Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
• • 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
• • 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
  • A61K31/16—Amides, e.g. hydroxamic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
  • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
  • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
  • C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C239/00—Compounds containing nitrogen-to-halogen bonds; Hydroxylamino compounds or ethers or esters thereof
  • C07C239/08—Hydroxylamino compounds or their ethers or esters
  • C07C239/16—Hydroxylamino compounds or their ethers or esters having nitrogen atoms of hydroxylamino groups further bound to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
• • 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
  • C07C279/12—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 being further substituted by nitrogen atoms not being part of nitro or nitroso groups
• • 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
  • C07C279/14—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 being further substituted by carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• This invention relates generally to the use of polyamine compounds as agents regulating ionic conductances in cellular membranes. More specifically, certain aspects of this invention relate to the use of certain polyamines in blocking, modulating, or activating calcium (as well as other cationic) channels in neuronal cell membranes and in blocking, modulating or activating calcium channels of a specific type in any cell membrane where such channels are present.
• channel and “channel protein” are used interchangeably without implying that a channel must necessarily consist of a single protein, although the channels that have been isolated are believed to be single proteins.
• channel proteins mediate the transport of one ionic species with substantially higher specificity than transport of other ionic species. It is common to name various channels after the ion for which they are specific: thus, there are sodium channels, potassium channels, calcium channels, etc.
• ionic channels specific for the transport of one cation may be further divided into various subcategories or channel types, based on the way they interact in response to electrical and/or chemical (pharmacological) stimuli.
• Calcium channels in particular which have been identified in a number of different cell types, including neurons, appear to have differences (as well as similarities) in morphology, properties and/or function. Some of these differences are ascribable to the morphology and function of the cells in which such channels occur: for example neuronal calcium channels are by far the most complex in all three aspects. Miller, R.J., infra. Other differences have not been directly related to cell types; in fact, different types of calcium channels are normally present in the same cell.
• Neuronal cells for example, display four operationally distinct types of calcium conductance and thus there are four "types" of neuronal calcium channels. (Some of the properties of each type of neuronal calcium channel, however, appear to be shared by calcium channels in other tissues.)
• neuronal calcium conductances were first divided into two categories based on the driving force that activates them: the high-threshold calcium conductance (or HTCC) and the low-threshold calcium conductance (LTCC). In central neurons, HTCC is more prominent in the dendrites and LTCC is more prominent in the soma or cell body. Later, neuronal calcium channels were grouped into three categories: the T-channels, which are believed to be responsible for LTCC; the N-channels, the conductance properties of which showed imperfect correspondence to HTCC; and the L-channels which are not commonly represented in central neurons but of which the conductance properties also showed correspondence to HTCC.
• the T-channels which are believed to be responsible for LTCC
• the N-channels the conductance properties of which showed imperfect correspondence to HTCC
• L-channels which are not commonly represented in central neurons but of which the conductance properties also showed correspondence to HTCC.
• the L-channels are dihydropyridine-sensitive, i.e. they are effectively blocked by dihydropyridines, such as nifedipine and nitrendipene, whereas the T- and N-channels are dihydropyridineresistant.
• the T-channels resist blockage by cadmium ions; more important, the T-channels are specifically blocked by alcohols (especially octanol) at 10 -4 M or lower concentrations.
• both the N- and L-channels are said to be blocked by omega-conotoxin, a toxin isolated from the venom of the marine snail Conus geographicus to which the T-channels are resistant. Miller, R.J. Science, 1987, 235: 46-52.
• HTCC both the calcium-dependent plateau potential component and the dendritic spike component of the HTCC
• Purkinje cells are activated at -50 mV, and are dihydropyridine-insensitive and also omega-conotoxin-resistant. These channels are specifically blocked by a low-molecular weight blocking agent isolated from the venom of funnel-web spiders. Llinas et al, PNAS. 1989, 86:1689-1693.
• the dihydropyridine- and conotoxin-resistant calcium channels appear to be absent from inferior olivary and thalamic neurons. These calcium channels have been called P-channels because they were first described in Purkinje cells. Llinas, R.R., et al., Ann. N.Y. Acad. Sci., 1989, 560:103-111. P-type channels have also been shown to exist in squid giant synapse and squid optic lobe.
• Sodium and potassium channels are also of various types. See generally Hille, B. [”Ionic Channels of Excitable Membranes” Sinauer Assoc. Inc. ,Sunderland, Mass. 1984.]
• TTX tetrodotoxin
• a second type of sodium channel responsible for the slow sodium current is blocked by local anesthetic agents such as lidocaine.
• a distinctly different sodium channel is located in non-electrically excitable tissue such as epithelium, and is not blocked by TXX or the local anesthetics but blocked by the diuretic amiloride.
• K + channels tetraethylamonium (TEA)
• TAA tetraethylamonium
• Agents that block calcium channels with high affinity and specificity as well as agents that activate calcium channels are useful as reagents in electrophysiological research. Availability of such agents is essential for understanding calcium channel properties and function. Such agents are especially useful in the design of prototype drugs and in drug screening.
• a blocking agent specific for P-channel permits the investigation of conductances of other channels (which coexist with the P-channel in various in vitro or in vivo experiments) without interference from the P-channel calcium conductance.
• a blocking agent can serve as a prototype drug which could be used, e.g., for regulating calcium transport through cellular membranes.
• such a drug would have potential applications in prevent ing cell death caused by ischemia or other anoxic cerebropathies or by other factors which disrupt the regulation of calcium transport, such as aging.
• Such drugs also would have potential applicability in the treatment of epilepsy, and memory and learning disorders.
• the P-channel in particular is resistant to the natural toxin, omega conotoxin; to dihydropyridine and its derivatives; and to alcohols.
• Calcium-channel activating agents i.e. agents that increase the influx of calcium through these channels
• a known L-channel-specific activator is available commercially under the tradename Bay-K8644.
• Bay-K8644 there are no prior art activators for N-,.T- or P-channels.
• Specific calcium-channel activators can also serve as useful research reagents, but unlike blockers, activators can be used to test different channel properties such as, for example, the limits of the capacity of P-channels to allow calcium influx into a cell. Activators can also be used to study synaptic transmitter release and other aspects of transmission more closely, e.g., by amplifying the driving force, namely the presynaptic inward calcium current.
• the Ca channel activators of the present invention also have potential uses as prototypic drugs exhibiting anticonvulsant (e.g. anti-epileptic), anxiolytic, tranquilizing, anti-Alzheimer's, and/or memory-improving properties. More generally activators are potentially useful as prototypic drugs in pathologies or behavioral alterations attributable to an insufficient ability of cells (especially neurons) to permit Ca ++ influx.
• Alteration of the function of Na + channels may be of therapeutic utility for the treatment of muscle spasms, torticollis, tremor, learning disorders, and Alzheimer's disease.
• Polyamines which block slow sodium channels would have additional utility as local anesthetic agents.
• Agents which act on the epithelial Na channel would be useful adjuncts in the treatment of cystic fibrosis, and asthma and as antihypertensive agents.
• Drugs which modulate the activity of K + channels would be useful as protective agents against the damaging effects of anoxic and ischemic disorders and hypertension, act to protect red blood cells against damage in malaria, and sickle-cell disease.
• Calcium activators and calcium blockers are expected to yield information about how the event of cell death is organized. For example, the influence of the presence of a calcium activator in the extracellular medium on the onset and rate of progression of the cytosolic calcium "flood" observed could be measured, eliciting information on whether an increased ability of the cell membrane to transport calcium influences the onset and the rate of release of intracellular calcium and, more important, whether this late-occurring manifestation of cell death can be reversed.
• spermidine may have a Ca ++ antagonist function and that spermidine and certain other polyamines may have a calcium channel blocking activity, "perhaps" similar to that of the known Ca 2+ -entry blocking drugs referred to above.
• spermine is said to inhibit spontaneous contraction of the uterus muscle, in a manner than can be counteracted by increasing extracellular Ca 2 + concentration;
• spermine and spermidine are said to have a relaxant effect on smooth muscles of the gut, uterus, respiratory tract and vasculature.
• L-type channels might be implicated.
• calcium channels were involved (many other factors or processes could influence the concentration of calcium in the cytosol).
• some of the results reported in this article are inconsistent with blockage of calcium channels, and (based on the disclosure of this article) cannot be attributed to activation of calcium channels because of the many other factors that may be at work, including actions on potassium channels or directly on smooth muscle (papaverine-like effects).
• the article discloses no determination on the specificity of these agents for sarcoplasmic reticulum (SR) calcium channels (compared to other SR ionic transport components and/or receptors) although it states that the small concentrations at which these agents are active would "suggest potential utility as probes of sarcoplasmic reticulum calcium channels".
• SR sarcoplasmic reticulum
• the authors admit that ruthenium red is not specific and neither is spermine.
• the experiments reported in this article measured only blockage of calcium release, they did not positively establish calcium channel involvement.
• Ar is an aromatic group
• R, R' are hydrogen or methyl
• m,n are various integer combinations within the range 5-14 have M-2 muscarinic receptor blocking activity in guinea-pig heart atria and intestinal ileum.
• R, R' are hydrogen or methyl
• m,n are various integer combinations within the range 5-14 have M-2 muscarinic receptor blocking activity in guinea-pig heart atria and intestinal ileum.
• Several of these compounds are said to selectively block the atrial muscarinic receptor with considerably higher affinity than the ileal receptor, and thus could possibly serve to distinguish between the two receptors.
• Nothing is disclosed about calcium channels, but a general synthetic scheme for the non-aromatic moieties of the disclosed polyamines is provided: J. Med. Chem.. 1989, 32:79-84; J. Med. Chem., 1985 , 28 : 1643-1647 ; and Can . J. Physiol . Pharmacol . , 1980 , 58:
• the present invention has the following objects:
• ionic channels including calcium, sodium, and potassium channels
• ionic channels including calcium, sodium, and potassium channels
• agents that have calcium, sodium, and/or potassium channel modulating functions other than activation or blockage.
• agents which act on the G-protein in cell membranes alter the activity of ionic channels indirectly.
• Figure 1A is a tracing of spontaneous firing activity observed in the guinea-pig Purkinje cell pursuant to applica tion of a direct square current pulse (0.3 nA, 62 msec) in the absence of any blocking agents (control).
• Figure 1B is a tracing of the response of the Purkinje cell to a 0.45 nA direct square current pulse after addition of spermidine: the calcium conductance is blocked.
• Figure 1C upper trace is a tracing of the response of a Purkinje cell to a 0.45 nA direct square current pulse after (i) the P-channel has been blocked with spermidine, (ii) the potassium conductance has been blocked with TEA, (iii) the sodium conductance has been blocked with TTX. The remaining spike is due to a calcium channel of the dihydropyridinesensitive type. This is demonstrated in Fig. 1C lower trace where the L-channel also is blocked by dihydropyridine or Fig. ID (lower trace) where the L-channel is blocked by streptomycin. (The upper trace in ID is of the same type as in lC.)
• Figure 2 is a graph of the presynaptic calcium current in the squid synapse observed after addition of various amounts of A. aperta venom in a voltage-clamp experiment demonstrating dose-dependence of this effect.
• FIG. 3 is a graph showing the fraction of the excitatory postsynaptic potential (EPSP) remaining (with the control taken as 1) after addition of various amounts of partially purified P-channel blocking factor from A. aperta venom in squid synapse. The results demonstrate the dose-dependence of the effect by the factor on the EPSP.
• EBP excitatory postsynaptic potential
• Figure 4 shows the postsynaptic action potential in squid synapse pursuant to direct stimulation of the presynaptic terminal. Tracing A was recorded prior to the addition of compound E in the bath (0.15 ml of the preparation of Example 2); tracing B was recorded 4 minutes after compound E addition; and tracing C was recorded 6 minutes after addition of compound E.
• FIG. 5 upper trace is the postsynaptic potential (EPSP) response in squid synapse in a voltage clamp experiment before (A) and after (B) application of spermidine in the bath (which already contained TTX and 3-aminopyridine).
• the middle trace is the presynaptic calcium current before (A) and after (B) application of spermidine in the bath.
• the lower trace is the applied voltage step (28 mV, 5.5 msec).
• Figure 6 is the trace of the postsynaptic potential again in squid synapse after application of a 38.2 mv/10 msec voltage step in a voltage clamp experiment in the presence of
• TTX and 3-aminopyridine Recordings were made 2, 6, 10 and 13 minutes after the addition of spermidine.
• Figure 7A and B are traces of the presynaptic and postsynaptic potential in squid synapse in the absence (upper trace) or presence (lower trace) of calcium blocker.
• Figure 8 is a superimposition of traces of the inward calcium channel (traces 1-1 through 1-4) of the presynaptic cell to which a 35 mv/ 1 msec voltage is applied (tracing V) and the corresponding EPSP's (tracings P-1 through P-4 ) in the presence of TTX and TEA and a calcium channel activating agent, a compound produced by the reaction of lysine ethyl ester and spermidine.
• One aspect of this invention is directed to a method for regulating cation transport across cellular membranes possessing cation channels comprising exposing a cell membrane possessing an ion channel of a specific type, to a nonaromatic polyamine compound specifically effecting the channel and having the formula.
• the present invention is directed to the compounds of the foregoing formula themselves.
• aspects of this invention are directed to a methods for blocking (or activating as the case may be) calcium channels and to methods for blocking (or enhancing) synaptic transmitter release using one or more of the compounds referred to above, and/or a polyamine P-channel blocking agent isolated from the venom of the funnel-web spiders.
• Another aspect of this invention involves methods for regulating ionic channels, either voltage or ligand-gated such as the glutamate-activated channel, by selecting a compound according to the present invention that is a specific calcium- sodium- or potassium blocker, activator or modulator, and exposing a cell to the presence of this compound to bring about the desired specific regulating result on the target channel.
• a compound according to the present invention that is a specific calcium- sodium- or potassium blocker, activator or modulator
• ionic channel regulating agents shall mean compounds and compositions which regulate cation flow by acting directly on a channel or by acting indirectly on it (e.g., by acting on another substance or cellular structure which in turn influences the function of an ionic channel).
• this calcium channel blocking agent more specifically a preparation of it isolated from A. aperta spider venom by deproteination (boiling) followed by removal of the precipitate by centrifugation and subjection of the supernatant to FPLC (high-pressure liquid chromatography - Pharmacia) under conditions of 0-1.0 M NaCl, pH 7.5 and a flow-rate of 1 ml/min using a cation-exchange column, e.g., Mono-S (5 ⁇ 50 mm) from Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) with the active fraction (resulting from 0.5 ml of venom) eluting at about 0.8 M NaCl, gave the following results:
• A. aperta P-channel blocker (hereafter sometimes referred to as "FTX") comprises a nonaromatic polyamine structure.
• straight or branched chain nonaromatic polyamines have the ability to block or to enhance P-type calcium channels (i.e. channels that display one or more of the characteristics associated with the P-channels first identified in Purkinje cells and described above).
• polyamine will refer to a compound that has at least three -NH 2 and/or -NH- groups.
• nonaromatic polyamines having an asymmetric methylene and/or nitrogen atom distribution, and most preferred are nonaromatic polyamines having a moiety bearing two or a plurality of amine groups on a single carbon atom or on neighboring carbon atoms.
• asymmetric methylene and/or nitrogen atom distribution is meant that the two moieties of the compound are not the same: for example, spermine (which is not within the scope of this invention) has a symmetric distribution in that the nitrogen atoms are separated by four methylene groups on each side.
• Compound P below, on the other hand has an asymmetric nitrogen atom distribution in that there is a higher number of N atoms (per number of carbon atoms) in the left moiety; compound H has an asymmetric methylene group distribution.
• polyamines useful as ionic channel regulating agents are the following compounds:
• Examples of compounds having ionic channel blocking and/or activating activity include derivatives or analogues of compounds A-W having, e.g., a dehydroxylated or decarboxylated lysine moiety.
• compounds having ionic channel blocking and/or activating activity include derivatives or analogues of compounds A-W having, e.g., a dehydroxylated or decarboxylated lysine moiety.
• many of the above compounds and their analogues are active on sodium or potassium channels.
• polyamines useful as such channel regulating agents are the following compounds:
• ion-channel blocking, modulating or activating polyamines useful in the present invention may be deemed to be encompassed by the formula
• the polyamines of the present invention have a molecular weight below 800, and most preferably between 200 and 400.
• the compounds in which R is linked to the polyamine chain via a alpha-aminomethylene group (with or without an intervening -CO-, or -O- or -CH 2 - group) and which have both x and y be positive integers within the above definition have calcium channel activity.
• this activity will be blocking activity if the left-hand moiety is arginine-based and calcium-activating activity if the left-hand moiety is lysine-based.
• Compound B is a calcium (P-channel) blocker;
• Compound BB on the other hand is a calcium (P-channel) activator.
• x,y are e.g., 3,4; 3,3; 4,3; or 4,4 do not block calcium in P-channels but instead are weak sodium-channel blockers.
• polyamine compounds used in the present invention can be synthesized using well-known and commercially available starting materials and synthetic schemes well-known in the art. Alternatively, polyamine compounds within the scope of the present invention may be obtained from commercial sources.
• decarboxylated arginine (agmatine), or arginine ethyl ester, decarboxylated lysine or lysine methyl or ethyl ester
• decarboxylated arginine (agmatine), or arginine ethyl ester
• decarboxylated lysine or lysine methyl or ethyl ester can be purchased from Sigma Chemical Co., St. Louis, MO.
• Other well known polyamines such as spermine, spermidine, 1,6 diaminohexane, putrescine, cadaverine can also be obtained from Sigma or other commercial sources.
• Those which are not themselves active (e.g., spermine) can be used to synthesize active compounds as follows:
• One protocol involves using an amine ester in aqueous solution and reacting it under basic conditions with an appropriate (usually equimolar) amount of a diamine or other polyamine also in aqueous solution (or in liquid form).
• One preferred synthetic scheme for compounds within the scope of this invention that contain a decarboxylated arginine moiety involves reducing the appropriate protected amine ester, e.g., Z-NH-(CH 2 ) 4 -CH(NH-Z)-CO-OEt, (wherein Z is an appropriate protective group such as Ph-CH 2 -O-CO- wherein Ph is phenyl) in the presence of THF (at -78 degrees C) and di-isobutylaluminum hydride to the corresponding aldehyde, aminating this product in the presence of sodium cyanoborohydride to produce the desired polyamine which can then be
• the protected amino acid (e.g. the free acid corresponding to the ethyl ester referred to above) can be first converted to the corresponding mixed anhydride by reacting with, e.g.,Et-O-CO-Cl (ethoxycarbonyl chloride) and TEA (triethylamine) and THF (tetrahydrofuran) at -20 degrees C.
• the anhydride can then be reacted with an alcohol, e.g., methanol at -10 degrees C in the presence of sodium borohydride to convert the mixed anhydride to the corresponding alcohol.
• the alcohol can then be converted to the corresponding halide according to well-known halogenation techniques.
• the halide can be condensed with the appropriate amine (in the presence of an acid scavenger such as K 2 CO 3 or dimethyl formamide) to yield the target protected compound which can be deprotected by e.g., hydrogenolysis (H 2 palladium/carbon 10%) or where the protecting group is Boc, with trifluoroacetic acid to yield the actual target compound.
• an acid scavenger such as K 2 CO 3 or dimethyl formamide
• R is linked to the NH group of the -N-C-..N chain via an -O- linkage
• the appropriate amino acid e.g., arginine
• the appropriate straight-chain polyamine e.g., spermine
• H 2 N-(CH 2 ) 4 -NH-(CH 2 ) 4 -NH 2 or H 2 N- (CH 2 ) 3 -NH-(CH 2 )3-NH 2
• an acid e.g., acetic acid
• a peroxide such as hydrogen peroxide
• the compounds of the present invention can be used in widely ranging units from nanomolar to no particular upper limit. It will be appreciated that the amount that needs to be used in each instance will depend on the activity of a particular compound (whether the compound is a potent blocker, activator, or modulator), on the sensitivity of the specific channel on which this compound is active (whether the channel properties are easily modifiable), on the specificity of the activity of the particular compound (whether the compound acts exclusively on one type of channel) and on other factors which are well-recognized in the art to be subject to optimization. Such optimization can be easily achieved without undue experimentation by well-known methods.
• the compounds of the present invention including pharmaceutically acceptable salts thereof are used for pharmaceutical purposes (for example to bring about the behavioral modifications or to treat the behavioral alternatives referred to in the Background section, above), they may be incorporated in pharmaceutical compositions in oral, enteral, topical, depot, or parenteral dosage forms containing one or more of the present compounds, in association with one or more pharmaceutically acceptable excipients, fillers, salts, coatings, carriers or diluents such as are customarily used with pharmaceutical preparations, e.g., tablets, sustainedrelease preparations, gelatin capsules, injectable solutions, suppositories, or coupled to a drug delivery system, etc.
• pharmaceutical preparations e.g., tablets, sustainedrelease preparations, gelatin capsules, injectable solutions, suppositories, or coupled to a drug delivery system, etc.
• the active ingredient dosage ranges will encompass the following preferred ranges: 10-100 mg/kg of a particular compound or compounds in rodents, and 0.05-50 mg/kg in man. It will of course be preferred to use the minimum amount which will accomplish the desired effect in order to avoid as many side effects as possible.
• the unit content of each dosage form need not by itself constitute an effective amount of active ingredient since a plurality of dosage forms may be administered to achieve an effective amount in combination.
• the amount used and the duration of the treatment will also be subject to optimization and will vary depending on the severity and responsiveness of the condition to be treated, the age, weight, and physical condition of the patient, and often also on the administration route.
• EXAMPLE 1 ASSAYS FOR P-CHANNEL BLOCKING ACTIVITY
• the tissue was then immediately immersed in aerated Krebs-Ringer solution containing 124mM NaCl; 55mM KCl; 1.2mM KH 2 PO 4 ; 2.4mM CaCl 2 ; 1.3mM MgS04; NaHCO 3 (26mM); and 10mM glucose.
• This solution was kept refrigerated at 6oC.
• the cerebellar mass was then transacted sagitally and a single cell slice about 2mm thick was isolated from the vermis or from one of the hemispheres.
• the slice was affixed with cyanoacrylate to the bottom of a plexiglass cutting chamber and agar blocks were used to surround the slice, thus providing side support.
• the tissue was immersed in Krebs-Ringer solution at 6oC and further sectioned with an Oxford G501 Vibratome (Ted Pella, Inc., Tustin, CA) to yield about six 200-(or 300-)micron thick cerebellar slices, containing sagittal sections of all the cerebellar folia in a given rostrocaudal plane as well as central white matter and cerebellar nuclear cells.
• the slices were incubated in oxygenated (95% O 2 ; 5% CO 2 ) Krebs-Ringer solution at 37oC for about one hour.
• a slice was transferred to a recording dish such as that described in Llinas, R. et al, J. Physiol., 305:171, 1980.
• the cerebellar slice was placed in a Sylgard plate (Corning Glass, Corning, New York) at the bottom of the recording chamber and secured with a bipolar stimulating electrode pressing lightly on the white matter.
• the experiments were conducted at a chamber temperature of 37°C maintained by a surrounding temperature-controlled water bath.
• the saline (Ringer's) solution used for continuous perfusion was also kept at 37oC.
• TTX 10 -6 M was used to block sodium conductance
• TAA triethylammonium chloride
• nitrendipme (10 -5 -10 -6 M) was used to block the L-channel.
• Purkinje cells were impaled with recording micropipettes under direct vision using Hoffman modulation microscopy (Hoffman, R., J. Microsc. 110:205-222. 1977). Intracellular recordings were obtained with micropipettes filled with 3M potassium acetate or 1M tetraethylammonium chloride (TEA), and having an average D.C. resistance of 60-80 megohms. Synaptic activation of the cells was effected with a bipolar stimulation electrode located on the white matter at the basis of the folium studied. Direct stimulation of the Purkinje cells was implemented with a high-input impedance (10 12 ohms) bridge amplifier.
• TAA tetraethylammonium chloride
• the neurons responded with firing having both sodium-dependent and calcium-dependent spikes in accordance with the normal electrical response of the Purkinje cells in the absence of a P-channel blocker (Fig. 1A).
• the cell responses were also measured at several time intervals after introduction of a polyamine compound in the recording chamber medium.
• a small depolarizing current (approximately 0.3 or 0.45 nA for 62 msec) generates a burst of potential spikes and a plateau potential, the latter due to the non-inactivating ("persistent") sodium conductance; calcium spikes are substantially reduced or extinguished and there is no calcium-dependent component to the plateau potential (Fig. 1B).
• Figure 1B was recorded after the addition of spermidine to a final concentration of 0.8mM.
• Action potentials were induced by direct electrical stimulation of the presynaptic nerve bundle of the giant synapse in squid stellate ganglia.
• the thus evoked postsynaptic action potential is shown in Figure 4 in the absence of any calcium-blocking agents in the bath (trace A).
• the postsynaptic action potential is markedly reduced (trace B) four minutes after such addition and completely extinguished at 6 minutes.
• the decrease and eventual extinction of the postsynaptic action potential is attributed to blockage by the P-channel blocking factor (here compound E) of the presynaptic calcium channels. That calcium channels are involved was demonstrated in another type of voltage clamp experiment (Fig. 6) in which the postsynaptic potential in squid synapse (in a preparation containing tetrodotoxin and 3-aminopyridine) is reduced over time after the addition of 0.4 ml of a 1mM solution of the P-channel blocker Compound B in the 3 cc bath.
• FIG. 5 shows yet another voltage clamp experiment in which a 28mV/5.5 msec voltage step is applied to the presynaptic terminal (trace C) in a squid synapse preparation, wherein sodium conductance was blocked with TTX and potassium conductance was blocked with 3-aminopyridine.
• the applied step voltage generates a compensating inward ionic current in the presynaptic cell (trace B) which is due solely to calcium since the other conductances are blocked.
• a postsynaptic potential (EPSP) is evoked, which is the normal response (upper curve, trace A) in the absence of a P-channel blocker (here Compound B).
• EBP postsynaptic potential
• the presynaptic ionic current is also reduced (trace B, upper curve, i.e. curve with the lesser amplitude).
• the P-channel blocker acts on the presynaptic calcium channels since the time relationship in Fig. 5 of the presynaptic calcium entry to the postsynaptic response remains constant and only the amplitude of the postsynaptic response is reduced in direct relationship to the reduction of the presynaptic calcium current.
• the P-channel blocker blocks presynaptic calcium channels which impedes calcium influx and consequently the expected transmitter release does not occur.
• the reduced transmitter release in turn causes a reduced postsynaptic response.
• the corresponding postsynaptic potential is depicted in Figure 7B (upper trace). Seven minutes after addition of P-channel blocker (here Compound B) the presynaptic calcium spike is reduced (7A, lower trace) and so is the EPSP (7B, lower trace). The presynaptic calcium spike after blockage varies in amplitude only. The evoked postsynaptic potential also varies in amplitude. Moreover, the time course of the EPSP after blockage is the same as in the absence of blocker.
• P-channel blocker here Compound B
• polyamine P-channel blocking agents act presynaptically and do not have a postsynaptic effect: if a postsynaptic blockade was present, the time course of the EPSP would also be different (in addition to its amplitude being lower).
• Figures 2 and 3 demonstrate that the effect of the P-channel blocking agent on the presynaptic calcium current and postsynaptic potential is dose-dependent: both decrease in amplitude with an increasing amount of P-channel blocker used in the extracellular medium.
• venom was used and in Fig. 3 partially purified A. aperta P-channel blocker was used).
• the partially purified preparation was processed by deproteination from venom (boiling), centrifugation, removal of the pellet, addition to original volume of water followed by twice butanol extraction in 20 volumes of butanol. The aqueous phase was used.
• Figure 8 is a superimposition of tracings from a voltage clamp experiment in squid (similar to that illustrated in Fig. 5, but with the background removed) in which a 35 mv/l msec voltage step (trace V) is applied to the presynaptic terminal in a squid synapse preparation of the type described above, wherein sodium conductance was blocked with TTX and potassium conductance was blocked with TEA.
• TTX 35 mv/l msec voltage step
• EXAMPLE 2 POLYAMINES USED FOR BLOCKING OR ACTIVATION
• Compound B were used in experiments with the assay system of Example 1.
• Compound A was synthesized in the same manner except that an equimolar amount of NH 2 -(CH 2 ) 6 -NH 2 was first dissolved in a minimum volume of water before adding to the arginine ester in 1N NaOH.
• Vancomycin (Eli Lilly Industries, Inc., Carolina,
• Amounts of up to 1.0 mM of the morpholine derivative and even larger amounts of PTX433 were used for electrophysiological evaluation of their P-channel blocking activity.
• the aqueous phase can be further purified by FPLC (high performance liquid chromatography-Pharmacia) under a 0-1.0 NaCl gradient, pH 7.5 and a flow rate of 1ml/min using a cation-exchange column such as Mono-S (5x50 mm) from Pharmacia.
• FPLC high performance liquid chromatography-Pharmacia
• a cation-exchange column such as Mono-S (5x50 mm) from Pharmacia.
• aqueous phase resulting from the above-described final ethyl acetate extraction can be used as is.
• the foregoing technique can be used to synthesize other lysine-based polyamines within the Formula I.
• Another synthetic technique that can be used to yield only compound (BB)' is to first react the lysine with 1,3 diamino propane using, e.g., the above carbodiimide method, and then reacting the product (preferably) after purification with 1,4 diamine butane using one of the methods described above for the compounds of Formula I.
• the order of use of the diamines can be reversed for synthesizing the (BB) product.
• the calcium blocking factor isolated from A.aperta venom or compound B or E are lethal to rats when administered at a dose of 50 to 100 microliters. Similar amounts of purified compound BB do not kill these animals. On observation, rats are placid but display no visible movement disorders, and do not extend their limbs when picked up. Compound BB (and/or BB') appeared to have a calming effect similar to a tranquilizing effect but without much muscle relaxation.
• Arginine was labelled by mixing with an equimolar amount of fluorescein in sodium borate buffer, pH 9.5 and allowing the mixture to react overnight in the dark under stirring. Excess fluorescein was extracted by washing three times with 10 volumes ethyl acetate per wash. The thus labelled arginine can be used to synthesize compounds of Formula I. Lysine ethyl ester can be similarly labelled. Thus, labelled Formula I compounds (both blockers and activators) can be used in fluorescence experiments, using methods well-known in the art.
• Example 1 in the absence and presence of various amounts of the compounds listed in Example 2.
• the results for the P-channel were as follows:
• Compounds A, B and E were potent P-channel blockers producing complete P-channel blockage at concentrations lower than 0.5 mM. None blocked the dihydropyridine-sensitive L-channel at the concentrations tested (well below 1 mM). The other channels (sodium, potassium) were not affected.
• the minimum effective concentrations of the P-channel blocking agents of this invention are micromolar or less; this can be determined by routine experimentation using serial dilutions of these agents. Preferred are concentrations up to 1mM but it should be appreciated that the choice of concentration of each agent is subject to optimization as is well-known in the art and also depends on whether partial or total P-channel blockage is desired.
• nonaromatic blocking polyamines of the present invention block calcium channels of the P-type specifically (i.e., without affecting sodium, potassium or L-type calcium channels).
• these agents block presynaptic calcium channels and thereby control transmitter release from the presynaptic to the postsynaptic neuron.
• Analogous specificity is expected of the activating agents of the present invention.
• the P-channel activating compounds of the present invention differ from the arginine-based blockers only in that arginine has been replaced by lysine. It can therefore be seen that polyamines can have different and in fact opposing effects on P-type channels depending on the structure of the group R. In fact, it is anticipated that some calcium channel activators (or blockers) would be specific to P-channels from cells of a particular animal species and could therefore serve as markers of additional distinguishing characteristics of subtypes of P- channels.
• EXAMPLE 6 EXAMPLE 6 :
• the compound BB was administered to a rat in amounts ranging between, 50 and 100 mg/kg.
• the rat was subsequently injected with 40mg/kg (actual volume: 0.2 ml) of sodium phenobarbital (NEMBUTAL), a dose which normally produces total anesthesia, and in many cases death.
• the BB-injected rat did not become unconscious upon injection. It took two additional doses of nembutal to anesthetize this rat. The experiment was repeated except this time two rats were injected with compound BB, one with the same dose as was admininstered in the previous experiment and one with twice that dose. Immediately after injection, the first rat appeared normal and the second exhibited reduced activity on observation.
• the first rat After each was injected with 40mg/kg of nembutal, the first rat showed no effect but the second appeared to wake up. Subsequent 40mg/kg injections of Nebutal caused the first rat to become anesthetized at a total dose of 160mg/kg but the second rat received a total of 200 mg/kg before he was anesthetized, and received 240 mg/kg before he was killed.
• the normal LD-50 for Nembutal in rats is about 60 mg/kg. Therefore this dose is enormous by comparison.
• compound BB which is a calcium-channel activator causes resistance to barbiturate action. Furthermore, it causes animals to be serene without being tranquilized, in that it does not cause much muscle relaxation and has potential utility as a prototype drug for anxiolysis.

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• 1990
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