The invention involves identification of a mechanism of .beta.-amyloid peptide cytotoxicity, which enables treatment of conditions caused by .beta.-amyloid peptide aggregates by administration of compounds which antagonize the mechanism of cytotoxicity. The invention includes the identification and isolation of compounds which can reduce the neurotoxic effects of such aggregates. Methods for treating conditions resulting from neurotoxic .beta.-amyloid peptide aggregates, such as Alzheimer's disease and pharmaceutical preparations are provided. Also provided are methods for selecting additional compounds which can reduce the neurotoxic effects of .beta.-amyloid aggregates.

DETAILED DESCRIPTION OF THE INVENTION

We have chosen the peptides A.beta.25-35 (GSNKGAIIGLM, SEQ ID NO:1) and A.beta.1-42 (SEQ ID NO:2) as model systems to explore the effect of .beta.-amyloid peptides on calcium homeostasis in neuronal cells, using quantitative estimation of the internal calcium concentration of the cells, and membrane depolarization, using voltage-sensitive fluorescent dyes.

Reports in the literature have shown that .beta.-amyloid peptides cause an influx of calcium into cells, using not only A.beta.25-35, but also A.beta.1-40 (SEQ ID NO:3) and A.beta.1-42. We have investigated the connection between .beta.-amyloid peptide aggregation and the influx of calcium into neuronal cells as the first molecular event in the cytotoxicity of neurons in Alzheimer's Disease.

Pollard has reported the formation of ionophores from A.beta.1-40 in artificial membrane which could be blocked by AlCl.sub.3 or Tromethamine (Arispe, 1993). Our attempts to reproduce aluminum blockage in our experiments have been inconclusive because we found that AlCl.sub.3 by itself powerfully induces calcium influx in hNT neuronal cells from external calcium sources. Thus, we turned to an alternative hypothesis, that aggregates of the .beta.-amyloid peptides modulate ligand-gated ion channels such as NMDA and non-NMDA channels. Previous patch-clamp experiments indicated that voltage-gated calcium channels were not involved, because CdCl.sub.2 did not block the calcium influx. We have also determined that the increased cytosolic calcium is derived entirely from the external medium. We have determined that calcium influx into hNT neuronal cells caused by A.beta.25-35 can be blocked by MgCl.sub.2, and by CNQX, but not by DL-AP5. hNT neuronal cells are known to express both NMDA and non-NMDA glutamate receptor channels. The blocking effect of CNQX, coupled with the lack of blocking effect of DL-P5, indicated that the effect on calcium influx by A.beta.25-35 aggregates in hNT cells is mediated by a non-NMDA cation channel. Since these observations involved the obligatory role of .beta.-amyloid peptide aggregates, we hypothesized that compounds capable of antagonizing the formation of A.beta.1-42 or A.beta.25-35 aggregates will alleviate neurotoxicity of Alzheimer's Disease. These observations also suggest a strategy for developing therapeutics which modulate the activity of non-NMDA channels affected by .beta.-amyloid peptide aggregates.

Peptides with a relatively high content of .beta.-sheet forming sequence are likely to form multimers or aggregates, often in the form of fibrils, in aqueous solutions. Such .beta.-sheet forming sequences are often present in intact globular proteins, but are embedded in other largely hydrophilic amino acid sequences and thus the proteins are kept in solutions. Once released from their precursor protein by proteolysis, peptides with .beta.-sheet forming sequences can aggregate. Relevant to Alzheimer's Disease is the "abnormal" proteolysis of APP (Amyloid Precursor Protein) which yields A.beta.1-40, A.beta.1-42, and possibly also A.beta.25-35. These peptides form aggregates, including fibrils, in aqueous solution which, as described above, may be causative agents of increased neuronal cell calcium influx.

Our aim was to design or select antagonistic peptides, which we call decoy peptides (DPs), which (i) reduce aggregate formation by either blocking aggregation of .beta.-amyloid peptides or, by incorporation into the nascent aggregate, make it inactive; (ii) are soluble in aqueous solutions but retain .beta.-sheet forming potential associated with the multimer-forming amyloid peptide; and (iii) contain amino acids with charged side chains that can interfere with the interaction between .beta.-amyloid aggregates and ligand-gated Ca.sup.2+ channels. Decoy peptides are unlikely to interact with .beta.-sheet regions of other biologically important proteins because, as noted above, such regions generally are buried in the tertiary structure of the protein and therefore inaccessible. Preferably, decoy peptides are resistant to proteolytic digestion, to increase usefulness of such peptides in therapeutic applications. Decoy peptides active against .beta.-amyloid neurotoxicity are described in U.S. Pat. No. 6,172,043.

It is believed that .beta.-amyloid peptides are neurotoxic at least in part because they bind together to form multimers, or aggregates, which may even be fibrils of .beta.-amyloid peptides linked together by binding of .beta.-sheet structures of the .beta.-amyloid peptides. Thus, compounds which prevent binding of .beta.-amyloid peptides, which reduce the formation or size of the aggregates, such as fibrils, or which alter the tertiary structure and/or calcium influx or depolarization stimulating properties of the aggregates can be useful for reducing the neurotoxicity of .beta.-amyloid peptides. It has been discovered that a certain class of peptides, decoy peptides, is effective in reducing neurotoxic .beta.-amyloid peptide aggregate formation.

The invention thus involves in one aspect the discovery of a mechanism of .beta.-amyloid peptide aggregate cytotoxicity, which in turn enables intervening to interfere with that aggregate cytotoxicity by administration of compounds which antagonize the mechanism of cytotoxicity. A number of compounds which antagonize the mechanism of cytotoxicity have been identified using the high-throughput methods of the invention. These compounds include organic molecules and inorganic molecules. In one aspect of the invention the compounds interfere with the ability of .beta.-amyloid peptide to form neurotoxic aggregates, which aggregates cause unwanted cytotoxic calcium influx into cells. The compounds can affect neurotoxic aggregates by inhibiting binding of .beta.-amyloid peptides to existing aggregates, by disrupting existing aggregates, by altering the structure of aggregates which incorporate the compound, by otherwise altering the structure of the aggregates (e.g. by capping) or by other mechanisms. Compounds useful in the invention also can interfere with unwanted calcium influx and/or membrane depolarization, e.g., by acting on the cell surface binding partner of the neurotoxic .beta.-amyloid peptide aggregate, by reducing .beta.-amyloid peptide aggregation, and the like. Examples of such compounds, discussed in greater detail below, include decoy peptides, which inhibit or interfere with neurotoxic .beta.-amyloid peptide aggregates, and non-NMDA channel antagonists.

Various changes may be made to such compounds including the addition of various side groups that do not affect the manner in which the compound, e.g., decoy peptide, binds to its binding partner, or which favorably affect the manner in which the compound binds to its binding partner. Such changes may involve adding or subtracting charge groups, substituting amino acids, adding lipophilic moieties that do not effect binding but that affect the overall charge characteristics of the molecule facilitating delivery across the blood-brain barrier, etc. For each such change, no more than routine experimentation is required to test whether the molecule functions according to the invention. One simply makes the desired change or selects the desired compound and tests it in accordance with standard procedures as described herein. For example, if the candidate molecule interferes with the ability of a .beta.-amyloid peptide to form neurotoxic aggregates that cause an increase in calcium influx, and/or alters membrane depolarization, in neuronal cells, then the candidate a decoy peptide or other compound useful in antagonizing the effects of .beta.-amyloid aggregates.

As used herein, a "decoy peptide" is one which binds to a .beta.-amyloid peptide, such as A.beta.1-40, A.beta.1-42, or A.beta.25-35, and thereby reduces the ability of .beta.-amyloid peptide to form neurotoxic aggregates. The decoy peptides may inhibit neurotoxic aggregate formation by inhibiting formation of new aggregates, inhibiting binding of .beta.-amyloid peptides to existing aggregates, disrupting existing aggregates, altering the structure of aggregates which incorporate the decoy peptides or by other mechanisms. While not being limited to any particular mechanism, it is believed that decoy peptides can inhibit .beta.-amyloid peptide aggregate formation by presenting a O-sheet secondary structure which is compatible with and binds to existing .beta.-amyloid peptide .beta.-sheet structures, but which does not permit binding of additional .beta.-amyloid peptides sufficient to form aggregates. Alternatively, decoy peptides can inhibit .beta.-amyloid peptide aggregate formation and/or cytotoxicity by altering the structure of the aggregate sufficiently to reduce its cytotoxic effects.

.beta.-amyloid peptide aggregate formation can be determined directly, e.g., by observation of the extent of .beta.amyloid peptide aggregate formation by microscopy, or indirectly, e.g., by determination of the effects of .beta.-amyloid peptide aggregate formation, such as a change in neuronal cell calcium influx or membrane depolarization. Other methods for determining the extent or effects of .beta.-amyloid peptide aggregate formation will be apparent to one of ordinary skill in the art.

Compounds that reduce unwanted calcium influx induced by .beta.-amyloid peptide aggregates also can be identifies. Calcium influx can be measured using indicator compounds which change a physical property (e.g., excitation/emission spectra) in response to a change in intracellular calcium concentration. Other methods for assaying changes in calcium influx useful in selecting compounds which oppose the effects of .beta.-amyloid peptide aggregates on calcium influx will be known to one of ordinary skill in the art.

Still other methods for determining the effectiveness of a compound in inhibiting the neurotoxic effects of .beta.-amyloid peptide aggregates can be used. For example, the effectiveness of compounds against damage in rat brain slices caused by neurotoxic .beta.-amyloid peptide aggregates can be determined. As another example, A.beta. fibrils can be injected into particular regions of rat brains to cause tissue damage which mimics the effects seen in Alzheimer's disease. Compounds can be administered to determine the sparing effect of the decoy peptides. All of the foregoing methods are known in the art and can be employed using no more than routine experimentation.

Compounds need not have both properties to be useful according to the invention. It is possible to identify compounds which do not inhibit .beta.-amyloid peptide aggregation but do reduce .beta.-amyloid-induced calcium influx or membrane depolarization, and vice versa. It is contemplated that compounds having only one of the desirable properties identified herein are useful, although it is preferable that a compound have more than one of such properties.

Selection of compounds which disrupt .beta.-amyloid peptide aggregate formation is particularly contemplated. Methods for selecting such compounds include binding assays with which the art is familiar, as well as functional assays for determining the effects of such compounds on a biological response to aggregate formation, such as neuronal cell calcium influx. Methods for selecting compounds which disrupt .beta.-amyloid peptide binding are provided in greater detail below.

Changes to the structure of a compound which disrupts .beta.-amyloid peptide aggregate formation to form variants or analogs of such a compound can be made according to established principles in the art. Such changes can be made to increase the therapeutic efficacy of the compound, reduce side effects of the compound, increase or decrease the hydrophobicity or hydrophilicity, and the like. Changes to the structure include the addition of additional functional groups, such as for targeting the compound to a particular organ of a subject, and substitution of one or more portions of the compound. In general, substitutions involve conservative substitutions of particular moieties or subunits of the compound. For example, when preparing variants of a compound which is a peptide, one of ordinary skill in the art will recognize that conservative amino acid substitutions will be preferred, i.e., substitutions which retain a property of the original amino acid such as charge, O-sheet forming potential, etc. Examples of conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Preferred substitutions include substitutions amongst P-branched amino acids. Of course, non-conservative substitutions can also be made to the peptide sequence of the decoy peptides, followed by testing the function of the substituted decoy peptide as described herein.

Preferably, peptide-based compounds are non-hydrolyzable. To provide such peptide compounds, one may select peptides from a library of non-hydrolyzable peptides, such as peptides containing one or more D-amino acids or peptides containing one or more non-hydrolyzable peptide bonds linking amino acids. Alternatively, one can select peptides which are optimal for disrupting .beta.-amyloid peptide aggregation, calcium influx and/or membrane depolarization and then modify such peptides as necessary to reduce the potential for hydrolysis by proteases. For example, to determine the susceptibility to proteolytic cleavage, peptides may be labeled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing it peptides and proteolytic fragments. Alternatively, potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of a peptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds which are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide. Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Non-hydrolyzable bonds include -psi[CH.sub.2NH]-- reduced amide peptide bonds, -psi[COCH.sub.2]-- ketomethylene peptide bonds, -psi[CH(CN)NH]-- (cyanomethylene)amino peptide bonds, -psi[CH.sub.2CH(OH)]-- hydroxyethylene peptide bonds, -psi[CH.sub.2O]-- peptide bonds, and -psi[CH.sub.2S]-- thiomethylene peptide bonds.

Peptides preferably are short enough to be synthesized and isolated readily, yet long enough to effectively disrupt .beta.-amyloid peptide aggregate formation. Preferred peptides thus are between four and twenty amino acids in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids. More preferably, peptides are between five and ten amino acids in length. Those skilled in the art are well-versed in methods for preparing and isolating such peptides, such as synthetic chemistry or even recombinant biological methods.

Peptides useful in the invention can be linear, or maybe circular or cyclized by natural or synthetic means. For example, disulfide bonds between cysteine residues may cyclize a peptide sequence. Bifunctional reagents can be used to provide a linkage between two or more amino acids of a peptide. Other methods for cyclization of peptides, such as those described by Anwer et al. (Int. J. Pep. Protein Res. 36:392 399, 1990) and Rivera--Baeza et al. (Neuropeptides 30:327 333, 1996) are also known to those of skill in the art.

Nonpeptide analogs of peptides, e.g., those which provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogs can be prepared based on a selected decoy peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural confirmation, or stabilize a preferred, e.g., bioactive, confirmation. One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al., Regul. Pept. 57:359 370 (1995). Peptide as used herein embraces all of the foregoing.

Decoy peptides are useful in the treatment of conditions which are characterized by .beta.-amyloid peptide aggregate formation. Decoy peptides also are useful for the selection of other compounds which interfere with neurotoxic .beta.-amyloid peptide aggregate formation, e.g., by use of a decoy peptide in competition assays to select compounds which bind to .beta.-amyloid peptides more avidly than the decoy peptide and which still interfere with neurotoxic .beta.-amyloid peptide aggregate formation. Decoy peptides are also useful in the design of other compounds for disrupting .beta.-amyloid peptide aggregate formation, such as small molecule inhibitors, based on the molecular structure of the decoy peptide. Thus, the decoy peptides can be used in vivo for the treatment of disease, as well as in vitro for the design and testing of compounds active in the disruption of .beta.-amyloid peptide aggregate formation.

In some circumstances, it may be preferred to conjugate the compound to a molecule which facilitates transport of the decoy peptide across the blood-brain barrier (BBB). As used herein, a molecule which facilitates transport across the BBB is one which, when conjugated to the compound, facilitates the amount of compound delivered to the brain as compared with non-conjugated compound. The molecule can induce transport across the BBB by any mechanism, including receptor-mediated transport, and diffusion. The compound can be conjugated to such molecules by well-known methods, including bifunctional linkers, formation of a fusion polypeptide, and formation of biotin/streptavidin or biotin/avidin complexes by attaching either biotin or streptavidin/avidin to the compound and the complementary molecule to the BBB-transport facilitating molecule.

Molecules which facilitate transport across the BBB include transferrin receptor binding antibodies (U.S. Pat. No. 5,527,527); certain lipoidal forms of dihydropyridine (see, e.g., U.S. Pat. No. 5,525,727); carrier peptides, such as cationized albumin or Met-enkephalin (and others disclosed in U.S. Pat. Nos. 5,442,043; 4,902,505; and 4,801,575); cationized antibodies (U.S. Pat. No. 5,004,697); and fatty acids such as docosahexanoic acid (DHA; U.S. Pat. No. 4,933,324).

For other uses of the compounds, it may be preferred to administer the compounds in combination with a molecule which increases transport of compounds across the blood-brain barrier (BBB). Such molecules, which need not be conjugated to a decoy peptide, increase the transport of the compound across the BBB into the brain. A molecule which increases transport across the BBB is one, for example, which increases the permeability of the BBB, preferably transiently. Coadministration of a compound with such a molecule permits the compound to cross a permeabilized BBB. Examples of such molecules include bradykinin and agonist derivatives (U.S. Pat. No. 5,112,596); and receptor-mediated permeabilizers such as A-7 (U.S. Pat. Nos. 5,268,164 and 5,506,206).

Compounds which reduce the ability of .beta.-amyloid peptides to form aggregates which increase neuronal cell calcium influx and/or membrane depolarization can be administered to a subject to treat a condition characterized by unwanted .beta.-amyloid peptide aggregates. Compounds are administered in an amount effective to reduce or inhibit formation of unwanted aggregates. By effective amount is meant an amount of a compound which inhibits formation of new unwanted .beta.-amyloid peptide aggregates, modifies the structure of new or existing unwanted aggregates so that the aggregates do not increase neuronal cell calcium influx, or destabilizes existing unwanted aggregates. .beta.-amyloid peptide aggregates can include one or more of A.beta.1-42, A.beta.1-40 and A.beta.25-35, as well as other components.

Conditions characterized by unwanted .beta.-amyloid peptide aggregate formation include Alzheimer's Disease. It will be apparent to one of ordinary skill in the art that cytotoxicity of certain neuronal cells is involved in such conditions. For example, neuronal cells involved in Alzheimer's Disease include cells from hippocampal neurons, cortical layer 3 neurons, amygdala neurons, locus coeruleus neurons, and others known to be involved in memory formation and storage. It is envisioned that the compounds described herein, particularly decoy peptides, can be delivered to neuronal cells by site-specific means. Cell-type-specific delivery can be provided by conjugating a compound to a targeting molecule, e.g., one which selectively binds to the affected neuronal cells. Methodologies for targeting include conjugates, such as those described in U.S. Pat. No. 5,391,723 to Priest. Another example of a well-known targeting vehicle is liposomes. Liposomes are commercially available from Gibco BRL. Numerous methods are published for making targeted liposomes. Liposome delivery can be provided by encapsulating a decoy peptide in liposomes which include a cell-type-specific targeting molecule. Methods for targeted delivery of compounds to particular cell types are well-known to those of skill in the art.

Methods for reducing .beta.-amyloid peptide induced neuronal cell calcium influx also are provided. The internal calcium concentration in neuronal cells can be affected by release of calcium from intracellular stores, influx of calcium from the extracellular milieu and possibly other sources. As described herein, .beta.-amyloid peptides increase internal calcium concentrations by influencing the permeability of certain ligand-gated ion channels, the non-NMDA channels. Non-NMDA channels are ordinarily activated by a combination of two factors: (1) the presence of the excitatory amino acid neurotransmitter glutamate, and (2) a lack of magnesium ions at the cell surface following depolarization of the cell. Non-NMDA channels include subtypes for which AMPA ((RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)-propionate) and kainate are agonists.

The discovery of a calcium influx mechanism by which .beta.-amyloid peptides induce neurotoxicity provides a basis for treating conditions characterized by .beta.-amyloid peptide induced calcium influx. Thus, subjects can be treated by administering any compounds which reduce the .beta.-amyloid peptide induced calcium influx. Such compounds can be inorganic or organic and can act on the .beta.-amyloid peptide, the neurotoxic .beta.-amyloid peptide aggregate or the cell surface binding partner of the neurotoxic .beta.-amyloid peptide aggregate to interfere with unwanted calcium influx. Examples of such compounds include decoy peptides which inhibit or interfere with neurotoxic .beta.-amyloid peptide aggregates, and non-NMDA channel antagonists. The compounds are administered in an effective amount, i.e., an amount which reduces the increased calcium influx. In neuronal cell types other than NT2-N cells differentiated with retinoic acid, .beta.-amyloid peptides may induce neurotoxicity via calcium influx through other means, such as NMDA channels. It is contemplated, therefore, that antagonists of calcium channels other than non-NMDA channels can be administered to treat conditions characterized by .beta.-amyloid peptide induced calcium influx.

Non-NMDA channel antagonists are well-known in the art. Such antagonists inhibit the calcium influx by inhibiting the opening of a non-NMDA channel in response to its ligand, such as glutamate, AMPA, kainate or, according to the invention, neurotoxic .beta.-amyloid peptide aggregates. Non-NMDA channel antagonists can act competitively or noncompetitively, and can block one or more subtypes of non-NMDA channels. Preferably, antagonists used are those which inhibit the function of only those channels opened by .beta.-amyloid peptide aggregates. Useful non-NMDA antagonists include 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX), 2,3-dihydroxy-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX), 1-(4-chlorobenzoyl)piperazine -2,3-dicarboxylic acid (CBPD), 6,7-dichloro-2(1H)-oxoquinoline-3-phosphonic acid (24c), Evans blue, 2,3-dihydroxy-7-sulfamoyl-benzo[f]quinoxaline (BQX), derivatives of 4-oxo-1,4-dihydroquinoline-2-carboxylic acid at the 6-position, 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA), 2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-- +++oxoisoxazolin-4-yl]propionic acid (AMNH), 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiazepine (GYKI 52466), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K), 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydr- o-5H-2,3-benzodiazepine (GYKI 53655), and (-)(3S,4aR,6R,8aR)-6-[2-(1 (2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a, 5,6,7,8,8a-decahydroisoquinoline -3-carboxylic acid monohydrate (LY326325).

Likewise, the discovery of a membrane depolarization mechanism by which .beta.-amyloid peptides induce neurotoxicity provides a basis for treating conditions characterized by .beta.-amyloid peptide induced membrane depolarization. Thus, subjects can be treated by administering any compounds which reduce the .beta.-amyloid peptide induced membrane depolarization. Such compounds can be inorganic or organic and can act on the .beta.-amyloid peptide, the neurotoxic .beta.-amyloid peptide aggregate or the cell surface binding partner of the neurotoxic .beta.-amyloid peptide aggregate to interfere with unwanted membrane depolarization. Exemplary compounds that decrease .beta.-amyloid peptide aggregate induced membrane depolarization are identified in the Examples.

The invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents useful in the treatment of conditions associated with .beta.-amyloid peptide aggregation or conditions associated with increased neuronal cell calcium influx induced by the presence of .beta.-amyloid peptide aggregates. Generally, the screening methods involve assaying for compounds which interfere with .beta.-amyloid peptide aggregation or neuronal cell calcium influx through non-NMDA channels as regulated by .beta.-amyloid peptide aggregates. Such methods are adaptable to automated, high throughput screening of compounds.

A wide variety of assays for pharmacological agents are provided, including, labeled in vitro peptide-peptide binding assays, Ca.sup.2+ influx assays, etc. For example, peptide binding screens are used to rapidly examine the effect of candidate pharmacological agents on the binding of decoy peptides to .beta.-amyloid peptide. The candidate pharmacological agents can be derived from, for example, combinatorial peptide libraries. Convenient reagents for such assays are known in the art. An exemplary cell-based assay involves contacting a neuronal cell with a mixture of .beta.-amyloid peptide and a candidate pharmacological agent. A reduction in the induction of calcium influx by resulting .beta.-amyloid peptide aggregates indicates that the candidate pharmacological agent disrupts .beta.-amyloid peptide aggregate formation or reduces the sensitivity of calcium channels to .beta.-amyloid peptide aggregates. Methods for determining changes in the intracellular calcium concentration are known in the art and are addressed elsewhere herein.

.beta.-amyloid peptides used in the methods of the invention are added to an assay mixture as an isolated peptide. .beta.-amyloid peptides can be produced recombinantly, or isolated from biological extracts, but preferably are synthesized in vitro. .beta.-amyloid peptides encompass chimeric proteins comprising a fusion of a .beta.-amyloid peptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, or enhancing stability of the .beta.-amyloid peptide under assay conditions. A polypeptide fused to a .beta.-amyloid peptide or fragment may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.

The assay mixture includes a .beta.-amyloid peptide, such as A.beta.1-42, A.beta.1-40, and A.beta.25-35 and can include a decoy peptide as described herein.

The assay mixture also comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate agents encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500. Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the agent is a nucleic acid, the agent typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.

A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.

The mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the .beta.-amyloid peptide forms aggregates and specifically binds the cellular binding target and induces neuronal calcium influx, and/or induces membrane depolarization. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4.degree. C. and 40.degree. C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.

After incubation, the presence or absence of specific binding between the .beta.-amyloid peptide and one or more binding partners is detected by any convenient method available to the user. For cell free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assays such as a calcium influx assay. The calcium influx resulting from .beta.-amyloid peptide aggregation and binding to a target molecule typically alters a directly or indirectly detectable product, e.g., a calcium sensitive molecule such as fura-2-AM. For cell free binding assays, one of the components usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.), or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). The label may be bound to a .beta.-amyloid peptide, decoy peptide or the candidate pharmacological agent.

A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, streptavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.

Thus the present invention includes automated drug screening assays for identifying compositions having the ability to inhibit ion influx in a cell induced by A.beta. aggregates, thus contributing to a detectable change in the cytoplasmic level of a predetermined ion in the cell, the cytoplasm of which cell contains an indicator which is sensitive to the ion. The method is carried out in an apparatus which is capable of delivering a reagent solution to a plurality of predetermined cell-containing compartments of a vessel and measuring the detectable change in the cytoplasmic level of the ion in the cells of the predetermined compartments, such as the apparatus and method described in U.S. Pat. No. 6,057,114. Exemplary methods include the following steps. First, a divided culture vessel is provided that has one or more compartments which contain viable cells which, when exposed to A.beta. aggregates, have a detectable change in the concentration of the predetermined ion in the cytoplasm. The cytoplasms of the cells include an amount of an ion-sensitive fluorescent indicator sufficient to detect a change, if any, in the concentration of the predetermined ion. A.beta. aggregates are added to the cells to induce calcium influx and/or depolarization. Next, one or more predetermined cell-containing compartments are aligned with a predetermined position (e.g., aligned with a fluid outlet of an automatic pipette) and an aliquot of a solution containing a compound or mixture of compounds being tested for its ability to modulate A.beta. fibril-induced calcium influx and/or depolarization is delivered to the predetermined compartment(s) with an automatic pipette. Finally, fluorescence emitted by the ion-sensitive indicator in response to an excitation wavelength is measured for a predetermined amount of time, preferably by aligning said cell-containing compartment with a fluorescence detector. Preferably, fluorescence also measured prior to adding A.beta. aggregates to the cells and/or prior to adding the compound to the wells, to establish e.g., background and/or baseline values for fluorescence.

In accordance with the various assays of the present invention, cells are employed which have ion channels and/or receptors, the activation of which by aggregated A.beta. peptides (i.e., A.beta. aggregates or fibrils) results in a change in the level of a cation or anion in the cytoplasm. The cytoplasm of the cells employed are loaded with a fluorescent indicator which is sufficiently sensitive to said ion. By the phrase "sufficiently sensitive fluorescent indicator" is meant a fluorescent compound which, in the presence of, and over a range of physiological concentrations of, a particular ion, is capable of producing distinguishable levels of fluorescence intensity. Preferably, a fluorescent indicator should be able to produce detectably different intensities of fluorescence in response to relatively small changes in ion concentration. The relative intensities of fluorescence when the receptors or ion channels have not been activated, as compared to when the receptors or ion channels have been activated, preferably differ by at least about 50% or more, more preferably by at least about 100 200%.

Any cell which is capable, upon exposure to A.beta. aggregates, of directly increasing the intracellular concentration of calcium, such as by permitting calcium influx through calcium channels or ion pores formed in accordance with the ionophore properties of A.beta. aggregates, or by causing release of calcium from intracellular stores, may be used in the assay. Preferably neuronal cell lines or cultured neurons are used. Such cells include, but are not limited to, the hNT neuronal cells used in the Examples.

Activation of cellular receptors and/or ion channels (e.g., AMPA/kainate-type channels) by incubation with A.beta. aggregates and/or ionophore formation by A.beta. aggregates, may result in a transient increase in the level of intracellular calcium (and/or other ions). The initial increase in calcium may be detected as a rapid increase in fluorescence (e.g., within one to two seconds) after the addition of the A.beta. aggregates. As shown herein, calcium influx is generally short-lived, but depolarization is longer lasting. Fluorescence levels in the cytoplasm resulting from calcium influx typically increase to a peak value and then typically decline as excess calcium ions are removed by normal cellular mechanisms. Fluorescence due to depolarization after A.beta. fibril exposure rapidly increases to a plateau value, and remains at this plateau. The speed at which the fluorescence can be analyzed is important for analysis of the kinetics of the reaction, if it is desired to measure kinetics.

The cells used in the assays of the invention are loaded with a fluorescent indicator which is sufficiently sensitive so as to produce detectable changes in fluorescence intensity in response to changes in the concentration of the ions in the cytoplasm. It is particularly preferred to use a fluorescent indicator which has such sensitivity in the presence of calcium ions, although indicators sensitive to other ions such as sodium ions, potassium ions, chloride ions, and the like may be employed depending on the type of ion flux induced by the A.beta. aggregates, as will be understood by the person of ordinary skill in the art. Among the fluorescent indicators which may be employed are the following compounds commercially available from, e.g., Molecular Probes, Inc., Eugene Oreg.: DiBAC.sub.4(3) (B-438), Quin-2 (AM Q-1288), Fura-2 (AM F-1225), Indo-1 (AM 1-1226), Fura-3 (AM F-1228), Fluo-3 (AM F-1241), Rhod-2, (AM R-1244), BAPTA (AM B-1205), 5,5'-dimethyl BAPTA (AM D-1207), 4,41-difluoro BAPTA (AM D-1216), 5,5'-difluoro BAPTA (AM D-1209), 5,5'-dibromo BAPTA (AM D-1213), Calcium Green (C-3011), Calcium Orange (C-3014), Calcium Crimson (C-3017), Fura-5 (F-3023), Fura-Red (F-3020), SBFI (S-1262), PBFI (P-1265), Mag-Fura-2 (AM M-1291), Mag-Indo-1 (AM M-1294), Mag-Quin-2 (AM M-1299), Mag-Quin -1 (AM M-1297), SPQ (M-440), and SPA (S-460).

It is contemplated that each of the individual wells contain the same cell type so that multiple compounds (obtained from different reagent sources in the apparatus or contained within different wells) can be screened and compared for modulating activity with respect to A.beta. fibril-induced calcium influx and/or depolarization.

In another of its aspects the invention entails automated antagonist assays. Antagonist assays, including drug screening assays, may be carried out by incubating the cells (e.g., neurons) with A.beta. aggregates to induce calcium influx and/or depolarization, in the presence and absence of one or more compounds added to the solution bathing the cells in the respective wells of the microtiter plate for an amount of time sufficient for the compound(s) to modulate calcium influx and/or depolarization, and measuring the level of fluorescence in the cells as compared to the level of fluorescence in either the same cell, or substantially identical cell, in the absence of the A.beta. aggregates.

As will be understood by the person of ordinary skill in the art, compounds exhibiting agonist or antagonist activity in an assay of calcium influx or depolarization will either increase or decrease intracellular ion levels (agonist) or inhibit (antagonist) an increase or decrease in the intracellular concentration of ions after incubation of cells with A.beta. aggregates. It is desirable to measure the amount of agonist or antagonist activity in a linear range of the assay system, such that small but significant increases or decreases in fluorescence relative to control well (e.g., devoid of the test compound) may be observed. It is well within the skill of the art to determine a volume and concentration of a reagent solution which causes a suitable activation response in cells so that modulation of the calcium influx and/or depolarization may be reliably detected.

At a suitable time after addition of the A.beta. aggregates to initiate calcium influx and/or depolarization, the plate is moved, if necessary, so that the cell-containing assay well is positioned for measurement of fluorescence emission. Because a change in the fluorescence signal may begin within the first few seconds after addition of test compounds, it is desirable to align the assay well with the fluorescence reading device as quickly as possible, with times of about two seconds or less being desirable. In preferred embodiments of the invention, where the apparatus is configured for detection through the bottom of the well(s) and compounds are added from above the well(s), fluorescence readings may be taken substantially continuously, since the plate does not need to be moved for addition of reagent. The well and fluorescence-reading device should remain aligned for a predetermined period of time suitable to measure and record the change in intracellular ion, e.g., calcium, concentration. In preferred embodiments of the invention the fluorescence after activation is read and recorded until the fluorescence change is maximal and then begins to reduce. An empirically determined time period may be chosen which covers the transient rise and fall (or fall and rise) of intracellular ion levels in response to addition of the compound. When the apparatus is configured to detect fluorescence from above the plate, it is preferred that the bottom of the wells are colored black to reduce the background fluorescence and thereby decreases the noise level in the fluorescence reader.

After finishing reading and recording the fluorescence in one well, the just described apparatus steps are repeated with the next well(s) in the series so as to measure pre-reagent fluorescence, add reagent and measure and record the transient change, if any, in fluorescence. The apparatus of the present invention is programmable to begin the steps of an assay sequence in a predetermined first well (or row or column of wells) and proceed sequentially down the columns and across the rows of the plate in a predetermined route through well number n.

In assays of cells treated with A.beta. aggregates to cause an increase in intracellular calcium ion concentration and/or depolarization, it is preferred that the fluorescence data from replicate wells of cells treated with the same compound are collected and recorded (e.g., stored in the memory of a computer) for calculation of fluorescence and/or intracellular calcium ion concentration.

In assays of compounds that inhibit calcium influx and/or depolarization, the results can be expressed as a percentage of the maximal response caused by A.beta. aggregates (e.g., A.beta.1-42 aggr.). The maximal fluorescence increase caused by A.beta. aggregates is defined as being 100% response. For compounds effective for reducing calcium influx and/or depolarization induced by A.beta. aggregates, the maximal fluorescence recorded after addition of a compound to wells containing A.beta. aggregates is detectably lower than the fluorescence recorded in the presence of only A.beta. aggregates.

The fluorescence indicator-based assays of the present invention are thus useful for rapidly screening compounds to identify those that modulate calcium influx and/or depolarization that ultimately results in an altered concentration of ions in the cytoplasm of a cell. For example, the assays can be used to test functional ligand interactions with A.beta. aggregates or ligand competition with decoy peptide binding of A.beta. aggregates.

Automation of the fluorescent dye-based assays of the invention can be performed as described in U.S. Pat. No. 6, 057,114. Automation can provide increased efficiency in conducting the assays and increased reliability of the results by permitting multiple measurements over time, thus also facilitating determination of the kinetics of the calcium influx or depolarization effects.

For example, to accomplish rapid compound addition and rapid reading of the fluorescence response, the fluorometer can be modified by fitting an automatic pipetter and developing a software program to accomplish precise computer control over both the fluorometer and the automatic pipetter. By integrating the combination of the fluorometer and the automatic pipetter and using a microcomputer to control the commands to the fluorometer and automatic pipetter, the delay time between reagent addition and fluorescence reading can be significantly reduced. Moreover, both greater reproducibility and higher signal-to-noise ratios can be achieved as compared to manual addition of reagent because the computer repeats the process precisely time after time. Moreover, this arrangement permits a plurality of assays to be conducted concurrently without operator intervention. Thus, with automatic delivery of reagent followed by multiple fluorescence measurements, reliability of the fluorescent dye-based assays as well as the number of assays that can be performed per day are advantageously increased.

The invention, in one aspect, identifies compounds which reduce the increased neuronal cell membrane depolarization induced by the presence of .beta.-amyloid peptide aggregates, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. These compounds are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving .beta.-amyloid peptide, e.g., .beta.-amyloid peptide aggregation, neuronal membrane depolarization associated with neurotoxic .beta.-amyloid peptide aggregates, etc.

Compounds which antagonize the formation of neurotoxic .beta.-amyloid peptide aggregates or which inhibit calcium influx and/or membrane depolarization may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the compounds in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the therapeutic compound in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

When used therapeutically, the compounds of the invention are administered in therapeutically effective amounts. In general, a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated. Therapeutically effective amounts specifically will be those which desirably influence the existence or formation of aggregates of .beta.-amyloid peptides that induce calcium influx in neuronal cells, and/or desirably influence the cytotoxic effects of such aggregates. Generally, a therapeutically effective amount will vary with the subject's age, and condition, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intracranial, intraperitoneal, intramuscular, intracavity, intrarespiratory, subcutaneous, or transdermal. The route of administration will depend on the composition of a particular therapeutic preparation of the invention.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active compounds of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

A long-term sustained release implant also may be used. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Such implants can be particularly useful in treating conditions characterized by aggregates of .beta.-amyloid peptides by placing the implant near portions of the brain affected by such aggregates, thereby effecting localized, high doses of the compounds of the invention.
 

Claim 1 of 3 Claims

1. A composition comprising DAPH1 (4,5-dianilinophthalimide), and one or more non-NMDA channel antagonists.
 

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