depolarization, with pathophysiological consequences.
  • pathophysiological circumstances situations producing adverse or undesirable bodily consequences, such as ischemic damage to the brain tissue in stroke or ischemic damage to the heart in myocardial infarction (heart attack).
  • Purkinje cell network of cardiac cells a specialized, electrically coupled network of excitable cells intercalating the muscle of the ventricles of the heart.
  • the purpose of this cell network is to rapidly conduct depolarizing impulses from the AV node of the heart throughout the "working" ventricular myocardium to ensure uniform and properly timed contraction of the ventricles to effectively pump blood.
  • Ischemic damage to the Purkinje cell network can result in depolarizing impulses originating within the ischemic myocardium at "ectopic foci", producing irregular and inappropriate contractions of the heart which may be life threatening.
  • Compounds of the invention should block these "ectopic beats" selectively, due to their ability to selectively block voltage-activated Na channels under circumstances of sustained or repetitive
  • secretion the release of substances from a cell or aggregate of cells (such aggregates of cells are found in secretory tissues such as the pituitary, pancreas, or adrenal medulla), for the purpose of influencing the behavior or functional state of other cells or tissues.
  • secretory processes are the release of catecholamines from the adrenal medulla, release of growth hormone from the pituitary, and release of insulin from the pancreas.
  • Ion channels comprising the targets of compounds of the invention, among them L-type voltage-activated Ca channels, play a central role in governing secretion.
  • secretion occurs via a process of Ca-dependent exocytosis of secreted substances that are packaged within small secretory granules; for example, catecholamines are concentrated in chromaffin granules within chromaffin cells, and catecholamines are released upon elevation of intracellular free Ca levels which is triggered by the opening of voltage-activated Ca channels.
  • SS1/SS2 ion-selectivity filter region the "ion-selectivity filter site” is a site critical to the function of a broad range of voltage-activated ion channels, and has been well characterized for voltage-activated Na and Ca channels. Said site governs the ability of specific ionic species, e.g.
  • SS1/SS2 regions
  • S5-S6 regions
  • the amino acid sequences of the SS1/SS2 regions of a representative family of voltage-activated Na and Ca channels is provided in Example 1.
  • Said channels possess four internally homologous repeat units, as illustrated in Figure 3.
  • Each of the four repeat units contain an SS1/SS2 site, as defined in Example 1 and further illustrated in Figure 4A .
  • the structural arrangement of the pore formed by the juxtaposition of the SS 1/SS2 regions of the four repeat units is illustrated in Figure 4B. Additional information on relevant properties of the SS 1/SS2 region is also provided in the "Detailed Description" below.
  • SS1/SS2 ion-selectivity filter region interaction with: certain compounds are known to block the conduction of ions through the pore of voltage-activated Na and Ca channels by binding to sites within the SS1/SS2 ion selectivity filter region.
  • these include the toxins tetrodotoxin and saxitoxin, which selectively block said Na channels, and the cone snail venom peptides ⁇ -conotoxin GVIA and ⁇ -conotoxin MVIIA
  • SNX111 which selectively block said Ca channels.
  • Compounds of the invention also block said channels by interacting with said ion selectivity filter, but in a use-dependent manner that, as detailed herein, makes them more attractive as therapies for disorders involving excessive or inappropriate activity of said channels (see definition of "use-dependent blocker”).
  • therapeutically effective as applied to the consequences of administration of a compound to an individual, able to produce significant alleviation of the
  • a use-dependent blocker is a compound with the ability to block the activity of voltage-activated Na and/or Ca channels in the conformational states of said channels favored during sustained or repetive depolarization of the excitable cell membrane; such a compound is relatively less effective in its ability to block channels under hyperpolarizing conditions similar to those in quiescent
  • Property 1 Direct interaction with the SS1/SS2 ion-selectivity filter region of voltage-activated Na and/or Ca channels.
  • Property 2 Use-dependent block of voltage-activated Na and/or Ca channels.
  • the prior art has identified compounds that satisfy either property 1 or property 2.
  • the invention claimed herein comprises the administration of compounds which satisfy both of the aforementioned properties. Compounds acting in this manner have not been previously identified or anticipated by the prior art, and constitute an improvement upon the prior art in terms of attractiveness as therapies for the indications described herein.
  • By blocking voltage-activated Na and Ca channels in a use-dependent manner through interaction with the SS1/SS2 ion-selectivity filter region of said channels By blocking voltage-activated Na and Ca channels in a use-dependent manner through interaction with the SS1/SS2 ion-selectivity filter region of said channels, excessive cellular Ca entry and/or cellular hyperexcitability will be inhibited by compounds of invention, and the concomitant pathophysiological consequences will be attenuated.
  • the object of this invention is to produce use-dependent compounds, administered as drugs, which selectively interact with said ion channels in the inactivated and/or the open state and result in attenuation of the ability of ions to move through said pore.
  • Property 1 may be established in either of two ways: (a) The ability to competitively displace suitably labelled ligands which bind to the SS1/SS2 site at concentrations which approximate those required to demonstrate biological activity .
  • Ligands may be employed which are themselves be outside the scope of the current invention by mechanistic criteria (i.e. lack of use-dependence) but which bind to said site.
  • An example described herein is the displacement of radiolabeled [ 3 H]-saxitoxin from binding to the SS1/SS2 site of voltage-activated Na channels (Example 2 and Figure 5).
  • Other examples of such a ligand include but are not restricted to tetrodotoxin, which binds to said site on certain classes of voltage-activated Na channels [Satin, J., et al.
  • Radiolabeled TTX and ⁇ conotoxin G-VI-A are commercially available.
  • radiolabeled [ 125 1] ⁇ conotoxin M-VII-A can and has been prepared [Valentino, K. et al. (1993) Proc. Nat. Acad. Sci. USA 90, 7894-7], and binding of both radiolabeled toxins to Ca channels of brain membranes has been described in the literature [Olivera, B.M. et al. (1987) Biochem. 26, 2086-2090; Miljanich, G.P. et al. (1991) U.S. Patent #5,051,403].
  • Valid tests for competition of said toxins with prospective compounds of the invention for binding to the SS1/SS2 site of Na or Ca channels could be readily developed by one skilled in the art.
  • Example 1 highlights the negatively-charged glutamate and aspartate residues shown by site-directed mutagenesis studies to be critically involved in regulating the tetrodotoxin and saxitoxin sensitivity of Na channels [Noda, M. et al. 19890 FEBS Lett. 259: 213-216; Terlau, H. et al. (1991) FEBS Lett. 293, 93-96] and the ion selectivity of voltage-activated Na and Ca channels [Yang, J. et al. (1993) Nature 366, 158-161; Kim, M.-K. et al. (1993) FEBS Lett. 318, 145-148; for review, see Yellen, G.
  • a preferred embodiment of the invention is the subset of use-dependent compounds whose affinity, as shown by site-directed mutagenesis, is changed by the same site-directed mutations of said glutamate and aspartate residues within the SS1/SS2 regions which affect tetrodotoxin affinity and/or cation selectivity of said channels.
  • An additional preferred embodiment is the subset of use-dependent compounds whose affinity, as shown by site-directed mutagenesis, is altered by site-directed mutations of residues immediately to the right of said glutamate residues diagrammed in Example 1, as that locus, in addition, corresponds to the sites of additional mutations which alter tetrodotoxin's affinity for the ion-selectivity filter [c.f. Satin, J., et al. ( 1992) Science 256, 1202-1205, and Figure 6b].
  • Compounds of the invention have the ability to block the activity of voltage-activated Na and/or Ca channels during sustained or repetitive depolarization of the excitable cell membrane, and are relatively less effective in their ability to block said channels under conditions found in quiescent, normally hyperpolarized excitable cells.
  • Property 2 may be established in either of two ways:
  • Variations and/or refinements of the electrophysiological methods described herein may also be employed, including but not restricted to: the use of single channel recording, a variant of the patch clamp electrophysiology technique that affords information on the activity of individual ion channels [Sakmann, B. Nad Neher, E. (1983) Single Channel Recording. New York, Plenum Press]; and measurement of the electrical activity within brain slices in vitro, for example the use of hippocampal slice preparations [Home, A. L., and Kemp, J.A. (1991) Br. J. Pharmacol. 103, 1733-39].
  • Example 4 Exemplified herein (Example 4) and further described below is the ability of compounds of the invention to accelerate the decay of neurotransmitter release from brain nerve terminals, measured in vitro using rapid kinetic techniques [Goldin S. M. (1990) U.S. Patent #4,891,185]. Said acceleration of decay results from use-dependent block of presynaptic Ca channels which govern neurotransmitter release from brain nerve terminals. Other examples of the acceleration of the decay of processes controlled by v ⁇ ltage-activated ion channels include the acceleration of the decay of the membrane depolarizations responsible for muscle contraction, a process also governed by voltage-activated ion channels [Cannon, S. C. et al. (1992), ibid.].
  • Additional methods, and/or variations and refinements of the methods described herein may be employed to measure neurotransmitter release or the activity of voltage-activated Na or Ca channels governing said release, among them measurement of the electrical activity induced by neurotransmitter release within hippocampal slices, fluorescence and/or enzymatic methods to analyze synaptosomal neurotransmitter release, and stopped-flow rapid kinetics [Cash, D.J., and Katragadda, S. (1987)
  • Some therapeutic applications may be more likely to be undertaken by compounds acting preferentially to block a voltage-activated channel comprising a relevant therapeutic target when said target is experiencing high stimulus frequencies. This would be a desirable property, for example, of an anticonvulsant agent, as epilepsy is known to involve rapid bursts of high frequency firing of action potentials at epileptic foci.
  • a second category of therapeutic applications such as limitation of ischemic brain damage, may be more appropriately treated by a compound which also exhibits frequency-dependent block of voltage-activated ion channels when the frequency of stimulation is relatively low.
  • Still a third category of therapeutic applications may benefit from compounds which act to block channel activity induced by sustained depolarization of the cell membrane. It has been demonstrated that in sustained brain ischemia, the depletion of cellular ATP levels [Shimizu, H. et al. (1993) Brain Res. 605, 33-42] causes dissipation of the Na and K gradients and concomitant sustained depolarization of the cell membrane [Zivin, J.A. and Choi, D.W. (July, 1991) Sci. Amer. 265, 36-43]. Said sustained depolarization generates hyperactivity of neuronal ion channels [Alzheimer, C. (1993) J. Neurosci. 13, 660-673].
  • the positively charged guanidinium group may interact with negatively charged residues in the filter, and high affinity block is imparted by interaction of other groups on that toxin with adjacent residues.
  • Figure 6a displays sequence data for the SS1/SS2 region of internal repeat I of a variety of Ca and Na channel clones from both rat and human cDNAs.
  • the negatively charged residue, glu (E) or asp (D) are the highly conserved amino acids hypothesized, in TTX-senstitive Na channels (e.g., brain I - III) to be the site of interaction of the positively charged guanidinium group of TTX with the selectivity filter.
  • the adjacent hydrophobic residue (tyr [Y] or phe [F]) confers TTX-sensitivity, as very recently reported by the independent studies of two different laboratories [Satin, J., et al. (1992) Science 256, 1202-1205; Backx, P.H.
  • Figure 6b illustrates repeat I of a generalized voltage-sensitive Na channel ( 85). The seven amino acid SS2 sequence is shown, and the differences between the TTX-resistant ("RHII") and TTX -sensitive ( 1 -Brain II ) isoforms are indicated.
  • Figure 4 displays salient structural information on voltage-activated Ca and Na channels.
  • Panel A is a Diagram of sodium channel transmembrane topology adapted from a description provided by Noda, M. et al. [(1986) Nature 320, 188-192.]. The
  • homologous internal repeats I - IV each contain six membrane spanning segments (S1 - S6) represented as cylinders.
  • the SS1/SS2 ion selectivity filter region is represented by the separation between S5 and S6 in each of the four internal repeats.
  • the sequence similarity in the SS1/SS2 site includes human as well as rat Ca and Na channel cDNA's (refer to sequence information in Example 1).
  • This information is of immense practical therapeutic utility, because drug candidates developed based on biological feedback from in vitro assays employing material derived from rats (e.g. rat brain nerve terminal preparations employed in examples 4 and 6 infra. ) and in vivo models of disease states (e.g. the rat kainate neurotoxicity model and the rat stroke model, examples 8 and 9 infra) are expected to exert similar actions in humans because the sites to which the drug candidates bind are structurally very similar if not identical.
  • This consideration has strongly influenced our decision to diligently pursue the creation of use-dependent blockers of the SS1/SS2 binding site of voltage-activated Na and Ca channels. 2. Creation of a compound series which interacts with the
  • NMR studies of the conformation in aqueous solution of these compounds suggested that II-II stacking of the two planar acenaphthyl groups forced the positively charged guanidinium group to extend away from the molecule and create a structure whose size and shape roughly resembles TTX. This is illustrated for the case of two compounds of the invention, with the aid of the computer-generated molecular models as shown in Figure 7. This result was supported by the results of molecular modelling/energy minimization studies, which indicated that a "stacked conformation" of these compounds was energetically accessible in aqueous solution.
  • Methods for generating use-dependent block at the ion-selectivity filter site of voltage-activated ion channels utilize the administration of particular substituted guanidines or analogs thereof.
  • Said substituted guanidines contain a positively charged guanidinium group which has been shown to interact with a negatively charged amino acid residue or residues within the ion-selectivity filter site.
  • Analogs of said substituted guanidines comprise compounds whose structural properties also predispose them to interact with the ion-selectivity filter site in a use-dependent manner.
  • Said analogs could include any compounds that contain groups that are likely to exist as a positively-charged ionic species under the conditions likely to pertain within voltage-activated ion channels.
  • Derivatives of the ammonium cation form a most numerous and diverse group of analogs of the guanidines.
  • Said analogs include, but are not limited to, substituted amidines, for example formamidines, substituted hydroxylamines, and substituted hydrazines. It is additionally pertinent with respect to the aforementioned chemical classes of compounds that there is evidence that the formamidinium cation, the hydroxylammonium cation, etc. interact with a negatively charged residue or residues within the ion-selectivity filter site (Hille, B., J. Gen Physiol. 58, 599-619; reviewed in Hille, B (1992) Ionic Channels of Excitable Membranes.
  • guanidinium moiety or analog thereof we demonstrate herein the creation of use-dependent blockers of the ion-selectivity filter site of voltage-activated ion channels.
  • Said substituents include but are not limited to acenaphthylenes, halogenophenyls, fluorenes, para- or meta-butylphenyls, para-terbutylbenzyls, anthracenes, and methoxynaphthylenes. 3.
  • Compounds of the invention which competitively displace [ 3 H ]- saxitoxin from the SS1/SS2 ion-selectivity filter site of voltage- activated Na channels
  • TTX analogue [ 3 H ]saxitoxin
  • said acenaphthyl guanidine derivatives were directly shown to interact with the TTX binding site on the ion-selectivity filter region of neuronal Na channels.
  • [ 3 HJSTX binding was measured in rat brain synaptosomes essentially as previously described [Gusovsky, F. et al., (1990) Brain Res. 518: 101-106].
  • the K d for [ 3 H]STX binding to the synaptosome preparation was found to be about 2.5 nM.
  • TTX inhibited the [ 3 H]STX binding with a K i of about 25 nM.
  • said guanidine derivatives provided as examples of compounds of the invention, at concentrations ranging from ⁇ 1 to 30 uM, were found to increase the Kd of [ 3 H]STX binding, with no significant change in the [ 3 H]STX binding site density, indicating a purely competitive mode of inhibition.
  • Said examples further establish that a subset of compounds of the invention have dual actions on two subclasses of Ca channels, based on direct measurement of the ability of compounds of the invention to block 45 Ca entry through said channels.
  • Example 6 demonstrates this for voltage- activated (P- and Q- type) Ca channels which control neurotransmitter release from brain nerve terminals.
  • Example 7 demonstrates this for L-type Ca channels, the channel subclass found in cardiovascular cells, cells of skeletal muscle, and secretory cells including those of the pituitary and the adrenal medulla.
  • a subset of compounds of the invention block both voltage-activated Na channels and Ca channels.
  • compounds of the invention block said Ca channels at concentrations comparable, on an order of magnitude basis, to those which block voltage-activated Type II Na channels (c.f Examples 6, 7 and 8).
  • Said broad spectrum of action is a desirable property for neuroprotective agents [Kucharczyk, J. et al. (1991) Radiology 179, 221-227], and should also be a desirable property for cardioprotective agents as well.
  • the aforementioned CHO cell line expressing Type II Na channels can be employed to assess the actions of compounds of the invention on said Na channels, directly demonstrating the use-dependent mechanism of action of compounds of the invention on voltage-activated ion channels constituting a therapeutically desirable target for drug action.
  • the actions of these compounds on sodium channels were examined using the whole-cell voltage clamp recording technique. As illustrated ( Figure 9a), CNS 1237 at low micromolar concentrations exhibits a high degree of depolarization-dependent block during stimulation at higher frequencies. These effects are similar to the actions of a number of local anesthetic, anticonvulsant and
  • CNS 1237 interacts preferentially with open and/or inactivated states of the channel, relative to its actions on channels in the closed but activatable state favored in resting, hyperpolarized cells.
  • compounds of the invention may be identified by rigorous examination of their interaction with voltage-activated ion channels using single-cell electrophysiological techniques. Below are described the specific criteria that may be used to identify compounds acting as use-dependent blockers of a specific voltage-activated ion channel. a. Repetitive channel activation enhances the potency of compounds of the invention for blocking said voltage-activated ion channel.
  • Figure 9a demonstrates the ability of one of several compounds of the invention, CNS 1237, to block voltage-activated Type II neuronal Na channels in a frequency-dependent manner. This characteristic is contrasted with the relative inability of one of the compounds of the prior art, tetrodotoxin, to produce the same phenomenon when tested as described ( Figure 9b).
  • the experimental protocols employed are detailed in Example 2. As illustrated, CNS 1237 is markedly more potent in its ability to block type II Na channels when the channels are repetitively opened by trains of depolarizing impulses.
  • the IC50 for tonic blockade of this channel is ⁇ 100 uM, whereas the IC50 for additional frequency-dependent blockade of this channel is about tenfold greater.
  • tetrodotoxin exhibits potent block of said Na channels irrespective of whether the channels are repetitively opened by trains of depolarization of the cell membrane containing said channel ( Figure 9b).
  • Repetitive channel activation accelerates the rate at which compounds of the invention block said voltage-activated ion channels.
  • the aforementioned Example entails stimulation protocols which demonstrate that CNS 1237 and other compounds of the invention achieve a significant degree of frequency-dependent block during merely several seconds of high-frequency
  • compounds such as CNS 1237 may be particularly advantageous for certain therapeutic indications
  • compounds of the invention at optimal therapeutic concentrations in mammals, may require minutes or even hours of repetitive stimulation in order for them to produce frequency-dependent block of a voltage-activated ion channel targets of the invention.
  • Such a relatively slow onset of block at therapeutic concentrations may be tolerable and even desirable, particularly for therapy of chronic indications such as LAS [Appel, S.H. (1993) Trends Neurosci. 16, 3-5].
  • Treatment with compounds of the invention may be delayed for at least a few hours, nonetheless producing a significant degree of neuroprotection.
  • FIG. 10 and 10b demonstrates the ability of repetitive nerve stimulation to accelerate the rate at which several of the compounds of the invention block a voltage-activated ion channel of the invention, the Type II neuronal sodium channel subclass of said channels, when the cloned gene for the ⁇ -subunit of said channel is expressed in a CHO cell line.
  • compounds of the invention CNS 1237 and CNS 5149 , are subjected to depolarizing stimuli at a relatively low frequency: 0.1 Hz, in contrast with the higher frequencies previously illustrated in Figure 9 and Example 2.
  • said low frequency of stimulation produces an additional component of blockade of Na current, due either to enhancement of the rate of onset of block or increase in the potency of the compound's ability to block said Na channels. when equilibrium between the blocked and unblocked states of the channel at this stimulus frequency has been reached.
  • a preferred embodiment of the current invention is the use of compounds of the invention to block, in a use-dependent manner, the voltage-activated Na and/or Ca channels which regulate neurotransmitter release, in particular glutamate release
  • the method involves first preloading rat brain synaptosomes with 3 H-glutamate via the Na-dependent glutamate uptake system.
  • the preloaded nerve terminals are retained in a superfusion chamber accessed by high-speed, solenoid- driven valves.
  • Microcomputer-operated circuitry controls the timing of valve operation; the valves control the delivery under nitrogen pressure of depolarizing pulses of high K + buffer, Ca, and/or drugs to the synaptosomes.
  • the 3 H-glutamate-containing effluent is continuously collected in a high speed fraction collector on a timescale as short as 30 msec.
  • the high solution flow rate and minimal dead volume of the superfusion chamber afford rapid solution changes and precise control of the chemical
  • Said use-dependent block is a therapeutically attractive property for an antiischemic agent, as such an agent would be more effective in blocking persistent, excessive glutamate release activity observed in ischemia than the brief, transient responses characteristic of "normal" release events.
  • an antiischemic agent would be more effective in blocking persistent, excessive glutamate release activity observed in ischemia than the brief, transient responses characteristic of "normal" release events.
  • CNS 1237 CNS 1237
  • compounds of the invention which exhibit depolarization-dependent block of 3 H-glutamate release from brain nerve terminal preparations also block the uptake of 45 Ca into synaptosomes prepared by the same method used to study 3 H-glutamate release from brain nerve terminals (Example 4).
  • depolarization-dependent block of presynaptic Ca channels is the mechanism responsible for the depolarization-dependence of 3 H-glutamate release from brain nerve terminal preparations by compounds of the invention.
  • Said toxin potently blocks the early kinetic (“phasic") component of glutamate release (measured during the first 600 msec of depolarization) , elicited by depolarization with 25 mM K, with much greater potency than displayed in its ability to block the "persistent" component (i.e., release measured during the time interval from 0.9 to 5.0 sec).
  • the toxin exhibits biphasic block of the phasic component, with an IC 50 of ⁇ 0.1 ⁇ M for block of the high affinity component.
  • the IC 50 for block of the persistent component was ⁇ 1 ⁇ M.
  • the current invention as defined in the present application identifies the binding site as the ion-selectivity filter/SSI -SS2 regions of the voltage-activated Na and/or Ca channels blocked by compounds of the invention.
  • the structural homology between voltage-activated Na channels and voltage-activated Ca channels and subclasses thereof See figure 3 and Catterall, W.A. (1988) Science 242, 50-52; Heinemann, S. H., H. Terlau, W. Stuhmer, K. Imoto, S. Numa (1992) Nature 356, 441-443; Backx et al., Science 257 (1992) 248-251; Zhang, J.-F. et al. (1993) Neuropharmacol.
  • a preferred embodiment of the current invention is the use-dependent blockade of disorders involving excessive and/or inappropriate activity of neuronal voltage-activated Na and/or Ca channels. Accordingly such compounds of the invention will be useful for the treatment of a variety of disorders involving inappropriate or excessive activity of said ion channels, including disorders of the nervous system such as produced by a hypoxic/ischemic insult to the brain or in epilepsy. In addition, it is appreciated that the structural homology between the neuronal
  • Na and Ca channels extends to their counterparts in other cells, among them cardiac cells, vascular and skeletal smooth muscle cells, pituitary cells, and secretory cells within the pancreas, pituitary, and adrenal medulla (see above and Hess, P., Ann. Rev. Neurosci. 13, 337-356).
  • cardiac cells vascular and skeletal smooth muscle cells
  • pituitary cells and secretory cells within the pancreas, pituitary, and adrenal medulla
  • a subset of compounds of the invention effectively block L-type Ca channels of clonal pituitary secretory cells; said L-type Ca channels are in turn closely related to cardiovascular L-type channels. Accordingly, compounds of the invention will be useful in therapy of a variety of additional disorders treatable by block of said Ca and Na channels.
  • cardiovascular disorders such as cardiac arrhythmias and hypertension
  • disorders of secretion such as acromegaly or diabetes insipidus.
  • compounds of the invention will be useful for other indications, among them relief of chronic pain and as local anesthetics.
  • Voltage-activated Na-channels to be blocked by said compounds include but are not limited to voltage-activated ('action potential'), tetrodotoxin-sensitive Type I and Type II sodium channels in nerve axons and nerve terminals, and voltage-activated tetrodotoxin-insensitive sodium channels in cardiac or skeletal muscle [Catterall, W.A. ( 1992) Physiol. Rev.
  • Voltage-activated Ca-channels to be blocked by said compounds include but are not limited to voltage-activated L-type calcium channels of heart, skeletal muscle, or brain; voltage-activated N-type calcium channels of neurons or neuroglial cells; and voltage-activated P or- Q-type calcium channels of nerve terminals, or nerve cell bodies [Snutch, T.P. and Reiner, P.B. (1992) Curr. Opin. Neurobiol. 2, 247-53; Tsien, R.W. et al (1991) Trends Pharmacol. Sci. 12, 349-54; Zhang, J.-F. et al. (1993) Neuropharmacol. 32, 1075-84].
  • compounds of the invention Under the circumstances of brain or tissue ischemia, which result in excessive and/or inappropriate depolarization of the excitable cell membrane, compounds of the invention will exhibit effective blockade of said ion channels. This results in reduced Ca and/or Na influx into the cells through said ion channels within the cell membrane, hence avoiding the concomitant pathophysiological consequences.
  • the avoidable consequences are cell injury and death induced by excessive Ca entry as occurs in brain ischemia, and inappropriately timed activation of contractions of the heart by Na channel activation as occurs in cardiac arrhythmias generated in ischemic cardiac tissue.
  • compounds of the invention are relatively ineffective in blocking said ion channels in healthy tissue, because the cell membrane in which said ion channels reside is more usually hyperpolarized, causing the ion channels to adapt conformational states which are resistant to interaction with said compounds of the invention.
  • Efficacy as therapy in stroke is exemplified by the actions of CNS 1237, which effectively reduces cell death in a rodent models of focal ischemia (stroke) ( Figure 12 and Example 9).
  • compounds of the invention with such dual actions are neuroprotective in an animal model relating to depolarization-induced neuronal hyperactivity such as occurs in epilepsy.
  • Said compounds may be administered prophylactically or within 72 hours of an acute ischemic insult to prevent cellular death and destruction.
  • treatment should preferably commence within 24 hours of said insult.
  • Example 7 documents the ability of compounds of the invention to block Ca channels in GH4C1 clonal pituitary cells. Said pituitary cells secrete prolactin and growth hormone [Tashjian, A.H. (1979) in Meth. Enyzmol. 58: 527-535, Acad. Press, N.Y.].
  • Example 7 Block of voltage-activated Ca channels of pituitary cells GH4Cl cells inhibits secretion of said peptides [Tan, K.N., and Tashjian, A.H. (1984) J. Biol. Chem. 259: 418-426 ]. Therefore, the results disclosed in Example 7 constitute a direct demonstration of the ability of compounds of the invention to function as effective antisecretory agents.
  • a hypersecretory disorder treatable by a suitably formulated compound of the invention could be the treatment of pheochromocytoma, which is a disorder resulting from the presence of a tumor of the chromaffin cells in the adrenal medulla [Bravo, E.L. and Gifford, R.W. (1984) New Eng. J. Med. 311: 1298-1300].
  • This disorder is characterized by the hypersecretion of catacholamines, resulting in hypertension which may be paroxysmal and associated with attacks of palpitation, headache, nausea, breathing difficulty, and anxiety.
  • Another hypersecretory disorder treatable by compounds of the invention may be pancreatitis, which is an inflammation of the pancreas leading to hypersecretion of hormones and enzymes from the acinar cells of the pancreas, among them hormones such as vasoactive intestinal peptide (VIP) and insulin; digestive enzymes and their inactive precursors, among them upases and proteases, deoxyribonucleases, ribonucleases, and amylase [Greenberger, N.J.
  • compounds of the invention particularly those which are charged and/or hydrophilic and otherwise do not cross the blood/brain barrier are believed to be clinically useful upon systemic and/or local administration.
  • Certain hypersecretory disorders may result from abnormal activity of cells within the central nervous system, among them cells of the pituitary gland, also termed the hypophysis, located at the base of the brain. Secretion of hormones and related substances from cells of the adenohypohysis is regulated by releasing factors, primarily those secreted by the hypothalamus [Cooper, P.E. and Martin, J.B. (1992) in Diseases of the Nervous System: Clinical Neurobiology, Asbury et al. eds, Saunders,
  • Substances secreted by the adenohypophysis include growth hormone, prolactin, thyroid stimulating hormone (TSH), and
  • adrenocorticotrophic hormone (ACTH). Hypersecretion of these substances from the pituitary can lead to a variety of disorders of growth (e.g. acromegaly due to
  • Secretion from the neurohypophysis of the pituitary is regulated by innervation from elsewhere in the CNS.
  • the release of oxytocin and vasopressin is regulated at least in part by the activity of neurons in the paraventricular nucleus of the hypothalamus, which innervates the neurohypophysis.
  • Compounds of the invention will be useful in disorders of secretion of substances from the neurohypophysis, among them dilutional hyponatremia, which is believed to be caused by inappropriate secretion of vasopressin [ see Martin, J.B., and Reichlin, S., Clinical Neuroendocrinology, 2nd Edition (1987) Philadelphia, Davis].
  • compounds of the invention will also be useful for treatment of disorders involving hypersecretion of substances produced by the hypothalamus, among them diabetes insipidus, which may be caused by
  • AVP hypersensitivity to, or excessive release of, AVP.
  • AVP is a peptide synthesized in and released from neurons of the supraoptic and paraventricular nuclei of the hypothalamus [see Cooper, P.E., and Martin, J.B. in Diseases of the Nervous System: Clinical Neurobiology, Asbury et al., eds, Saunders, Philadelphia (1992), pp. 567-583].
  • a current means of treatment of diabetes insipidus is surgical destruction of most of the cells in the supraoptic nucleus.
  • Pharmacotherapy with compounds of the invention may in some instances obviate the need for such neurosurgery.
  • D Use of compounds of the invention for treatment of cardiovascular disorders
  • Example 7 a subset of compounds of the invention show equal or greater potency for block of L-type Ca channels when compared with the ability of verapamil or diltiazem to block said channels using the same assay protocol.
  • Example 8 indicate that compounds of the invention will find utility in therapy of cardiovascular disorders treatable by blockers of Na channels.
  • a major indication for such Na channel blockers is cardiac arrhythmias, which are currently treated by blockers of Na channels, among them quinidine, procainamide, lidocaine, and diphenylhydantoin (phenytoin).
  • cardiac arrhythmias successfully treatable by said Na channel blockers are ventricular tachycardia; ventricular premature depolarizations; digitalis-induced atrial tachycardia and atrial and ventricular arrhythmias; paroxysmal supraventricular tachycardia; atrial fibrillation; and
  • compounds of the invention should find utility in treatment of cardiac arrhythmias treatable by blockers of cardiac Na channels.
  • compounds of the invention particularly those which are charged and/or hydrophilic and otherwise do not cross the blood/brain barrier, are believed to be clinically useful upon systemic and/or local administration.
  • Compounds of the invention can be employed, either alone or in combination with one or more other therapeutic agents as discussed above, as a pharmaceutical composition in mixture with conventional excipient, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
  • conventional excipient i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • solutions preferably oil
  • suppositories are convenient unit dosages.
  • a syrup, elixir or the like can be used wherein a sweetened vehicle is employed.
  • Sustained release compositions can be formulated including those wherein the active component is protected with
  • differentially degradable coatings e.g., by microencapsulation, multiple coatings, etc.
  • Intravenous or parenteral administration e.g., sub-cutaneous, intraperitoneal or intramuscular administration are generally preferred. It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.
  • a suitable effective dose of one or more compounds of the invention will be in the range of from 0.5 to 500 milligrams per kilogram body weight of recipient per day, preferable in the range of 1 to 100 milligrams per kilogram bodyweight of recipient per day.
  • the desired dose is suitable administered once daily, or in several sub-doses, e.g., 2 to 4 sub-doses, are administered at appropriate intervals through the day, or other appropriate schedule.
  • Such sub-doses may be administered as unit dosage forms, e.g., containing from 0.25 to 25 milligrams of compound(s) of the invention per unit dosage, preferably from 0.5 to 5 milligrams per unit dosage.
  • compounds of the invention may be administered continuously for a period of time, for example by an intravenous infusion or by means of a transdermal mode of delivery using, for example, a patch incorporating and releasing said compounds.
  • Said method of drug delivery should result in selective binding of the liposomes to the target tissue, and release of the compound of the invention near the abnormally functioning skeletal muscle Na channels, where said compound will inhibit the persistent activation of muscle Na channels which constitutes the molecular abnormality underlying the disease.
  • the use of site-directed pro-drugs or related chemical modifications of compounds of the invention may enable a lower dosage, and/or result in fewer undesirable side effects.
  • Another instance in which such a targetted drug delivery method may be desirable is in the case of pheochromocytoma or another abnormality which results in hypersecretion of catecholamines into the blood. Because the Ca channels of chromaffin cells are closely related to those of nerve, cardiac cells, and muscle (Neher, E. and Zucker, R.S. (1993) Neuron 10, 21-30; Bean, B.P. (1989) Ann. Rev. Physiol. 51: Example 1
  • Amino acid sequences of the SS1/SS2 ion-selectivity filter domains for a family of Na and Ca channel targets comprising sites of
  • catecholamines from chromaffin cells may produce undesirable side effects resulting from block of, for example, neuronal and cardiovascular Ca channels. Accordingly, delivery of compounds of the invention to chromaffin cells may be enhanced by their incorporation into liposomes containing a monoclonal antibody targetted to specific antigens on or near the surface of the chromaffin cells of the hypersecreting adrenal medulla. This method of liposome-mediated drug targetting is currently being developed for delivery of a variety of agents, to be used for indications such as cancer
  • the assay utilizes the whole cell configuration of the voltage clamp recording technique (Hamill et al. , 1981) which allows a direct measurement of inward sodium current.
  • the assay takes advantage of the fact that varying the timing of stimuli relative to the application Na channel antagonists provides a methodology for distinguishing between tonic (resting state) and frequency-dependent (open state) blockade of sodium channels.
  • the protocol of the assay is described below.
  • the CNaIIA-1 cells are grown in RPMI 1640 (MediaTech) supplemented with 5% FCS (Hyclone), 200 ug/ml G418 (Sigma) and 5.75 mg/ml Proline (Sigma).
  • EDTA-free trypsin (1:250, Sigma) is used to split the cells IX per week and cells are seeded @ 1:100 into a T75 flask (CoStar) and 1:200 - 1:6400 into a 24-well plate containing glass coverslips (Fisher).
  • the media is changed once a week in the flasks. Since a single 24-well plate is prepared each week, the media is not changed. Cells whose passage number exceeds 20-25 are not used.
  • the external recording solution contains 150 mM NaCl, 5 mM KCl, 1.5 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Glucose, 5 mM HEPES; pH 7.4, adjusted with NaOH; -310 mOsm.
  • Working solutions are prepared from 10X stock solutions.
  • the internal pipette solution contains 150 mM CsF, 10 mM EGTA, 10 mM HEPES; pH 7.4, adjusted with CsOH; ⁇ 300 mOsm.
  • Whole cell currents are measured using an Axopatch 200A integrating patch clamp amplifier (Axon Instruments; Foster City, CA. Capacity transients are cancelled and series resistance is compensated 90%.
  • Isolated cells are used whenever possible to eliminate artifacts caused by gap junctions and/or poor spatial clamping.
  • Relative current from each trial (1 trial 10 pulses @ 1 Hz or 40 pulses @ 10 Hz) was then normalized by either dividing the current amplitude recorded from the first pulse of the test train (I firs t ) by the average current recording during the control train (I ave. control ) for determination of tonic block (Eq. 1) or by dividing all the current amplitudes of a given trial (I pulse ) by the highest amplitude of that trial (I max pulse ) for frequency-dependent block (Eq. 2).
  • Figure 9 demonstrates the ability, enhanced by depolarization, of one of the compounds of the invention, CNS 1237, to block voltage-sensitive type II neuronal Na channels.
  • CNS 1237 is considered to preferentially block the open state of the Na channel over the resting state. Therefore, it is concluded that CNS 1237 is better at producing frequency-dependent block than it is at producing tonic block. This characteristic is contrasted with the inability of one of the compounds of the prior art, tetrodotoxin (TTX), to produce the same phenomenon when tested in an identical manner.
  • TTX tetrodotoxin
  • the tonic block curve for TTX is shifted to the left of the frequency-dependent curves, which overlap each other. Therefore it is concluded that TTX is better at producing tonic block of Na channels than it is at producing frequency-dependent block.
  • the above tonic/frequency-dependent block relationship generates a fingerprint identifying compounds on the basis of their relative preference for producing one type of Na channel blockade over another.
  • Figure 13 which provides examples of each of slow and fast depolarization-dependent blockade of sodium current.
  • Panel A Figure 13.
  • depolarizations is as high as 10 Hz .
  • Patent #4,891,185; Turner, T.J. Bruce, L.B., and Goldin (1989) Anal. Biochem. 178:8-16] to measure depolarization-induced 3 H-glutamate release from brain nerve terminals.
  • the depolarizing stimulus opens presynaptic voltage-activated ion channels as the key step required to initiate Ca-dependent exocytosis of glutamatergic synaptic vesicles.
  • the method involves preloading rat brain synaptosomes with 3 H-glutamate via the Na-dependent glutamate uptake system.
  • the preloaded nerve terminals are retained in a superfusion chamber accessed by high-speed, solenoid-driven valves.
  • Microcomputer- operated circuitry controls the timing of valve operation; the valves control the delivery under nitrogen pressure of pulses of depolarizing buffer, Ca, and/or drugs to the synaptosomes.
  • the 3 H-glutamate-containing effluent is continuously collected in a high speed fraction collector on a subsecond timescale as short as 30 msec (300 msec fractions were employed herein, and in Figures 8 and 11).
  • the high solution flow rate and minimal dead volume of the superfusion chamber afford rapid solution changes and precise control of the chemical microenvironment of the nerve terminal preparation.
  • the method is, more specifically, that described in Goldin et al.,
  • Veratridine is known to stimulate neurotransmitter release by opening voltage-activated Na channels, which results in depolarization of the nerve terminal plasma membrane and in turn, secondarily, opens presynaptic Ca channels to directly trigger 3 H-glutamate release via Ca-dependent exocytosis [Leach et al., Epilepsia 27, 490-497].
  • the use of veratridine-induced glutamate release was employed to detect compounds of the invention which may block neurotransmitter release by blocking voltage-activated presynaptic Na channels.
  • FIG. 8a The ability of compounds of the invention block glutamate release from brain nerve terminal preparations is illustrated in Figure 8.
  • a compound of the invention, CNS 1237 accelerates the decay and reduces the amplitude of Ca-dependent 3 H-glutamate release.
  • substituted guanidine compounds of the invention show use dependence in blocking Ca-dependent glutamate release.
  • Initial amplitude and decay constants of Ca-dependent release in the presence of drug were expressed as % of control (no CNS compound in superfusion). The results are from a minimum of 3 different experiments of each condition.
  • CNS compounds were tested at 2 M with the exception of 5149 (1 M) and 5118 (3 M).
  • ⁇ -Aga IV A and w-Cm toxin MVIIC were tested at 0.3 M.
  • Non-specific binding was determined in the presence of 10 M TTX, and was generally about 10% of the total binding.
  • the K d for [ 3 H]STX binding to the synaptosome preparation was found to be about 1.5 nM.
  • TTX inhibited the [ 3 H]STX binding with an IC50 of ⁇ 32 nM; using the Chang Prussoff analysis, this corresponds to a K i of about 6 nM.
  • CNS 1145 was found to inhibit the binding of [ 3 H]STX to synaptosomes with an IC50 of -22 M, and CNS 1237 inhibited with an IC50 of -6 M.
  • CNS 1145 was used in further experiments to determine whether the inhibition was competitive or non- competitive.
  • CNS 1 145 (20 ⁇ M) was found to increase the Kd of [ 3 H]STX binding in competitive manner (p ⁇ 0.02), with no significant change in the [ 3 H]STX binding site density, indicating a purely competitive mode of inhibition.
  • Table 1A shows that the listed compounds competitively inhibit the binding of [ 3 H]STX, because they decrease the [ 3 H]STX affinity but not the number of [ 3 H]STX binding sites. Graphically this is shown in the accompanying representative Scatchard plot ( Figure 5), which demonstrates that CNS 1145, 1212, 1237 and 5044 decrease the slope of the binding curve, but not its X-axis intercept.
  • Table IB Displays the IC50 values and chemical names for the above compounds and several additional compounds of the invention:
  • PCT/US92/01050 The principle of the method involves opening ion permeation through synaptosomal calcium channels by high K + - induced depolarization of the synaptosomal preparation. The rapid component of 45 Ca uptake measured by this procedure is mediated by presynaptic calcium channels.
  • synaptosomes are prepared by the method of Hajos [Brain Res., 93:485-489 (1975)]. Freshly prepared synaptosomes (8 ⁇ l) were suspended in low potassium "LK” buffer (containing 3 mM KCl). Test compounds in 8 ⁇ l LK buffer were added to synaptosomes to final concentrations ranging from 0.3 ⁇ M to 100 ⁇ M, and the mixture was preincubated for 5 minutes at room temperature. 45 Ca uptake was then initiated by adding isotope in either LK or in buffer (“HK”) containing high [potassium] (150 mM KCl). After 5 seconds, the 45 Ca uptake was stopped by adding 0.9 ml quench buffer (LK + 10 mM EGTA). This solution was then filtered under vacuum and the filters washed with 15 ml of quench buffer.
  • LK low potassium
  • Test compounds in 8 ⁇ l LK buffer were added to synaptosomes to final concentrations ranging from 0.3 ⁇ M to 100 ⁇ M,
  • Washed filters were subjected 10 scintillation spectrophotometry to determine the extent of 45 Ca uptake. Net depolarization-induced 45 Ca uptake was determined for each concentration of each compound tested, as the difference between 45 Ca uptake in HK and LK buffers. Results were plotted as % inhibition of depolarization-indueed 45 Ca uptake vs. [compound] for each compound tested. Representative IC 50 for inhibition of depolarization-induced 45 Ca uptake are presented below in the accompanying tables.
  • L-type calcium channels Compounds of the invention representative of each of the major classes of agents claimed herein were tested to determine their ability to inhibit voltage-activated, dihydropyridine-sensitive L-type calcium channels in clonal GH4Cl pituitary cells.
  • Said voltage-activated L-type calcium channels are found in cardiac muscle, vascular smooth muscle, and the cardiac Purkije cell conduction system ( see references in Background and Description of Invention). They are the sites of action of the major classes of Ca antagonists employed to treat hypertension, angina, cardiac arrhythmias, and related disorders.
  • L-type Ca channels are also the sites of action of certain neuroprotective dyhydropyridine Ca antagonists such as nimodipine.
  • the uptake of 45 Ca into GH4Cl cells was performed by an adaptation of the method of Tan, K., and Tashjian, A.H. [J. Biol. Chem. , 259: 418-426 (1984)].
  • the principle of the method involves activating ion permeation through synaptosomal calcium channels by high K + - induced depolarization of the synaptosomal preparation.
  • the uptake of 45 Ca measured by this procedure is mediated by presynaptic L-type calcium channels, and is sensitive to dihydropyridine, phenylalkylamine, and benzothiazipine Ca antagonists at therapeutically relevant concentrations [Tashjian and Tan, ibid, and unpublished data, Cambridge NeuroScience].
  • the adaptation of the aforementioned method involves growing GH4Cl cells in 96-well culture plates, and is designed to provide a rapid and quantitative determination of the potency of various compounds in inhibiting 45 Ca uptake through L-type Ca channels.
  • GH4 cells stored in liquid nitrogen, are suspended in 15 ml growth medium (Ham's F-10 medium plus 15% heat-inactivated horse serum and 2.5% heat-inactivated fetal bovine serum). The cells are centrifuged, resuspended, and then added to T-75 flasks containing 12-15 mls Growth Medium, and incubated at 37 °C for approximately 1 week. The cells are them removed from the T75 flask after dissociation from the walls of the flask by treatment for 5 minutes at 37°C with 1 mg/ml Viocase. The Viocase is decanted, and the cells are resuspended in ⁇ 200 ml of Growth Medium.
  • growth medium Ham's F-10 medium plus 15% heat-inactivated horse serum and 2.5% heat-inactivated fetal bovine serum.
  • the cells are centrifuged, resuspended, and then added to T-75 flasks containing 12-15 mls Growth Medium, and incubated at 37
  • the cells are then aliquoted (200 ⁇ l/well) into each well of several 96 well plates.
  • the cells are then grown under the aforementioned conditions for 3-4 weeks, with replacement of Growth Medium occurring twice per week. Cells are fed growth medium 24 hours before they are employed for 45 Ca uptake determinations.
  • media is aspirated from each 96-well plate using a manifold designed to allow 50 ⁇ L of liquid to remain in each well.
  • Each plate is washed and aspirated twice with a low K + buffer solution "LKHBBS" (in mM 5 KCl, 145 NaCl, 10 Hepes, 1 MgCh, 0.5 MgCl 2 , 10 glucose, pH 7.4), 200 ⁇ l/well.
  • LKHBBS low K + buffer solution
  • 50 ⁇ l of HBBS containing the drug to be tested in twice the final concentration is added.
  • the plates are incubated for 10 minutes at room temperature.
  • 50 ⁇ l of either of two solutions are added:
  • HKHBBS a high K + buffer containing 150 mM KCl and no NaCl, but otherwise identical to LKHBBS.
  • Each plate is then incubated for 5 minutes at room temperature, aspirated as above, and quenched with 200 ⁇ l/well of Quench Buffer (Ca-free LKHBBS containing 10 mM Tris-EGTA). Each plate is aspirated and rinsed with Quench Buffer a second time, then carefully aspirated to dryness. To each well of each plate 100 ⁇ l of High Safe II scintillation fluid is added. The plates are sealed, shaken, and subjected to scintillation spectrophotometry on a Microbeta 96-well Scintillation Counter (Wallac, Gaithersburg, MD, USA).
  • Net depolarization-induced 45 Ca uptake was determined for each concentration of each compound tested, as the difference between 45 Ca uptake in HKBBS and LK buffers. Results were plotted as % inhibition of depolarization-induced 45 Ca uptake vs. [compound] for each compound tested. Representative IC 50 for inhibition of depolarization-induced 45 Ca uptake are presented below in the accompanying tables.
  • Antagonists of the neuron-specific type II subclass of voltage-activated Na channels are neuroprotective [Stys, P. K., S. G. Waxman, and B. R. Ransom (1992) J. Neurosci. 12, 430-439].
  • the ability of compounds of the invention to block voltage - activated Type II Na channels was determined in a functional assay employing a Chinese Hamster Ovary ("CHO") cell line expressing cloned Type II Na channels derived from rat brain [West, J. W., T. Scheuer, L. Maechler and W. A. Catterall (1992) Neuron 8, 59-70].
  • CHO Chinese Hamster Ovary
  • the assay is based on the observation that veratridine, an alkaloid neurotoxin, causes persistent activation of sodium channels, and tetrodotoxin, a heterocyclic agent derived from puffer fish, is a potent and highly specific blocker of several major subclasses of voltage-activated sodium channels, including the said Type II subclass. It further takes advantage of the finding that guanidinium cation will permeate through tetrodotoxin-sensitive Na channels when said channels are opened, either by membrane depolarization [Hille, B., Ionic Channels of Excitable Membranes 2nd Edition, Sinauer Associates, Sunderland, MA (1992) pp. 349-353 ] or by exposure to veratridine [Reith, M.E.A., Eur. J. Pharmacol. 188 (1990), 33-41]
  • the assay entails measuring veratridine-stimulated, tetrodotoxin-sensitive influx of [ 14 C]-guanidinium ion through cloned Type II Na channels expressed in CHO cells.
  • the protocol of the assay is described below. Assay protocol:
  • the aforementioned CHO cell line is grown by standard cell culture techniques in RPMI 1640 medium (Media Tech), supplemented with 5% fetal calf serum
  • Cultures are rinsed 3 times with 200 1 of "preincubation buffer” (5.4 mM KCl, 0.8mM MgSO4, 50 mM Hepes, 130 mM choline chloride, 0.1 mg/ml BSA, 1 mM guanidine HCI, 5.5 mM D-glucose, pH7.4) and incubated with 200 1 preincubation buffer at 37 °C for 10 minutes.
  • preincubation buffer 5.4 mM KCl, 0.8mM MgSO4, 50 mM Hepes, 130 mM choline chloride, 0.1 mg/ml BSA, 1 mM guanidine HCI, 5.5 mM D-glucose, pH7.
  • CNS test compounds Different concentrations of CNS test compounds are prepared by dilution into "uptake buffer” (preincubation buffer plus -2.5 mCi/ml [ 14 C]-guanidinium HCI, ⁇ 40 mCi/mmol) containing veratridine (100 M). Aliquots (50 1) of these working stocks is added to the 96-well plates and incubated at room temperature for 1 hour. The veratridine-induced [ 14 C]-guanidinium uptake was linear with time and a good signal (4-8 fold basal uptake) was obtained following a 1 hour incubation.
  • uptake buffer preincubation buffer plus -2.5 mCi/ml [ 14 C]-guanidinium HCI, ⁇ 40 mCi/mmol
  • veratridine 100 M
  • results herein demonstrate the neuroprotective efficacy of a compound of the invention, CNS 1237, in the rat permanent MCA occlusion model of focal ischemia.
  • the results indicate that the compound, dosed i.p. as a single bolus prior to MCA occlusion affords significant neuroprotection with relatively modest effects on blood pressure.
  • the data offer encouragement that we are solidly on track towards
  • Focal ischemia was induced in isoflurane-anaesthetized rats by occlusion of the MCA, using the technique of Shiraishi et al. (1989) 1 .
  • Male Sprague-Dawley rats (275-300 g) were induced with 5% isoflurane, intubated, and ventilated with 1.5%
  • isoflurane The femoral artery and vein were cannulated. Animals were maintained on a heating pad and under a heating lamp. Blood pressure was monitored continuously during and for up to 30 minutes following the surgery for selected animals. An incision was made over the temporal scalp, the temporalis muscle was retracted, and a portion of the maxilla was removed to expose the foramen ovale. The foramen oyale was enlarged to expose the origin of the MCA. The MCA was then coagulated under direct vision from its origin to the olfactory tract. Care was taken to coagulate the penetrating arteries that arise from the MCA and supply the lateral caudate at this level. The operator was blinded as to which animal received drug. Compound was administered in 0.3 M mannitol as an i.p. bolus 5 minutes before MCA occlusion. An equal volume of vehicle was infused i.v. to control animals at corresponding times.
  • Hemispheric infarct area in each section was calculated by subtracting the area of normally TTC staining brain in the ipsilateral ischemic hemisphere from the contralateral non-ischemic area.
  • the volume of infarction for the total brain hemisphere was calculated by summing the infarct area in each section measured and multiplying by the distance between sections. This technique minimizes the effect of edema upon measurement of infarct size 1 1 .
  • the infarct size results were also subjected to One-way Analysis of Variance (AVOVA) and the Bonferroni Multiple comparisons Test to determine P value. Differences between control groups and each dose were regarded as statistically significant (*) if the P value in the latter analysis was ⁇ 0.05.
  • the lower dose (6 mg/kg) displayed a comparable degree of neuroprotection: 29% (S.E.M. 6.3%, P ⁇ 0.05).
  • the highest dose, 24 mg/kg displayed a lower level of neuroprotection that was below the level of statistical significance ( 16%, P > 0.05).
  • Glutamate is believed to be the most abundant excitatory neurotransmitter in the brain, and at the elevated levels known to be present in cerebral ischemia, trauma and other pathologic states, is highly neurotoxic.
  • glutamate receptors Several major classes of glutamate receptors have been identified including NMDA, AMPA and kainate receptors.
  • Kainate receptors are present in a number of brain regions (including the hippocampus) that are critical for memory function. Numerous investigators have examined the lesions that occur following kainate administration and have determined that kainate's excitotoxic effects are not only due to direct neuroexcitation, but also to enhanced release of glutamate which then generates a neurotoxic cascade. Thus the destructive effects of kainate are reduced by agents (such as lamotrigine) that inhibit the synaptic release of glutamate, and intracranial kainate injection can serve as an assay for identifying more effective glutamate-release blockers.
  • agents such as lamotrigine
  • This example demonstrates the use of a kainate neurotoxicity model to identify compounds with in vitro glutamate-release inhibiting properties to determine whether they are neuroprotective in vivo and therefore of potential value in the treatment of disorders associated with excessive glutamate release.
  • young adult rats 150g
  • kainate in 0.3M mannitol
  • they are treated with potentially neuroprotective glutamate-release inhibiting compounds that are either co-injected with the kainate (intracranial administration) or injected intraperitoneally.
  • the compounds identified with this assay are believed to be neuroprotective as a result of their ability to block glutamate release. These compounds and others of their class have strong potential as therapeutics for the wide range of neurologic disorders in which excessive glutamate release leads to neuronal death and cerebral dysfunction.

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Abstract

L'invention concerne des procédés de traitement de maladies et de diverses autres affections corporelles qui impliquent l'activité excessive inappropriée et/ou prolongée de canaux ioniques activés par une tension, procédés qui permettent une amélioration par l'administration de composés qui bloquent ces canaux. Ils consistent à administrer à des mammifères des composés qui bloquent une ou plusieurs classes de canaux ioniques sodium et/ou calcium activés par tension, d'une façon définie opérationnellement comme 'dépendante de l'utilisation'. On peut citer à titre d'exemples précis de pathologies qu'on peut traiter avec des composés relevant de cette invention, entre autres, les ischémies cérébrales et cardiaques, l'épilepsie et la sclérose latérale amyotrophique (SLA).
PCT/US1995/002301 1994-02-23 1995-02-22 Agents bloquants des canaux ioniques et leur procedes d'utilisation Ceased WO1995023132A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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RU2211032C2 (ru) * 1996-06-03 2003-08-27 Хехст Акциенгезелльшафт Применение ингибиторов клеточного натрий-водородного обмена для получения лекарственного средства для нормализации уровня липидов сыворотки
US6780866B2 (en) * 2001-05-18 2004-08-24 Wex Medical Instrumentation Co., Ltd. Analgesic composition and method
US6949567B2 (en) 2001-02-26 2005-09-27 4Sc Ag Compounds for the treatment of protozoal diseases
US7084116B2 (en) 2003-03-10 2006-08-01 Dynogen Pharmaceuticals, Inc. Methods for treating lower urinary tract disorders and the related disorders vulvodynia and vulvar vestibulitis using Cav2.2 subunit calcium channel modulators
US7125848B2 (en) 2003-06-13 2006-10-24 Dynogen Pharmaceuticals, Inc. Methods of treating non-inflammatory gastrointestinal tract disorders using Cav2.2 subunit calcium channel modulators
US7223754B2 (en) 2003-03-10 2007-05-29 Dynogen Pharmaceuticals, Inc. Thiazolidinone, oxazolidinone, and imidazolone derivatives for treating lower urinary tract and related disorders

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WO1990014067A2 (fr) * 1989-05-02 1990-11-29 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Procedes de traitement de l'angoisse a l'aide de ligands de recepteur sigma
WO1991018868A1 (fr) * 1990-05-25 1991-12-12 STATE OF OREGON, acting by and through the OREGON STATE BOARD OF HIGHER EDUCATION, acting for and onbehalf of the OREGON HEALTH SCIENCES UNIVERSITY Guanidines substituees ayant un coefficient de liaison eleve avec le recepteur sigma et utilisation de ces substances
WO1992014697A1 (fr) * 1991-02-08 1992-09-03 Cambridge Neuroscience, Inc. Guanidines substituees et leurs derives utilises comme modulateurs de liberation de neurotransmetteurs et nouveau procede d'identification d'inhibiteurs de liberation de neurotransmetteurs
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WO1990014067A2 (fr) * 1989-05-02 1990-11-29 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon Procedes de traitement de l'angoisse a l'aide de ligands de recepteur sigma
US5262568A (en) * 1990-03-02 1993-11-16 State Of Oregon Tri- and tetra-substituted guanidines and their use as excitatory amino acid antagonists
WO1991018868A1 (fr) * 1990-05-25 1991-12-12 STATE OF OREGON, acting by and through the OREGON STATE BOARD OF HIGHER EDUCATION, acting for and onbehalf of the OREGON HEALTH SCIENCES UNIVERSITY Guanidines substituees ayant un coefficient de liaison eleve avec le recepteur sigma et utilisation de ces substances
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2211032C2 (ru) * 1996-06-03 2003-08-27 Хехст Акциенгезелльшафт Применение ингибиторов клеточного натрий-водородного обмена для получения лекарственного средства для нормализации уровня липидов сыворотки
US6949567B2 (en) 2001-02-26 2005-09-27 4Sc Ag Compounds for the treatment of protozoal diseases
US6780866B2 (en) * 2001-05-18 2004-08-24 Wex Medical Instrumentation Co., Ltd. Analgesic composition and method
US7084116B2 (en) 2003-03-10 2006-08-01 Dynogen Pharmaceuticals, Inc. Methods for treating lower urinary tract disorders and the related disorders vulvodynia and vulvar vestibulitis using Cav2.2 subunit calcium channel modulators
US7223754B2 (en) 2003-03-10 2007-05-29 Dynogen Pharmaceuticals, Inc. Thiazolidinone, oxazolidinone, and imidazolone derivatives for treating lower urinary tract and related disorders
US7459430B2 (en) 2003-03-10 2008-12-02 Dynogen Pharmaceuticals, Inc. Methods of using ziconotide to treat overactive bladder
US7125848B2 (en) 2003-06-13 2006-10-24 Dynogen Pharmaceuticals, Inc. Methods of treating non-inflammatory gastrointestinal tract disorders using Cav2.2 subunit calcium channel modulators

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