WO1994016327A1 - Methods and compositions for blocking amyloid protein ion channels - Google Patents

Abstract

Therapeutic agents, modalities and methods for treating amyloid protein related pathologies involving amyloid protein ion channels. The invention further relates to methods for determining the effectiveness of such therapeutic agents or modalities.

Description

Methods and compositions for blocking amyloid protein ion channels

FIELD OF THE INVENTION This invention relates to the field of therapeutics and, more particularly to therapeutic agents, modalities and methods for treating amyloid protein related pathologies involving amyloid protein ion channels. The invention further relates to methods for determining the effectiveness of such therapeutic agents or modalities.

BACKGROUND OF THE INVENTION

Several amyloid diseases have been characterized that involve or may involve amyloid protein ("AP") accumulation in the tissues, termed "amyloidosis". The cause of amyloidosis is unknown, but has been characterized by different biochemical types having different etiologic mechanisms. One form of AP, AL, has an N terminal sequence that is homologous to a portion of the variable region of an i munoglobulin light chain, and is found usually in normal tissue. The presence of such an AL form is designated primary or idiopathic amyloidosis, but Al is also found to be associated with a few pathologies, such as multiple myeloma.

A second form of AP, AA, does not have an N terminal homology to immunoglobulin light chain and is associated with the secondary or reactive amyloidosis, as clinical diseases such as infectious diseases (e.g., tuberculosis, bronchiectasis, osteomyelitis, leprosy) or inflammatory diseases (e.g., rheumatoid arthritis, granulomatous ileitis) . See, e.g., Berkow et al., eds., The Merck Manual , 16th edition, Merck and Co., Rahway, N.J., 1992.

Other forms of amyloid proteins, including beta forms, are also found in pathologies, such as in Alzheimer's disease. Alzheimer's disease (AD) a chronic dementia is produced and is associated with production and accumulation of amyloid β proteins (AjSP) . AD affects increasingly large numbers of the aging population. Pathologically, the brain is locally characterized by extracellular amyloid plaques, intraneuronal neurofibrillary tangles, and by vascular and neuronal damage (Blessed, et al, 1968; Neve, et al, 1990; Katzman and Saito, 1991; Selkoe, 1991; McKee, et al, 1991; Hardy and Higgins, 1992; Kosik, 1992) . The major component of brain amyloid is a 42 residue peptide AβP (Roth, et al, 1966; Terry, et al, 1981; Glenner and Wong, 1984; Masters, et al, 1985; Joachim, et al, 1988), which is a proteolytic product of amyloid precursor protein (APP) . APP is a widely distributed membrane glycoprotein, defined by a locus on chromosome 21 (Goldgaber, et al, 1987; Tanzi, et al, 1987), in which mutations have been demonstrated in several cases of familial AD (Goate, et al., 1991; Jones and St. Clair, quoted in Kosik, 1992) .

However, in spite of the circumstantial evidence linking AjδP with AD, the mechanism by which A0P might cause cell damage remains poorly understood. It has been widely presumed that the APP, or the A3P peptide, or a fragment of APP containing AJP, could be neurotoxic (Hardy and Higgins, 1992) . However, direct demonstration of this presumed toxicity has proved elusive. A C-terminal fragment of APP containing the AjSP domain has been reported to be toxic to neurons in culture (Neve, et al, 1990; Yankner, et al, 1989) . Alternatively, it has been claimed that AβP is not itself toxic, but potentiates neuronal sensitivity to excitotoxins (Koh, et al, 1990; Mattson, et al, 1992; Pike, et al. 1991). Finally, it has been claimed that toxicity may be mediated by interaction between AjSP and neuronal serpin receptors (Joslin, et al,1991), although such interactions do not appear to have intrinsic toxic consequences (Kosik, 1992) .

AβP could well be toxic in some way, since in addition to the senile plaques, AD is also characterized by dystrophic neurites, both in the vicinity of the plaques, as well as elsewhere (Kosik, 1992) . The dystrophic neurites are said to be characterized by degenerative features, including accumulation of filaments as well as ubiquitin i munoreactivity (Mori, et al, 1987; Perry, et al, 1987) . Due to the lack of understanding of the causes of brain cell destruction in Alzheimer's disease, or the relationship to deposition of AβP, there is a long felt need to provide a suitable model and accompanying mechanism of the role of AβP for evaluating therapeutic agents for treating AD and related diseases involving amyloid proteins.

Tromethamine Clinical Aspects. Tromethamine (tris (hydroxymethyl)aminomethane; "TRIS" or "THAM") is a buffering compound and has been in clinical use as a treatment for respiratory and metabolic acidosis since the early 1960's (Manfredi, et al, 1960; Nahas, 1963; Goodman and Gilman,

1975) . According to Nahas (1962) , "thus far this compound is the only amine that can be used in doses sufficient to have a buffering effect on the organism without any marked toxicity. The low toxicity of THAM is one of its striking characteristics..."

Initial studies on humans were performed by Berman, et al (1960) , who administered 3-5 mmol/kg to 10 healthy volunteers for 30-60 minutes, without ill effects. This study was repeated by Brown, et al (1959), on three healthy males. The latter investigators found that toxic manifestations could be detected at 8.8 mmol/kg (about lgm/kg) for lhr, and that tromethamine was excreted in urine over several days. This was interpreted to indicate that the drug accumulated in the body. Consistently, Robin, et al (1961) observed that administration of 150 mmol tromethamine to a dog caused the DMO-defined intracellular pH to rise from 7.08 to 7.27.

Tromethamine was also given to children in the early phase of studies on this compound. Physiological studies had indicated that tromethamine alkalinized the urine, possibly by action on the H⁺<=>Na⁺ exchanger in the kidney. Taking advantage of the fact that an alkaline urine enhances excretion of weak acids, two clinical investigators successfully administered tromethamine (500 mg/kg in 1 hour) to children suffering from salicylate intoxication (Clark, 1960; Israels, 1961) . Prompt elevation of urinary salicylate was observed.

Swim (1961) subsequently showed that 10-20 mM tromethamine was suitable for inclusion in the culture medium of Hela cells, mouse L cells, human foreskin fibroblasts, and others, and that the additive could be present for "many months." However, above 30 mM, tromethamine was deleterious to the cells.

According to Goodman and Gilman (1976) , Tromethamine N.F. (THAM) is available as 0.3M solution, adjusted to pH 8.6 with acetic acid. A powder form, THAM-E, is available which is to be dissolved in 1 liter sterile water. Each liter of dissolved THAM-E contains 300 mmole (36 grams) tromethamine, 30 mmoles NaCl, 5 mmoles KC1. The recommended route of administration to modify human electrolyte balance is i.v., because at the pH of the small intestine, only 30% of ingested tromethamine is in the uncharged form, suitable for diffusion across the intestinal wall into the body. Furthermore, the unabsorbed ionic tromethamine causes typical osmotic catharsis (Nahas, 1962) . Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents is considered material to the patentability of the claims of the present application. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents. SUMMARY OF THE INVENTION

The present invention is intended to overcome one or more of the deficiencies of the related art.

The present invention also provides therapeutic agents, modalities and methods for the treatment of animals suffering from amyloid related animal pathologies including mammal, fish or bird, preferably human, including but not limited to neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease), nerphrodegenerative diseases, neurological trauma, amyloid cancers, and other amyloid related pathologies.

The invention can also provide pharmaceutical compositions containing AP ion channel blocking agents or modalities, as presented herein and as would be apparent to one skilled in the art, based on the teaching and guidance presented herein, in amounts sufficient for use in one or more of the embodiments of this invention.

The present invention also provides blocking agents, modalities and methods for blocking amyloid protein ion channels ("AP ion channels") in biological membranes or lipid bilayers, by administration of a blocking effective amount of an AP ion channel blocking agent or modality.

The present invention further provides methods for determining the effectiveness of such therapeutic agents or modalities. Such "blocking" agent or modality can be used in vitro, in si tu, and/or in vivo, based on quantitative and/or qualitative channel blocking activity, to inhibit or block the ion conduction through AP ion channels in model lipid bilayers or cell membranes. The present invention also provides methods of using

AP ion channels in lipid bilayers or biological membranes, in vi tro or in si tu, at sufficient concentrations for determining effects of agents or modalities for blocking amyloid ion channels, such as by measuring conductance or cell viability compared to suitable controls. While the blocking of AP ion channels has been discovered to be useful for therapeutic treatment of AP related pathologies, the present invention is also contemplated to include other types of modulation of AP ion channels, such as enhancement, stimulation, modification of structure or specificity, or other change in channel transport by chemical or protein AP modulators.

Other objects of the invention will be apparent to skilled practitioners from the following detailed description and examples of the invention provided below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical representation showing ion channel activity of AβP in a planar lipid bilayer. Transmembrane potential is given in mV above the corresponding current record. Symmetrical CsCl (75 mM, l mM C1C1₂, 2 CsHEPES, pH 7) solutions were used. To determine the conductance of the AβP channel, a linear I-V curve with a slope of 206 pS was drawn by eye to intercept the potential axis at zero mV (not shown) „ At the bottom of the figure, the segment a-b, from the second record, is shown on an expanded time base, where the mark representing 10 s on the upper traces now represents 2.2 s. Vertical arrows indicate 10 mV step changes in membrane potential. The dotted lines marked by the letter C represent the closed state. Inset: diagram of the bilayer chamber (top view) .

Figure 2A-B is a graphical representation demonstrating that the AβP Channel is ion-specific. 2A. The upper record of AjSP channel activity was made in a symmetric system of 40 mM KC1 (1 mM CaCl₂, 2 mM Na-HEPES, pH 7) . The transmembrane potential (in mV) is indicated next to the corresponding current record. The lower record was made after the increase in (KCl)^, from 40 to 60 mM. 2B. The amplitude of the A/?P channel current (in pA) is plotted as a function of the transmembrane potential (in mV) . Each point of the I-V curve represents the mean value of at least three readings of the current amplitude at the potential indicated. Channel conductance is ca. 325 pS (β) in symmetrical (40 mM KC1) and 346 pS (o) in asymmetrical (40 vs 60 mM KC1) solutions, respectively. Intercepts are at 0 (•) and 8.5 mV (o) , respectively.

Figure 3A-C presents graphical representations of calcium ion permiability of APs. 3A. Segments of a continuous record are shown at different potentials. The solution in the cis chamber contains (in mM) : 37.5 CsCl, 1

CaCl₂, 1 CsHEPES, pH 7) .The trans chamber contains (in mM) : 25 CaCl₂, 2 mM Na-HEPES, pH 7) . 3B. Records labelled 1-3 (upper and lower panels) are continued from those in 3A. Ca²⁺ is the charge carrier at negative potentials and Cs⁺ at positive potentials. The numbers to the left of each record indicate the order in which the same voltage step was applied (either from 0 to -40 or to -60 mV) «. 3C. The amplitude of the current (in pA) is plotted as a function of the transmembrane potential (in mV) . Each point on the I-V curve represents either the mean value of 2-3 readings of the amplitude of the A/JP channel current at the positive potentials (Cs⁺ current) or single readings at negative potentials (Ca²⁺ current) .

Figure 4A-B presents graphical representations of lithium ion permiability of APs. 4A. Sample records of the AβP channel activity are shown at different potentials. Composition of the solution in the cis chamber is 37.5 mM CsCl, 1 mM CaCl₂, 2 Na-HEPES, pH 7, while that in the trans chamber is 37.5 mM LiCl, 1 mM aCl₂, and 2 Na-HEPES, pH 7. The vertical arrows represent a step change in membrane potential. 4B. Each point on the I-V curve represents the mean value of 2-3 readings of the current amplitude at the indicated potential. The slope conductance at positive potentials is for Cs⁺ current, while that for the negative potentials is for Li⁺ current. FIGURE 5A-B presents graphical representations of sodium and potassium ion permiability of APs. Initially, channel activity was recorded with the asymmetrical system of a KCl solution in the trans compartment (in mM: 40 KC1,1 CaCl₂ and 2 mM Na-HEPES, pH 7) , and a NaCl solution in the the cis compartment (in mM: 40 NaCl,l CaCl₂ and 2 mM Na-HEPES, pH 7) . Keeping the composition of the solution in the cis compartment constant, the solution in the trans compartment was replaced by a CaCl₂ solution (in mM: 25 CaCl_2>1 mM CaCl₂ and 2 mM Na-HEPES, pH 7) . A few minutes later, channel activity was recorded at different potentials, 5A. Sample records of the AjSP channel activity with either K⁺ (top pair) , Ca²⁺ (middle pair) or Na⁺ (lower pair) as charge carrier. 5B„ The amplitude of the current (in pA) is plotted as a function of membrane potential (in mV) . Each point on the I-V curve represents the mean value of 2-3 readings of the amplitude of the AjSP channel current for the KCl//NaCl system (B, •) , and for the CaCl₂// NaCl system (B, o) . Note the lines joining the experimental points intercept the horizontal axis at ca. 7 mV.

FIGURE 6A-B presents graphical representations of calcium effects on ion permiability of APs. 6A. The AjSP Channel activity was recorded using the symmetrical system of 200 mM CsCl₂ (, 1 mM CaCl₂, and 2 mM Na-HEPES,pH 7). Control current records were gathered at ±60, ±40, and ±20 mV. 6B. The concentration of CaCl₂ in the cis compartment was then increased from 1 to 10 mM, and another series of records collected 10 minutes later. Representative records from ±60 mV are shown. FIGURE 7A-C presents graphical representations of tromethamine and aluminum ion effects on ion permiability of APs. For panels A, B and C, we used the asymmetrical system of 37.5 mM CsCl ( and 1 mM CaCl₂, 2 Na-HEPES, pH 7 ) in the cis chamber, and 25 mM CaCl₂ (and 2 Na-HEPES, pH 7) in the trans chamber. For panel D we used the asymmetrical system of 40 NaCl (and 1 mM CaCl₂, 2 mM Na-HEPES, pH 7.0) in the cis chamber, and 40 KCl (and 1 mM CaCl₂, 2 mM Na-HEPES, pH 7.0) in the trans chamber. 7A. Control AjSP channel activity was measured at 0, ±10, and ±20 mV, prior to the addition of tromethamine. Tromethamine (10 mM, as Tris-HCl, pH 7) was added to the cis side, and remaining channel activity recorded after 1-2 minutes at ±20 and -40 mV. 7B. Sample records of control activity gathered at -40 and -60 mV (upper two records) under the same conditions as for part A. Al₂(SO₄)₃(10 μM) was added to the cis chamber, and channel activity was recorded ca. 3 minutes later. The lower two records are representative of remaining channel activity at -60 and -80 mV. 7C. Control AβP channel activity is shown at -40 mV, under the same conditions as parts A and B. A1₂(S0₄)₃ (20 μM) was added to the cis side, and records were made cji. 3-5 minutes later. No channel activity could be detected at -40 mV, and the records shown for -60 and -100 mV illustrate the potency of aluminum as a blocker of the AjSP channel. 7D. Control AjSP channel activity is shown at 0 mV, in the asymmetrical system of Na//K, described above. Upon the addition of A1₂(S0₄)₃ (1 mM) , channel activity was substantially attenuated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that amyloid proteins may form pathologic ion channels in biological membranes, such that therapeutic agents or modalities and methods are provided for treating amyloid protein related pathologies, as well as methods for determining the therapeutic effectiveness of such therapeutic agents and/or modalities.

When present in sufficient concentration, transmembrane domains of amyloid proteins may associate in the lipid bilayer of cell membranes to form amyloid ion channels with pathological effects. The presence of such amyloid channels can result in cell damage or death due to abnormal influx or outflux of ions in the cell.

APs of the present invention can be used to provide biological membrane models of AP ion channels in phospholipid bilayers, cell membranes, intact cells or cell extracts. The use of alternative isolated, synthesized or recombinantly expressed APs, as described herein, provides the ability to model AP ion channel formation in various forms of amyloid related pathologies, such as AD or tumor cells in multiple myeloma or carcinomas, using membrane models having AP ion channels.

In the context of the present invention, "modulate", "blocking" or "blocking" of AP ion channels refers to inhibition, stimulation or alteration of ion transport through the amyloid ion channels. An amyloid protein blocker acts by associating with an AP ion channel so as to modulate the transport, movement or transmission of ions through the ion channel. Such blockers are preferably specific for AP ion channels and may bind one or more types of amyloid ion channels.

"AP ion channel" refers to the formation of an ion channel by association of at least one transmembrane domain of an amyloid protein, in a cell membrane, a biological membrane or lipid bilayer. Such an ion channel is capable of transmitting at least one type of ion, such as a cation or an anion, across the membrane or bilayer, with or against an ion gradient, as active or passive transport. A "transmembrane domain" of an amyloid protein or polypeptide refers to hydrophobic portions of the protein which span the membrane and are capable of associating with other transmembrane domains on a same or different protein.

AP ion channel blockers may be used according to one aspect of the present invention, as therapeutic and/or diagnostic agents which possess AP ion channel ligand specificity, consistent with clinical utility in the treatment of AP ion channel related pathologies, such as Alzheimer's disease and other disorders associated with amyloidosis or other AP related pathologies. As a non-limiting example, AP ion channel blockers may be used as therapeutic agents for neurodegenerative diseases, which blockers lack, or have substantially reduced, side effects relative to known treatment regimes.

AP related pathologies may include those involving amyloidosis, such as infective diseases (tuberculosis, bronchiectasis, osteomyelitis, leprosy) or inflammatory diseases (rheumatoid arthritis, granulomatous ileitis) ; multiple myeloma; neurodegenerative diseases; neurological trauma, nephrodegenerative diseases; Hodgkin's disease; other tumors; Mediterranean fever; Hereditary amyloidosis may include peripheral sensory and motor neuropathy, autonomic neuropathy, and cardiovascular and renal amyloidosis. Carpal tunnel syndrome and vitreous abnormalities may also occur.

Neurodegenerative diseases include, but are not limited to, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supra-nucleo palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph) ; systemic disorders (Refsum's disease, abetalipoprotemia,ataxia, telangiectasia, and mitochondrial multi.system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis Hallerrorden-Spa z disease; and Dementia pugilistica. Nephrodegenerative diseases include, but are not limited to, AIDS-associated nephropathy, immunologically related mediated renal diseases, glomerular diseases, tubulointerstitial disease, nephrotoxic disorders and hereditary chronic nephropathies. Non-limiting examples of neurological trauma of the central nervous system, including head injury, postconcussion syndrome and spinal cord injury. See, e.g., Berkow et al, eds., The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Katzung, ed. Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992) , which references and references cited therein, are entirely incorporated herein by reference for preparation and administration of pharmaceutical companies.

AP ion channel blocking activity of AP ion channel blocking compounds according to the present invention may be readily determined by of one of ordinary skill in the art without undue experimentation, based on the guidance and teachings presented herein. Appropriate AP blockers may be provided and used in therapeutic, diagnostic and research applications, based on the relationship between the structure and pharmacological activity of the various blockers in each case, based on pharmacological characterization, using known techniques, of a particular AP blocker.

As a non-limiting example, AβP forms AP ion selective channels in lipid bilayers and is expected to cause neuronal and/or vascular damage leading to chronic dementia or other amyloid pathologies. Ion conduction through the AjSP channel is also found to be blocked by Al³⁺ and tromethamine, as non-limiting examples of AP ion channel blockers.

Other compounds expected to have AP ion channel blocking activity include, but are not limited to TEA, tacrine, dihydropyridine drugs, diphenylhydantoin, amiloride, adenosine antagonists, antidepressant drugs, neuroleptics, neuroactive peptides, tachykinins, sedative hypnotics, anesthetics, antipsychotics or derivatives or analogs thereof. See Berstein Clinical Pharmacology Littleton, Mass.:PSG Publishing (1978) ; Usdin et al Clinical Pharmacology in Psychiatry New York: Elsevier North-Holland (1981) ; and Baldessarini, supra, (1985) ; and Katzung, Basic and Clinical Pharmacology, 5th edition, Appleton and Lange, Norwalk, Conn., p295-450 (1992) , which references are herein entirely incorporated by reference.

Non-limiting representative examples of neuroleptics include phenothiazine derivatives (e.g., chlorpromazine) ; thioxanthine derivatives (e.g., thiothixene) ; butyrophenone derivatives (e.g., haloperidol) ; dihydroindolone (e.g., molindone) ; dibenzoxazepine derivatives (e.g., loxapine) ; and "atypical" neuroleptics (e.g., sulpiride, remoxipiride pimozide and clozapine) . Opiates include benzomorphans, such as N-allylnormetazocine (NANM, SKF 10047) , The basis of neuronal and endothelial toxicity in AP related pathologies may thus include the ion channel activity of residual AP-related peptides in target membranes. As a non-limiting example, synthetic amyloid- β-protein (AjSP, 1-40) forms cation selective channels across planar lipid bilayers. The permeability sequence (P_Caas reference) is: P_Ca = (0.6)P_C8 = (0.96)P_Li = P_κ = (1.3)P_Na, while taking P_Na as reference, the sequence is P_Na = (0.46) P_Cs = (0.74)P_Li = (0.77)P_κ. These relationships can be summarized by the following selectivity series for all the cations as, [P_Ce > P_u > P_C(, > P_κ > P_Na] . The AβP channel is therefore permeable to all the monovalent metal ions tested, a property not uncommon in other classical Ca⁺ channels, including the voltage-gated L-type Ca⁺ channel present in the plasma membrane (Tsien, et al. 1987) and the calcium release channel of the endoplasmic reticulum (Suarez-Isla, et al. 1991) .

However, permeation by these monovalent cations through conventional Ca²⁺channels, when studied in Ca²⁺-free solutions, ceases if calcium in the μM range is added (Tsien, et al. 1987). In contrast, in the presence of Ca²⁺ (1 mM) the A P channel is permeable to K⁺, Cs⁺, Na⁺, and Li⁺. However, increasing (Ca²⁺) from l to 10 mM in one compartment leads to a voltage-dependent blockade of AβP channel activity (Figure 6B) . Therefore, at least on a qualitative basis, both types of calcium channels share a common mode of interaction with monovalent cations and calcium, but the AjSP channel is a novel and distinct ion channel.

Based on the available data, and not limiting the present invention to any particular theory or mode of action, the multi-ion channel model (Tsien, et al. 1987) can be used to explain the behavior of the A0P channel. In this model,ion permeation involves interactions with hypothetical sites within the pore of the channel, and ion permeability is chiefly determined by the relative fractional occupancy of these sites by separate or contending ions. The multi-ion channel model predicts, as found here for the AjSP channel, that there will be a ion selectivity sequence based on the putative relative affinities of different cations for the sites of interaction within the channel. This sequence is summarized above. The model further predicts, as shown for the AβP channel in Figures 1, 2 and 6, that the channel conductance will be dependent upon the ion concentration. Furthermore, inasmuch as raising the driving force will increase the concentration of calcium and other ions within the multi-ion channel, there should be a voltage dependence of blockade by calcium. Indeed, this was found (Figures 6 and 7) . Finally, the multi-ion channel model predicts that current amplitude should be less when driving permeant ions from the side containing a blocking ion such as calcium. This was found for the AβP channel, as shown in Figure 5. A similar interpretation can be ventured to explain the slightly asymmetric blockade of AβP channels by either tromethamine or aluminum (Figure 7) .

A consistent and related phenomenon, common to both the classical Ca⁺⁺ channel and the Aj3P Ca⁺⁺ channel, the observation that the high conductance of a monovalent cation, such as Cs⁺, can be blocked by a low concentration of Ca⁺⁺, and that upon raising the calcium concentration, the Ca⁺⁺ ion itself becomes the permeant ion (Tsien, et al. 1987).

AP ion channel blockers of the present invention can be determined by routine experimentation using known method steps for in vitro or in situ membrane models. According to the present invention a method is thus provided wherein a membrane model may be used to evaluate the blocking activity of potential AP ion channel blockers, followed by animal and clinical testing to determine in vivo efficacy. As would be clear to one skilled in the art, once an AP is discovered to associate in membranes to form an ion channel, known method steps can be used to determine appropriate blockers, without undue experimentation. As a non-limiting example, a phospholipid bilayer, as described herein, having AjSP associated as a cation channel, can be conductance tested in the presence of various ions, to determine which compounds modulate A P ion conductance. For example, tromethamine and Al⁺³ are shown to block transport of calcium ions using such a model system with an Aj3P.

Alternatively, cultured cells, such as recombinant cells, expressing an AP in the plasma membrane can also be used to test the effectiveness of a putative ion channel blocker by testing the effect of the blocker on the cultured cells. As a non-limiting example, a cell expressing an AβP protein on its surface could be treated with tromethamine and then viability should be increased relative to the presence of tromethamine at concentrations that are insufficient to block Ca⁺⁺ from transport through AP ion channels.

Accordingly, an AP ion channel, to be used, tested or blocked according to methods of the present invention, is provided as a naturally occurring, synthesized or recombinantly expressed AP ion channel, which may form by association of APs. An Ap ion channel may be formed from the association in a membrane or lipid bilayer of 1 to 10 APs corresponding to known AP amino acid sequences (as presented in St. George-Hyslop, 1987; Kang et al. , 1987; and Goldgaber, 1987) or fragments thereof of 11-66 amino acids corresponding to transmembrane domains of AP proteins.

Such APs have amino acid sequences which substantially correspond to at least one amino acid fragment and/or consensus sequence of a known AP or group of AP transmembrane domains, wherein the AP has homology of at least 80%, such as 100% homology, while maintaining AP ion channel forming activity, wherein an AP. Preferably, an AP substantially corresponds to a transmembrane domain of an AP or group of APs as a consensus sequence of known AP transmembrane domains.

Also preferred are APs wherein the AP amino acid sequence is 11 to 50 amino acids in length, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, amino acids, or any range therein

An amino acid or nucleic acid sequence of an AP of the present invention is said to "substantially correspond" to another amino acid or nucleic acid sequence, respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one AP transmembrane domain, or which may be synergistic when two or more transmembrane domains, consensus sequences or homologs thereof are present.

Additionally or alternatively, such "substantially corresponding" sequences of APs include conservative amino acid or nucleotide substitutions, or degenerate nucleotide codon substitutions wherein individual amino acid or nucleotide substitutions are well known in the art.

Alternatively or additionally, substantially corresponding refers to APs having amino acid sequences having at least 80% homology or identity to an amino acid sequence of SEQ ID NO:l, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology or identity.

Accordingly, APs of the present invention, or nucleic acid encoding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T.E., Proteins : Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al, supra, at §§ A.l.l-A.1.24, and Sambrook et al, supra, at Appendices C and D, incorporated herein by reference.

However, when the exact effect of the substitution, deletion, or insertion is to be confirmed one skilled in the art will appreciate that the effect of the at least one deletion, insertion or substitution will be evaluated by routine screening assays, either immunoassays or bioassays to confirm biological activity, such as AP ion channel transport and blocking by Ap ion channel blockers. See, e.g., Maranges et al. , eds.. for example, a substituted polypeptide typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a chemically derivatized column or immobilized membranes or hollow fibers (to absorb the mutant by binding to at least one epitope) . Ions suitable for transport include any biologically compatible ions, such as, but not limited to ions of sodium, potassium, chlorine, calcium, carbonates, phosphates, hydrogen, rubidium, cesium, magnesium, manganese, barium, cobolt, fluorine, iodine, bromine, nickel, zinc, N0₃, acetate, ammonium, CH₃NH₃ ⁺, TMA⁺, TEA^"1", S0₄, or other ions, e.g., as presented in Hille, 1984.

AP blocking compounds having the structural and chemical features suitable as amyloid ion channel therapeutics and diagnostics provide compounds with selective AP ion channel affinity. Molecular modeling studies of AP ion channel blockers using a program such as MACROMODEL^®, INSIGHT^®, and DISCOVER^® provide such spatial requirements and orientation of the AP ion channel and blockers in AP ion channel blockers according to the present invention. Such AP ion channel blockers of the present invention thus provide selective qualitative and quantitative potency at AP ion channels in vitro, in si tu and in vivo.

Organic synthesis of AP ion channel blockers may be achieved by methods of the present invention using known method steps, without undue experimentation, based on the teaching and guidance presented herein. Alternatively, such blockers are commercially available. Biological activity, including blocking of AP ion channel functions, of AP ion channel blocking compounds and compositions of the present invention, at a variety of AP ion channels, may be determined by one of ordinary skill according to the present invention without undue experimentation, based on the guidance and teaching presented herein.

An AP of 35-50, such as 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, amino acids is expected to form a calcium channel with characteristic properties by association of at least two A?P molecules, e.g., since, in water, synthetic A/3P forms stable dimers (Hilbich, et al, 1991) , or dimers, trimers and tetramers (Barrow, et al, 1992) . Thus, any these forms could constitute a conductive unit, and these different possibilities could lead to the different kinetic and conductive properties of the AβP channel. Consistently, both the spectroscopic and molecular data on natural and synthetic A/3P emphasize the structural flexibility of the Aj3P molecule, and, furthermore, explicitly emphasize the possibilities of membrane interactions.

For example, natural AjSP from AD brain associates in fibrils which have a cross β sheet conformation (Kirschner, et al, 1986) , and even shorter segments from the AβP peptide contain the β sheet conformation (Burdick, et al, 1992;

Gorevic, et al 1987; Castano, et al, 1986; Hilbich, et al, 1991; Kirschner, et al , 1987) . Hilbich, et al (1992) report that synthetic AβP has a central (8 turn, while Barrow, et al (1992) emphasize that synthetic A P can express different proportions of a-helix and β-sheet, depending on physiologically relevant environmental variables such as ionic strength, pH, and hydrophobicity. Finally, Kang, et al (1987) have also suggested on the basis of molecular considerations that Aj3P could span the membrane through a C-terminal hydrophobic on-helical domain, leaving free the N-terminal jS-sheet domain. Perhaps not coincidentally, Lansbury (1992) reports that a synthetic C-terminal fragment of AJP, (034-42) forms an insoluble fibril lacking the cross -β structure.

The finding that AjSP molecules also form calcium channels has lead us to re-examine the primary structure for some functional hints as to the structural basis of possible conductance pathways. The most recent studies on structure of other channels have focussed on amphipathic, antiparallel β-sheet domains as the selectivity filter. Examples include the shaker K⁺ channel (Durrell and Guy, 1992), other voltage sensitive cation channels (Guy and Conti, 1990) , and annexin channels (Guy, et al, 1990; Pollard et al, 1992) . Upon inspection of the AβP sequence, we can observe that within the putative N-terminal 3-sheet domain, alternate residues [1, 3, 5, 7, (9), 11, 13, and 15,] are mostly charged or neutral, and that these residues are separated by mostly hydrophobic or neutral residues [2, 4, (6), 8, 10, 12, and (14) ]. A minimum of 11 residues in anti-parallel /3 pleated sheet conformation are needed to span the membrane twice. Thus although the structure of the AjSP molecule may be different in its filamentous, water soluble, and channel conformations, it can be concluded that the AβP molecule has the capacity to form the amphipathic, antiparallel jS-sheet domains predicted by Guy's general "TIM barrel" models for channel structure. The finding that AjSP has intrinsic calcium channel activity provides a basis for the cause of A/3P-derived neuronal or endothelial injury in Alzheimer's Disease (AD). Hardy and Higgins (1992) have viewed Alzheimer's disease as a cascade of insults to cells deriving from a primary toxic effect of AjSP on calcium homeostasis. Mattson, et al (1992) report that prolonged exposure to AβP alone causes a slight elevation in cytosolic calcium concentration, and that AβP potentiates the action of excitotoxins on intracellular calcium after a two day exposure. Thus the calcium channel property could actually be the molecular basis of the toxic action of AβP on target cells.

However, inasmuch as Alzheimer's Disease is selective, and takes time to be manifest as a syndrome, it is possible that some cells have evolved methods of resisting the action of AjSP. Resisting cells would have to resist the ever immanent catastrophe of a slow leak, and some might not be so robust. It is also possible that there may be a biosynthetic or assembly requirement to place AjSP channels into target membranes, and that some cells may have evolved local inactivation mechanisms. For example, Sisodia, et al (1990) have suggested that accumulation of AβP may be due to aberrant processing of APP, and not a product of normal degradative processes. Furthermore, it has been reported that AjSP accumulation in the brain begins long before the onset of Alzheimer's Disease symptoms (eg., McKee, et al, 1991), and that neuronal injury, either from trauma (Roberts, et al. 1991) , or defined chemical insults such as kainic acid (Siran, et al. 1989; Kawarabayashi, et al, 1991), cause acute increases in the high molecular weight amyloid /3/A4 protein precursor (APP) . Thus, the production of APP may be a normal response to local injury, and genesis of the toxic Aj3P product may not necessarily be obligatory.

Aluminum is often thought to be toxic to the brain (eg, Klatzo, et al. 1965; Terry and Pena, 1965), so its therapeutic potential could be considered problematic. For example, a lethal encephalopathy can be induced in rabbits by daily subcutaneous injections of aluminum lactate for 2 or 3 weeks (DeBoni, et al, 1976; Crapper, et al, 1978) . An encephalopathy is also associated with increased cerebral aluminum is found in some patients undergoing chronic renal dialysis (O'Hare, et al 1983) . Nonetheless, the average plasma aluminum level in humans is between 0.7 μM(Crapper, et al, 1978) and 6 μM (Doull, et al„ 1980), while normal human brain levels of Al are 1-2 μg/g dry weight. This corresponds to ca. 15 μM, which we could compare favorably with the 10-20 μM dose of Al³⁺ used in our experiments to block A/3P channels. A three-fold higher concentration of Al in human brain can be tolerated without evident toxicity (Crapper, et al, 1978), while toxicity is associated with concentrations of ca . 150 μM. In AD, serum aluminum is similar to controls (Crapper, et al, 1978) , while aluminum in AD brain can occur locally at 10-30 -fold higher levels than in equivalent regions in control brains. Presumably, much of the aluminum in AD brain is associated with A/3P in densely precipitated form in neuronal plaques. From these results we can conclude that the action and function of aluminum in nervous tissue is unclear, and that the presently available data do not rule out the possibility that lower concentrations may actually provide some advantages to the organism. AP ion channel blocking assays, based on the teaching and guidance presented herein may be used to determine which types of AP ion channels or other related receptors or associated molecules having a biological function, may be blocked by specific AP blockers of the present invention. Such assays may be performed, as a non-limiting example, calcium channels of AjSP in lipid bilayers, non-specific binding defined using non-blocking ions, such as Na⁺. As a further non-limiting subexample, AP ion channel blocking assays can be performed on mammalian cells expressing an AP ion channel in the cell surface membranes, such as AβP, such as from bovine, rat, mouse, human or rabbit, using an appropriately labeled AP ion channel ligand. Additionally, such assays may be performed on cell lines containing recombinant DNA in which the message for a particular AP ion channel has been inserted, according to known method steps. See, e.g., Montal and Mueller (1972); Wonderlin et al. (1990).

Molecular modeling may be used in the context of the present invention to correlate molecular shape and key functional group features of AP ion channel blockers of the present invention for particular AP ion channel blocking biological activities. As non-limiting example, a modeling program, such as MACROMODEL, may be used for empirical minimization of small molecules using Allinger's potentials. Alternatively, in a preferred embodiment, a molecular dynamics simulation type of modeling program may be used. As a non-limiting example, whichever computational algorithm is used, it is preferred that a shell for display of the computational results be used.. Examples include the implementation of the Karplus algorithm such as the programs INSIGHT and DISCOVER. Alternatively, additional algorithms may be used to transform the output of the various computational algorithms into the shell file format, such as mCHARM, which is preferred. Using such a molecular modeling system as AP ion channel blocker, e.g., tromethamine or Al⁺³, may be easily drawn in a low energy conformation upon which molecular dynamics may be performed with an appropriate number of cycles, until appropriate convergence is obtained. Repetition of such a modeling procedure may then be used to determine if the same or a similar conformational energy minimum is obtained, such that a representative conformational global energy minimum may be defined.

Alternatively, as another non-limiting example, known algorithms maybe used which utilize molecular annealing. See, e.g., Wilson et al. J. Comput . Chem. 12:342-349 (1991) and Drug News Perspect 4:325-331 (1991) the contents of which are entirely incorporated herein by reference. Preferably, molecular annealing type algorithms are combined with molecular dynamics to provide complementary methods to define global energy minima for AP blockers according to the present invention.

Accordingly, the determination using molecular modeling of AP blockers of the present invention may utilize minimized structures of AP blockers to define surfaces of the molecules and tabulate critical dimensions, which may then, be correlated with similar values for any alternative AP blockers of the present invention to calculate AP blockers having the expected greatest biological activity, in terms of both specificity and degree of blocking of AP ion channels. Pharmaceutical Preparations and Administration

Preparations of AP ion channel blockers for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.

By the term "protection" from infection or disease as used herein is intended "prevention, " "suppression" or "treatment." "Prevention" involves administration of a AP ion channel blocker, prior to the induction of the disease. "Suppression" involves administration of the composition prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term "prophylaxis" as distinct from "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection, " as used herein, is meant to include "prophylaxis." At least one AP ion channel blocker of the present invention may be administered by any means that achieve their intended purpose, for example, to treat an AP related pathology, as described infra, by inhibition or blocking of an AP ion channel using a AP ion channel blocker in the form of a pharmaceutical composition.

For example, administration of such a composition may be by various parenteral routes such as oral, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. Preferably, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.

A typical regimen for preventing, suppressing, or treating an amyloid related pathology, comprises administration of an effective amount of a AP ion channel blocker, administered over a period of one or several days, up to and including between one week and about 24 months, or as a long term preventive therapy. It is understood that the dosage of a AP ion channel blocker of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the inventors and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. The total dose required for each treatment may be administered by multiple doses or in a single dose, a AP ion channel blocker may be administered alone or in conjunction with other therapeutics directed to AP related pathologies, such as an amyloidosis related pathology as a non limiting example, or directed to other symptoms of the disease. Effective amounts of the a AP ion channel blocker or composition are from about 0.01 μg to about 1200 mg/kg body weight, and preferably from about 10 μg to about 100 mg/kg body weight, such 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9, 1, 2, 5, 10, 20, 25, 30, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or 1200 mg/kg.

As a non-limiting example, tromethamine can be administered at a dose of 0.1 g to 1200 mg/kg, such as 1-100, 10-500, 50-400, 80-200, 80-400, 80-150, 90-200, 90-150, 100- 400, 100-200, 100-150 kg/mg for treatment of AD, preferably from 10 mg to 200 mg/kg.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods. See, e.g., Parrott, Pharmaceutical Technology, Burgess Publishing Co., Minneapolis, Minn., (1970) ; Barker, supra, Goodman, supra, Avery, supra and Katzung, supra, which are entirely incorporated herein by reference, including all references cited therein.

Pharmaceutical compositions comprising at least one AP ion channel blocker of the present invention may include all compositions wherein the AP ion channel blocker is contained in an amount effective to achieve its intended purpose. In addition to the AP ion channel blocker, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions include suitable solutions for administration intravenously, subcutaneously, dermally, orally, mucosally, rectally or may by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e. the antibody) together with the excipient. Pharmaceutical compositions for oral administration include tablets and capsules. Compositions which can be administered rectally include suppositories.

Having now generally described the invention, the same will be further understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting.

Example I:

MATERIALS AND METHODS

Bilayer setup and recording system. The experimental chamber (made of plexiglass) consisted of two compartments separated by a thin teflon film. During experiments, the solutions were simultaneously stirred by two teflon-coated magnets placed in a restricted space at the bottom of each compartment. Ag/AgCl pellet electrodes were immersed in a small pool containing a 0.5 M KCl, and were electrically connected to the solutions in each compartment via agar bridges (2% agar in 0.5M KCl) . Single channel currents were recorded using a patch clamp amplifier (Axopatch-ID, equipped with a CV-4B bilayer headstage, Axon Instruments, Foster City, CA) , and were stored on magnetic tape using a PCNM/VCR digital system (Digital-4, Toshiba, Japan) with a frequency response in the range from DC to 25,000 Hertz. Records were made from playbacks through a lowpass filter (8-pole Bessel 902 LPF, Frequency devices) set in the range from 200 to 500 Hz.

Planar bilayers were formed by applying a suspension of palmityloleolyl-phosphatidyl-ethanolamine (POPE) and phosphatidylserine (PS), 1:1, 50 mg/ml in n-decane. A small glass rod was used to deliver the lipids to a hole of ca.100-150 μm in diameter in a teflonfilm separating two compartments that contained the required salt solutions. The A P peptide was first incorporated into a suspension of pure phosphatidylserine liposomes by a method described elsewhere (Arispe, et al. 1992). In brief, 20 μl of phosphatidylserine (Avanti Polar Lipids, Alabaster, PA) , dissolved in chloroform (lOmg/ml) , were placed in a microfuge tube. After evaporation of the chloroform by blowing nitrogen gas, 30 μl of 1M K-aspartate (pH adjusted to pH 7.2) were added, and the resulting mixture was sonicated for 5 minutes. Next, 20 μl of the A3P stock solutions (2 mg/ml) in water were added and the adduct was sonicated for 2 further minutes. For channel studies, 5 μl of the liposome preparation containing the AjSP peptide ( c_a. 5 μg AjSP) were to the cis side of the chamber. To facilitate fusion of the liposomes with the bilayer, CaCl₂ (1 mM) was added to the solutions in both compartments.

Data analysis. Analysis of the records was carried out using a digital oscilloscope (Nicolet Oscilloscope Division, Madison, WI) . In the majority of the incorporations, ion channel activity occurred in more than one conductance state. To ascertain whether the duration of each event satisfied a criterium of open-state, we measured the amplitude of every discernable level at each transmembrane potential. The minimum acceptable time interval for defining an open-state level was taken as 20 msec.

The electrical potential of the solution in the cis compartment is referred to that in the trans compartment which was electrically connected to ground. Positive charge moving through the open channel from trans to cis side represents negative current.

Chemicals. Amyloid jS Peptide (AjSP) was obtained from Bachem, Inc. (Torrance, CA) as jS-Amyloid (1-40) with the following primary structure: D- A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F- F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V (SEQ ID NO: 1) . The peptide was dissolved in milli-Q water at a concentration of 2 mg/ml (0.46 mM) , and stored at -20°C. In a few experiments, the peptide was solubilized in water containing 0.1% TFA and 20 % acetonitrile, which were of the highest grade commercially available. Aluminum sulfate was obtained from Fisher (Fisher Scientific Co., NJ) „ Tromethamme was from BRL (Bethesda Research Laboratories, MD) .. The concentration of free calcium in the solutions was measured using a calcium electrode (CAL-1, W-P Instruments, Inc. New Haven, CN) RESULTS

The AβP peptide forms ion selective channels across bilayer membranes. Discrete conductance changes, characteristic of ion channel activity, were always observed a few minutes after the addition of liposomes containing ASP to the cis compartment of the bilayer chamber. As illustrated in Figure 1, in symmetrical CsCl solutions (in mM: 75 CsCl, 1 CaCl₂, 2 Na-HEPES, pH 7) changing the potential had no noticeable effects on the kinetics of the channel activity or on the number of levels at each transmembrane potential.

Since AjSP-channel closures from any one level occurred at -50, -30 and -20 mV, and frequent displacements of the current trace between different levels occurred at all potentials, we conclude that only one channel with multiple conductance levels was active in the bilayer (cf, Fox, 1987; Meves and Nagy, 1989) .

To identify the charge on the ion carrying the current, we measured the shift in the reversal potential V* (or potential at which the net current through the open channel is zero) induced by a change from a symmetrical (i.e. V* = 0) to an asymmetric solution system (i.e. V* = 0) . First, we incorporated the AβP-channel in symmetrical 40 mM KCl solutions and recorded the current at ±20, ±10 and 0 mV (Figure 2A; upper part) . As expected, at zero membrane potential no net current could be detected in symmetrical (KCl) (Figure 2A; upper part) . Next, the (KCl) in the trans side was augmented to 60 mM by adding the appropriate volume of a 1 M KCl solution. After stirring the solutions in the chamber, another series of measurements of the A/SP-channel current at ±20, ±10 and 0 mV was made. As shown in Figure 2A (lower part) the kinetics of the A/?P-channel activity was not affected by this change in [KCl] . However, at zero membrane potential a net negative current (positive charges moving from the trans to the cis side) of ca. -3 pA was measured (Figure 2A, lower part) . This result demonstrates that the ion carrying the bulk of the current is indeed K⁺„

The linear character of the permeation mechanism in the AβP channel is made evident by drawing a straight line through the mean value of the current at each potential (Figure 2B) . With the symmetrical system of 40 mM KCl, the I-V straight line is expected to intercept the potential axis at V* = 0, which it does (Figure 2B, •) . For the asymmetrical system of 40 and 60 mM KCl, although the linear characteristics of the I-V curve was maintained, the potential V* at which the net current through the open AβP-channel is zero was shifted to 8.5 mV. The value of V* may be used to estimate the permeability ratio P_κ/P using the following equation,

V*= RT/F In { P_K(K)_t + P_C1(C1)_C}/{P_K(K)_C + P_α(Cl)_t}, (1)

where (X)_t and (X)_care the concentrations of the ion species X in the trans and cis compartments, respectively; and F, R and T have their usual meanings (Lakshminarayanaiah, 1984) . Inserting the values for the concentrations ((K)_t = 60 mM, (K)_c = 40 M, (Cl)_t = 60 mM, and (Cl)_c = 40 mM) into equation (1) we get a P_κ/P_Cι ratio of ca. 11.

If flow of K⁺ ions through the channel were independent of the number of ions present in the hypothetical pore, the channel conductance should increase with (K) . However, the measured increase in channel conductance from 325 pS (symmetrical: 40 mM KCl) to 346 pS (asymmetrical: 40 vs 60 mM KCl) was less than the expected conductance of ca. 487 pS (= 325 X 60/40) Ca²⁺ permeates the open AjSP channel. To test whether

Ca ²⁺ permeates the open AβP P-channel we used the asymmetrical system of 37.5 mM CsCl in the cis compartment and 25 mM CaCl₂ in the trans compartment. At negative transmembrane potentials (-20, and -30 mV in Figure 3A; -40 and -60 mV in Figure 3B) distinct, discrete jumps in the current record between different levels were observed. At potentials negative to -4 mV, currents representing Ca²⁺ flowing from the trans to the cis side through the open Aj3P-channel were recorded. Figure 3B illustrates another interesting property of the A0P channel. Repetitive applications of step changes in potential from 0 to either -40 mV (Figure 3B, upper records 1-3) or -60 mV (lower records 1, 2A and B, and 3) always induced the appearance of different Ca²⁺ current levels. In all cases, the size of the initial current jump was higher than that eventually attained. Furthermore, the duration of the initial current , although varied from pulse to pulse, was inversely proportional to the absolute size of the potential (Figure 3B) . This behavior was only seen if Ca²⁺ were the charge carrier. Also apparent in Figure 3B are the nearly complete AjSP-channel closures from any one level (see Figure 3B, records labelled A and B) , and the frequent displacements of the Ca²⁺ current trace between different levels, suggesting that only one channel with multiple conductance levels was active in the bilayer. At positive membrane potential, with Cs⁺ carrying the current, the conductance of the AjSP-channel was estimated to be 83 pS (Figure 3C, o) . By contrast, in symmetrical 75 mM CsCl solutions (Figure 1) the conductance was estimated as c_a. 206 pS. Permeability sequence for cations. To determine the sequence of permeabilities for different cations, we recorded Aj3P-channel currents at different transmembrane potentials under conditions of solution asymmetry. We then measured the amplitude of channel events at each membrane potential and constructed I-V curves. To estimate the permeability ratio P_Ca/P_Cs from the data in Figure 3C, we used the following equation (26) :

V* = RT/F {4P'_Ca(Ca)_t}/{ P_c,(Cs)_c + 4P'_Ca(Ca)_ce^v*FRT}, (2)

where P'_ca = P_Ca/ {l + e^v*FRT} . From the data in Figure 3C (V* = -4 mV, (Ca), = 25 mM, (Ca)_c = 1 mM, (Cs)_c = 37.5 mM) we calculate the P_Ca/^p _c» ratio as 0.6.

As expected for a cation selective channel, the conductance of the AjSP-channel in the asymmetrical system depends on the cation carrying the current. Figure 4A shows A/3P-channel current records made with 37.5 mM LiCl in the trans compartment and 37.5 mM CsCl in the cis compartment. At zero potential across the bilayer a net positive current is observed (Figure 4A) . Since the A/3P-channel is selective for cations, the current must be carried by Cs⁺ moving from the cis to the trans side of the AβP channel. Furthermore, with Cs⁺ as the charge carrier the conductance is ca. 264 pS (Figure 4B, straight line through the points at 0, 10, and 20 mV) and ca. 181 pS with Li⁺ as charge carrier (Figure 4B, points at -40, -30, 20, -14 mV) . Thus, the conductance of the AjSP-channel with Cs⁺ as charge carrier is ca . 1.5 times greater than that with Li⁺ as the charge carrier. The meaning of these results is that the AjSP-channel is more permeable to Cs⁺ than Li⁺. Consistent with the estimate of the permeability ratio based on conductance measurements, using the reversal potential V^* of 14 mV from Figure 4B and equation (1) we calculate a P_C8/P_Li ratio of ca. 1.6. Preferential flow of Ca⁺and K⁺ over Na⁺ through the open AjSP channel is illustrated in Figure 5. Three pairs of current records made under two asymmetrical cationic systems are depicted in Figure 5A. The recording of the channel activity was started in the presence of a KCl solution in the trans compartment ( in mM: 40 KCl, 1 CaCl₂, 2 Na-HEPES, pH 7) and a NaCl solution in the cis compartment (in mM: 40 mM NaCl,

1 CaCl₂, 2 Na-HEPES, pH 7) . Under these conditions, records were made at different potentials (Figure 5A, two sample records at the top) . Keeping the composition of the solutions in the cis compartment constant, the solution in the trans compartment was replaced by a CaCl₂ solution (in mM: 25 CaCl₂,

2 Na-HEPES, pH 7) . A few minutes after the solution change, another series of records was made (Figure 5A, middle pair of records at -10 mV and lower pair of records at 20 mV) . Under these new conditions, channel activity is characterized by frequent AjSP-channel closures and multiple current levels. These properties of the AjSP-channel activity are present when Ca²⁺ (lower pairs) or K⁺(top pair) or Na⁺ is the charge carrier through the open channel. The corresponding I-V curves for the two systems are shown in Figure 5B. For the KCl/NaCl system (Figure 5) , the I-V curve is clearly non-linear. Taking the reversal potential V^* as £____. 7 mV for both systems, using equations (1 and 2) we get the value of 1.3 for both P_K/P_Na and P_ca/P_Na ratios.

In symmetrical 75 mM CsCl (Figurel) and asymmetrical 37.5 mM CsCl/LiCl (Figure 4) solutions, the average conductance of the A0P-channel (with Cs⁺as the charge carrier) was found to be ca. 235 pS. In contrast, in the asymmetric system 37.5 mM CsCl/25 mM CaCl₂ (Figure 3), with Cs⁺ as the charge carrier, the conductance was only 83 pS. This apparent blockade by Ca²⁺ of the flow of Cs⁺ through the AjSP-channel was further studied directly in the experiment illustrated in Figure6. Control records of the channel activity were made at ±60, ±40 and ±20 mV (Figure 6A) „ Next, the concentration of Ca²⁺ in the cis compartment was augmented from 1 to 10 mM and a second family of records was made. As shown in Figure 6B, both the amplitude of the channel current and the frequency of openings were drastically reduced by Ca⁺. Figure 6B also shows that the current flowing through the open, but blocked,

AβP-channel is rectified, i.e. at -60 mV the magnitude of the Cs+ current is larger than at 60 mV. Making the potential of the cis side negative favors the entry of trans Cs⁺ into the open A0P-channel. This electrical force drives Cs⁺ through the channel, and these Cs⁺ ions might be able to displace Ca²⁺ from binding sites. However, in the absence of this electrical force, high affinity binding of Ca²⁺to the site might impede the occupancy of the site by Cs^+tthereby causing the block of cation flow. The permeability ratios obtained so far can be used to establish a permeability sequence for the different cations tested. We know that P_K/P_Na = ^p _ca ^p _N» = 1-3 (see Figure 5) . It follows that PQ, = P_κ = 1.3 P_Na. Furthermore, we also know that ^p _c ^p _cs = 0.6 (or, PCs/Po, = 1.67), and P_Cs/Pi = 1-6 (from Figures 3 and 4) . The product of these two ratios eliminates P_c, and gives P_Ca/^pι_i = 0.96. Eliminating P_Cafrom the ratios P_Ca/^p _cs = 0.6 (Figure 3) and Pc«/^pN = -■ - -■ (Figure 3), we obtain P_Na/P_Cs = 0.46. Thus, the complete permeability sequence is:

Blockade of the AβP-channel activity by tromethamine and Al³⁺. As part of our studies of the cationic permeability sequence through the A/5P-channel, we noted that tromethamine not only was impermeable, but actually blocked the channel currents. Figure 7A illustrates the blockade by tromethamine (10 mM) of the channel currents with either Ca²⁺ (records at -20, -10 and 0 mV) or Cs⁺ (records at 10 and 20 mV) carrying the current. Addition of tromethamine (10 mM) to the cis side drastically reduced the amplitude of the currents as well as the frequency of the channel events (see Figure 7A, 6th-10th records from the top) at ±20 and -40 mV.

Since AβP in amyloid plaques is known to bind Al³⁺, we also tested the AjSP-channel for sensitivity to aluminum. As shown in Figure 7 (B and C) , addition of Al³⁺ (10 or 20 μM) blocked the channel activity. Blockade of the AjSP-channel by a high dose of Al³⁺ (1 mM, Figure ID) was rapid, and the blockade persisted at high potentials (± 60 mV) .

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, and known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.

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SUBSTITUTESHEET

Claims

WHAT IS CLAIMED IS:

1. A method for blocking an amyloid protein ion channel in a phospholipid containing bilayer, comprising contacting said ion channel with an amount of an amyloid protein ion channel blocking agent or modality which is effective for blocking ion transport through said ion channel.

2. A method according to claim 1, wherein said channel blocking compound is tromethamine or an ion or salt thereof.

3. A method according to any of claims 1 or 2, wherein said is tromethamine is administered to provide said compound in an amount of 0.001 to 10 mM.

4. A method according to claim 2, wherein said channel blocking compound is an aluminum ion or salt.

5. A method according to any of claims 3 or 4, wherein said is aluminum is administered to provide said compound in an amount of 0.01 to 100 μM.

6. A method for treating an animal suffering from an amyloid protein related pathology, comprising administering an ion transport blocking effective amount of an amyloid protein ion channel blocking compound.

7. A method according to claim 6, wherein said amyloid protein related pathology is selected from an amyloidosis related pathology, a nephrodegenerative disease, a neurological trauma or a neurodegenerative disease.

8. A method according to any of claims 6 and 7, wherein said pathology is Alzheimer's disease.

9. A method according to any of claims 6, 7 and 8, wherein said ion channel blocking compound is tromethamine or a salt or ion thereof.

10. A method according to claim 9, wherein said is trometnamine is administered to provide said compound in an amount of 0.1 to 1200 mg/kg/day.

SUBSTITUTESHEET

11. A method according to any of claims 6, 7 and 8, wherein said ion channel blocking compound is an aluminum ion or salt thereof.

12. A method according to claim 11, wherein said is aluminum is administered to provide said compound in an amount of 0.01 to 10 mg/kg.

13. A method according to any of claims 8-12, wherein said animal is selected from a mammal, a fish or a bird.

14. A method according to claim 13, wherein said mammal is a human.

15. A method for testing the effectiveness of a compound for blocking an amyloid protein ion channel, comprising, providing a phospholipid bilayer containing an amyloid protein ion channel; measuring the conductance of the bilayer relative to the ion transport with and without the presence of said compound of an ion transportable by said ion channel; and determining the blocking effectiveness of said compound on said ion channel.

16. A method according to claim 15, wherein said phospholipid bilayer is provided in a form selected from in vitro, in situ, or in vivo.

17. A method according to any of claims 15 and 16, wherein said amyloid protein is expressed in said bilayer in a cell.

18. A method according to any of claims 15 and 16, wherein said amyloid protein is provided in said bilayer in an artificial membrane.

19. A pharmaceutical composition for use in si tu and in vivo for blocking ion channel activity of an amyloid protein ion channel, comprising a compound having amyloid channel blocking activity, and a pharmaceutically acceptable carrier.

SUBSTITUTE SHEET

20. A composition according to claim 19, wherein said compound is tromethamine or an ion or salt thereof.

21. A composition according to claim 19, wherein said compound is an aluminum ion or salt thereof.

22. Tromethamine, for use in manufacturing a pharmaceutical composition for the treatment of an amyloid protein related pathology.

23. Tromethamine according to claim 22, wherein said amyloid protein related pathology is selected from an amyloidosis related pathology, a nephrodegenerative disease, a neurological trauma or a neurodegenerative disease.

24. Tromethamine according to any of claims 22 and 23, wherein said pathology is Alzheimer's disease.

25. An aluminum ion or salt, for use in manufacturing a pharmaceutical composition for the treatment of an amyloid protein related pathology.

26. An aluminum ion or salt according to claim 22, wherein said amyloid protein related pathology is selected from an amyloidosis related pathology, a nephrodegenerative disease, a neurological trauma or a neurodegenerative disease.

27. An aluminum ion or salt according to any of claims 25-26, wherein said pathology is Alzheimer's disease.

SU_BSTITUTESHEET