Compounds that modulate the aggregation of amyloidogenic proteins or peptides are disclosed. The modulators of the invention can promote amyloid aggregation or, more preferably, can inhibit natural amyloid aggregation. In a preferred embodiment, the compounds modulate the aggregation of natural .beta. amyloid peptides (.beta.-AP). In a preferred embodiment, the .beta. amyloid modulator compounds of the invention are comprised of an A.beta. aggregation core domain and a modifying group coupled thereto such that the compound alters the aggregation or inhibits the neurotoxicity of natural .beta. amyloid peptides when contacted with the peptides. Furthermore, the modulators are capable of altering natural .beta.-AP aggregation when the natural .beta.-APs are in a molar excess amount relative to the modulators. Pharmaceutical compositions comprising the compounds of the invention, and diagnostic and treatment methods for amyloidogenic diseases using the compounds of the invention, are also disclosed.

Description of the Invention

This invention pertains to compounds, and pharmaceutical compositions thereof, that can modulate the aggregation of amyloidogenic proteins and peptides, in particular compounds that can modulate the aggregation of natural .beta. amyloid peptides (.beta.-AP) and inhibit the neurotoxicity of natural .beta.-APs. A compound of the invention that modulates aggregation of natural .beta.-AP, referred to herein interchangeably as a .beta. amyloid modulator compound, a .beta. amyloid modulator or simply a modulator, alters the aggregation of natural .beta.-AP when the modulator is contacted with natural .beta.-AP. Thus, a compound of the invention acts to alter the natural aggregation process or rate for .beta.-AP, thereby disrupting this process. Preferably, the compounds inhibit .beta.-AP aggregation. Furthermore, the invention provides subregions of the .beta. amyloid peptide that are sufficient, when appropriately modified as described herein, to alter (and preferably inhibit) aggregation of natural .beta. amyloid peptides when contacted with the natural .beta. amyloid peptides. In particular, preferred modulator compounds of the invention are comprised of a modified form of an A.beta. aggregation core domain, modeled after the aforementioned A.beta. subregion (as described further below), which is sufficient to alter (and preferably inhibit) the natural aggregation process or rate for .beta.-AP. This A.beta. aggregation core domain can comprises as few as three amino acid residues (or derivative, analogues or mimetics thereof). Moreover, while the amino acid sequence of the A.beta. aggregation core domain can directly correspond to an amino acid sequence found in natural .beta.-AP, it is not essential that the amino acid sequence directly correspond to a .beta.-AP sequence. Rather, amino acid residues derived from a preferred subregion of .beta.-AP (a hydrophobic region centered around positions 17-20) can be rearranged in order and/or substituted with homologous residues within a modulator compound of the invention and yet maintain their inhibitory activity (described further below).

The .beta. amyloid modulator compounds of the invention can be selected based upon their ability to inhibit the aggregation of natural .beta.-AP in vitro and/or inhibit the neurotoxicity of natural .beta.-AP fibrils for cultured cells (using assays described herein). Accordingly, the preferred modulator compounds inhibit the aggregation of natural .beta.-AP and/or inhibit the neurotoxicity of natural .beta.-AP. However, modulator compounds selected based on one or both of these properties may have additional properties in vivo that may be beneficial in the treatment of amyloidosis. For example, the modulator compound may interfere with processing of natural .beta.-AP (either by direct or indirect protease inhibition) or by modulation of processes that produce toxic .beta.-AP, or other APP fragments, in vivo. Alternatively, modulator compounds may be selected based on these latter properties, rather than inhibition of A.beta. aggregation in vitro. Moreover, modulator compounds of the invention that are selected based upon their interaction with natural .beta.-AP also may interact with APP or with other APP fragments.

As used herein, a "modulator" of .beta.-amyloid aggregation is intended to refer to an agent that, when contacted with natural .beta. amyloid peptides, alters the aggregation of the natural .beta. amyloid peptides. The term "aggregation of .beta. amyloid peptides" refers to a process whereby the peptides associate with each other to form a multimeric, largely insoluble complex. The term "aggregation" further is intended to encompass .beta. amyloid fibril formation and also encompasses .beta.-amyloid plaques.

The terms "natural .beta.-amyloid peptide", "natural .beta.-AP" and "natural A.beta. peptide", used interchangeably herein, are intended to encompass naturally occurring proteolytic cleavage products of the .beta. amyloid precursor protein (APP) which are involved in .beta.-AP aggregation and .beta.-amyloidosis. These natural peptides include .beta.-amyloid peptides having 39-43 amino acids (i.e., A.beta..sub.1-39, A.beta..sub.1-40, A.beta..sub.1-41, A.beta..sub.1-42 and A.beta..sub.1-43). The amino-terminal amino acid residue of natural .beta.-AP corresponds to the aspartic acid residue at position 672 of the 770 amino acid residue form of the amyloid precursor protein ("APP-770"). The 43 amino acid long form of natural .beta.-AP has the amino acid sequence

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

(also shown in SEQ ID NO: 1), whereas the shorter forms have 1-4 amino acid residues deleted from the carboxy-terminal end. The amino acid sequence of APP-770 from position 672 (i.e., the amino-terminus of natural .beta.-AP) to its C-terminal end (103 amino acids) is shown in SEQ ID NO: 2. The preferred form of natural .beta.-AP for use in the aggregation assays described herein is A.beta..sub.1-40.

In the presence of a modulator of the invention, aggregation of natural .beta. amyloid peptides is "altered" or "modulated". The various forms of the term "alteration" or "modulation" are intended to encompass both inhibition of .beta.-AP aggregation and promotion of .beta.-AP aggregation. Aggregation of natural .beta.-AP is "inhibited" in the presence of the modulator when there is a decrease in the amount and/or rate of .beta.-AP aggregation as compared to the amount and/or rate of .beta.-AP aggregation in the absence of the modulator. The various forms of the term "inhibition" are intended to include both complete and partial inhibition of .beta.-AP aggregation. Inhibition of aggregation can be quantitated as the fold increase in the lag time for aggregation or as the decrease in the overall plateau level of aggregation (i.e., total amount of aggregation), using an aggregation assay as described in the Examples. In various embodiments, a modulator of the invention increases the lag time of aggregation at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold or 5-fold. In various other embodiments, a modulator of the invention inhibits the plateau level of aggregation at least 10%, 20%, 30%, 40%, 50%, 75% or 100%.

A modulator which inhibits .beta.-AP aggregation (an "inhibitory modulator compound") can be used to prevent or delay the onset of .beta.-amyloid deposition. Moreover, as demonstrated in Example 10, inhibitory modulator compounds of the invention inhibit the formation and/or activity of neurotoxic aggregates of natural A.beta. peptide (i.e., the inhibitory compounds can be used to inhibit the neurotoxicity of .beta.-AP). Still further, also as demonstrated in Example 10, the inhibitory compounds of the invention can be used to reduce the neurotoxicity of preformed .beta.-AP aggregates, indicating that the inhibitory modulators can either bind to preformed A.beta. fibrils or soluble aggregate and modulate their inherent neurotoxicity or that the modulators can perturb the equilibrium between monomeric and aggregated forms of .beta.-AP in favor of the non-neurotoxic form.

Alternatively, in another embodiment, a modulator compound of the invention promotes the aggregation of natural A.beta. peptides. The various forms of the term "promotion" refer to an increase in the amount and/or rate of .beta.-AP aggregation in the presence of the modulator, as compared to the amount and/or rate of .beta.-AP aggregation in the absence of the modulator. Such a compound which promotes A.beta. aggregation is referred to as a stimulatory modulator compound. Stimulatory modulator compounds may be useful for sequestering .beta.-amyloid peptides, for example in a biological compartment where aggregation of .beta.-AP may not be deleterious to thereby deplete .beta.-AP from a biological compartment where aggregation of .beta.-AP is deleterious. Moreover, stimulatory modulator compounds can be used to promote A.beta. aggregation in in vitro aggregation assays (e.g., assays such as those described in the Examples), for example in screening assays for test compounds that can then inhibit or reverse this A.beta. aggregation (i.e., a stimulatory modulator compound can act as a "seed" to promote the formation of A.beta. aggregates).

In a preferred embodiment, the modulators of the invention are capable of altering .beta.-AP aggregation when contacted with a molar excess amount of natural .beta.-AP. A "molar excess amount of natural .beta.-AP" refers to a concentration of natural .beta.-AP, in moles, that is greater than the concentration, in moles, of the modulator. For example, if the modulator and .beta.-AP are both present at a concentration of 1 .mu.M, they are said to be "equimolar", whereas if the modulator is present at a concentration of 1 .mu.M and the .beta.-AP is present at a concentration of 5 .mu.M, the .beta.-AP is said to be present at a 5-fold molar excess amount compared to the modulator. In preferred embodiments, a modulator of the invention is effective at altering natural .beta.-AP aggregation when the natural .beta.-AP is present at at least a 2-fold, 3-fold or 5-fold molar excess compared to the concentration of the modulator. In other embodiments, the modulator is effective at altering .beta.-AP aggregation when the natural .beta.-AP is present at at least a 10-fold, 20-fold, 33-fold, 50-fold, 100-fold, 500-fold or 1000-fold molar excess compared to the concentration of the modulator.

Various additional aspects of the modulators of the invention, and the uses thereof, are described in further detail in the following subsections.

I. Modulator Compounds

In one embodiment, a modulator of the invention comprises a .beta.-amyloid peptide compound comprising the formula -- see Original Patent.

Preferably, .beta.-amyloid peptide of the compound has an amino-terminal amino acid residue corresponding to position 668 of .beta.-amyloid precursor protein-770 (APP-770) or to a residue carboxy-terminal to position 668 of APP-770. The amino acid sequence of APP-770 from position 668 to position 770 (i.e., the carboxy terminus) is shown below and in SEQ ID NO: 2 -- see Original Patent.

More preferably, the amino-terminal amino acid residue of the .beta.-amyloid peptide corresponds to position 672 of APP-770 (position 5 of the amino acid sequence of SEQ ID NO: 2) or to a residue carboxy-terminal to position 672 of APP-770. Although the .beta.-amyloid peptide of the compound may encompass the 103 amino acid residues corresponding to positions 668-770 of APP-770, preferably the peptide is between 6 and 60 amino acids in length, more preferably between 10 and 43 amino acids in length and even more preferably between 10 and 25 amino acid residues in length.

As used herein, the term ".beta. amyloid peptide", as used in a modulator of the invention is intended to encompass peptides having an amino acid sequence identical to that of the natural sequence in APP, as well as peptides having acceptable amino acid substitutions from the natural sequence. Acceptable amino acid substitutions are those that do not affect the ability of the peptide to alter natural .beta.-AP aggregation. Moreover, particular amino acid substitutions may further contribute to the ability of the peptide to alter natural .beta.-AP aggregation and/or may confer additional beneficial properties on the peptide (e.g., increased solubility, reduced association with other amyloid proteins, etc.). For example, substitution of hydrophobic amino acid residues for the two phenylalanine residues at positions 19 and 20 of natural .beta.-AP (positions 19 and 20 of the amino acid sequence shown in SEQ ID NO: 1) may further contribute to the ability of the peptide to alter .beta.-AP aggregation (see Hilbich, C. (1992) J. Mol. Biol. 228:460-473). Thus, in one embodiment, the M-AP of the compound consists of the amino acid sequence shown below and in SEQ ID NO: 3:

DAEFRHDSGYEVHHQKLV(Xaa.sub.19)(Xaa.sub.20)AEDVGSNKGAIIGLMVGGVVIAT

(or an amino-terminal or carboxy-terminal deletion thereof), wherein Xaa is a hydrophobic amino acid. Examples of hydrophobic amino acids are isoleucine, leucine, threonine, serine, alanine, valine or glycine. Preferably, F.sub.19F.sub.20 is substituted with T.sub.19T.sub.20 or G.sub.19I.sub.20.

Other suitable amino acid substitutions include replacement of amino acids in the human peptide with the corresponding amino acids of the rodent .beta.-AP peptide. The three amino acid residues that differ between human and rat .beta.-AP are at positions 5, 10 and 13 of the amino acid sequence shown in SEQ ID NOs: 1 and 3. A human .beta.-AP having the human to rodent substitutions Arg.sub.5 to Gly, Tyr.sub.10 to Phe and His.sub.13 to Arg has been shown to retain the properties of the human peptide (see Fraser, P. E. et al. (1992) Biochemistry 31:10716-10723; and Hilbich, C. et al. (1991) Eur. J. Biochem. 201:61-69). Accordingly, a human .beta.-AP having rodent .beta.-AP a.a. substitutions is suitable for use in a modulator of the invention.

Other possible .beta.-AP amino acid substitutions are described in Hilbich, C. et al. (1991) J. Mol. Biol. 218:149-163; and Hilbich, C. (1992) J. Mol. Biol. 228:460-473. Moreover, amino acid substitutions that affect the ability of .beta.-AP to associate with other proteins can be introduced. For example, one or more amino acid substitutions that reduce the ability of .beta.-AP to associate with the serpin enzyme complex (SEC) receptor, .alpha.1-antichymotrypsin (ACT) and/or apolipoprotein E (ApoE) can be introduced. A preferred substitution for reducing binding to the SEC receptor is L.sub.34M.sub.35 to A.sub.34A.sub.35 (at positions 34 and 35 of the amino acid sequences shown in SEQ ID NOs: 1 and 3). A preferred substitution for reducing binding to ACT is S.sub.8 to A.sub.8 (at position 8 of the amino acid sequences shown in SEQ ID NOs: 1 and 3).

Alternative to .beta.-AP amino acid substitutions described herein or known in the art, a modulator composed, at least in part, of an amino acid-substituted .beta. amyloid peptide can be prepared by standard techniques and tested for the ability to alter .beta.-AP aggregation using an aggregation assay described herein. To retain the properties of the original modulator, preferably conservative amino acid substitutions are made at one or more amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), .beta.-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, a modulator composed of a .beta. amyloid peptide having an amino acid sequence that is mutated from that of the wild-type sequence in APP-770 yet which still retains the ability to alter natural .beta.-AP aggregation is within the scope of the invention.

As used herein, the term ".beta. amyloid peptide" is further intended to include peptide analogues or peptide derivatives or peptidomimetics that retain the ability to alter natural .beta.-AP aggregation as described herein. For example, a .beta. amyloid peptide of a modulator of the invention may be modified to increase its stability, bioavailability, solubility, etc. The terms "peptide analogue", "peptide derivative" and "peptidomimetic" as used herein are intended to include molecules which mimic the chemical structure of a peptide and retain the functional properties of the peptide. Approaches to designing peptide analogs are known in the art. For example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues, derivatives and peptidomimetics include peptides substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942), peptides with methylated amide linkages and "retro-inverso" peptides (see U.S. Pat. No. 4,522,752 by Sisto). Peptide analogues, peptide derivatives and peptidomimetic are described in further detail below with regard to compounds comprising an A.beta. aggregation core domain.

In a modulator of the invention having the formula shown above, a modulating group ("A") is attached directly or indirectly to the .beta.-amyloid peptide of the modulator (As used herein, the term "modulating group" and "modifying group" are used interchangeably to describe a chemical group directly or indirectly attached to an A.beta. derived peptidic structure). For example, the modulating group can be directly attached by covalent coupling to the .beta.-amyloid peptide or the modulating group can be attached indirectly by a stable non-covalent association. In one embodiment of the invention, the modulating group is attached to the amino-terminus of the .beta.-amyloid peptide of the modulator. Accordingly, the modulator can comprise a compound having a formula:

##STR00005## Alternatively, in another embodiment of the invention, the modulating group is attached to the carboxy-terminus of the .beta.-amyloid peptide of the modulator. Accordingly, the modulator can comprise a compound having a formula:

##STR00006##

In yet another embodiment, the modulating group is attached to the side chain of at least one amino acid residue of the .beta.-amyloid peptide of the compound (e.g., through the epsilon amino group of a lysyl residue(s), through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s) or other suitable reactive group on an amino acid side chain).

The modulating group is selected such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. Accordingly, since the .beta.-AP peptide of the compound is modified from its natural state, the modulating group "A" as used herein is not intended to include hydrogen. In a preferred embodiment, the modulating group is a biotin compound of the formula -- see Original Patent.

The term "aryl" is intended to include aromatic moieties containing substituted or unsubstituted ring(s), e.g., benzyl, naphthyl etc. Other more complex fused ring moieties also are intended to be included.

The term "lower alkyl or alkylenyl moiety" refers to a saturated, straight or branched chain (or combination thereof) hydrocarbon containing 1 to about 6 carbon atoms, more preferably from 1 to 3 carbon atoms. The terms "lower alkenyl moiety" and "lower alkynyl moiety" refer to unsaturated hydrocarbons containing 1 to about 6 carbon atoms, more preferably 1 to 3 carbon atoms. Preferably, R.sub.2 contains 1 to 3 carbon atoms. Preferably, R.sub.1 contains 4 carbon atoms.

The spacer molecule (Y) can be, for example, a lower alkyl group or a linker peptide, and is preferably selected for its ability to link with a free amino group (e.g., the .alpha.-amino group at the amino-terminus of a .beta.-AP). Thus, in a preferred embodiment, the biotin compound modifies the amino-terminus of a .beta.-amyloid peptide.

Additional suitable modulating groups may include other cyclic and heterocyclic compounds and other compounds having similar steric "bulk". Non-limiting examples of compounds which can be used to modify a .beta.-AP are shown schematically in FIG. 2 (see Original Patent), and include N-acetylneuraminic acid, cholic acid, trans-4-cotininecarboxylic acid, 2-imino-1-imidazolidineacetic acid, (S)-(-)-indoline-2-carboxylic acid, (-)-menthoxyacetic acid, 2-norbornaneacetic acid, .gamma.-oxo-5-acenaphthenebutyric acid, (-)-2-oxo-4-thiazolidinecarboxylic acid, tetrahydro-3-furoic acid, 2-iminobiotin-N-hydroxysuccinimide ester, diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonyl chloride, 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride, 5-(and 6-)-carboxyfluorescein (succinimidyl ester), fluorescein isothiocyanate, and acetic acid (or derivatives thereof). Suitable modulating groups are described further in subsection II below.

In a modulator of the invention, a single modulating group may be attached to a amyloid peptide (e.g., n=1 in the formula shown above) or multiple modulating groups may be attached to the peptide. The number of modulating groups is selected such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. However, n preferably is an integer between 1 and 60, more preferably between 1 and 30 and even more preferably between 1 and 10 or 1 and 5.

In another embodiment, a .beta.-amyloid modulator compound of the invention comprises an A.beta. aggregation core domain (abbreviated as ACD) coupled directly or indirectly to a modifying group such that the compound modulates the aggregation or inhibits the neurotoxicity of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. As used herein, an "A.beta. aggregation core domain" is intended to refer to a structure that is modeled after a subregion of a natural .beta.-amyloid peptide which is sufficient to modulate aggregation of natural .beta.-APs when this subregion of the natural G-AP is appropriately modified as described herein (e.g., modified at the amino-terminus). The term "subregion of a natural .beta.-amyloid peptide" is intended to include amino-terminal and/or carboxy-terminal deletions of natural G-AP. The term "subregion of natural .beta.-AP" is not intended to include full-length natural G-AP (i.e., "subregion" does not include A.beta..sub.1-39, A.beta..sub.1-40, A.beta..sub.1-41, A.beta..sub.1-42 and A.beta..sub.1-43).

Although not intending to be limited by mechanism, the ACD of the modulators of the invention is thought to confer a specific targeting function on the compound that allows the compound to recognize and specifically interact with natural .beta.-AP. Preferably, the ACD is modeled after a subregion of natural .beta.-AP that is less than 15 amino acids in length and more preferably is between 3-10 amino acids in length. In various embodiments, the ACD is modeled after a subregion of .beta.-AP that is 10, 9, 8, 7, 6, 5, 4 or 3 amino acids in length. In one embodiment, the subregion of .beta.-AP upon which the ACD is modeled is an internal or carboxy-terminal region of .beta.-AP (i.e., downstream of the amino-terminus at amino acid position 1). In another embodiment, the ACD is modeled after a subregion of .beta.-AP that is hydrophobic. In certain specific embodiments, the term A.beta. aggregation core domain specifically excludes .beta.-AP subregions corresponding to amino acid positions 1-15 (A.beta..sub.1-15), 6-20 (A.beta..sub.6-20) and 16-40 (A.beta..sub.16-40).

An A.beta. aggregation core domain can be comprised of amino acid residues linked by peptide bonds. That is, the ACD can be a peptide corresponding to a subregion of .beta.-AP. Alternatively, an A.beta. aggregation core domain can be modeled after the natural A.beta. peptide region but may be comprised of a peptide analogue, peptide derivative or peptidomimetic compound, or other similar compounds which mimics the structure and function of the natural peptide. Accordingly, as used herein, an "A.beta. aggregation core domain" is intended to include peptides, peptide analogues, peptide derivatives and peptidomimetic compounds which, when appropriately modified, retain the aggregation modulatory activity of the modified natural A.beta. peptide subregion. Such structures that are designed based upon the amino acid sequence are referred to herein as "A.beta. derived peptidic structures." Approaches to designing peptide analogues, derivatives and mimetics are known in the art. For example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. See also Sawyer, T. K. (1995) "Peptidomimetic Design and Chemical Approaches to Peptide Metabolism" in Taylor, M. D. and Amidon, G. L. (eds.) Peptide-Based Drug Design: Controlling Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995) J. Am. Chem. Soc. 117:11113-11123; Smith, A. B. 3rd, et al. (1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993) J. Am. Chem. Soc. 115:12550-12568.

As used herein, a "derivative" of a compound X (e.g., a peptide or amino acid) refers to a form of X in which one or more reaction groups on the compound have been derivatized with a substituent group. Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatized (e.g., peptidic compounds with methylated amide linkages). As used herein an "analogue" of a compound X refers to a compound which retains chemical structures of X necessary for functional activity of X yet which also contains certain chemical structures which differ from X. An examples of an analogue of a naturally-occurring peptide is a peptides which includes one or more non-naturally-occurring amino acids. As used herein, a "mimetic" of a compound X refers to a compound in which chemical structures of X necessary for functional activity of X have been replaced with other chemical structures which mimic the conformation of X. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942), peptides in which all L-amino acids are substituted with the corresponding D-amino acids and "retro-inverso" peptides (see U.S. Pat. No. 4,522,752 by Sisto), described further below.

The term mimetic, and in particular, peptidomimetic, is intended to include isosteres. The term "isostere" as used herein is intended to include a chemical structure that can be substituted for a second chemical structure because the steric conformation of the first structure fits a binding site specific for the second structure. The term specifically includes peptide back-bone modifications (i.e., amide bond mimetics) well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the .alpha.-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including .psi.[CH.sub.2S], .psi.[CH.sub.2NH], .psi.[CSNH.sub.2], .psi.[NHCO], .psi.[COCH.sub.2], and .psi.[(E) or (Z) CH.dbd.CH]. In the nomenclature used above, .psi. indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets. Other examples of isosteres include peptides substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942)

Other possible modifications include an N-alkyl (or aryl) substitution (.psi.[CONR]), backbone crosslinking to construct lactams and other cyclic structures, substitution of all D-amino acids for all L-amino acids within the compound ("inverso" compounds) or retro-inverso amino acid incorporation (.psi.[NHCO]). By "inverso" is meant replacing L-amino acids of a sequence with D-amino acids, and by "retro-inverso" or "enantio-retro" is meant reversing the sequence of the amino acids ("retro") and replacing the L-amino acids with D-amino acids. For example, if the parent peptide is Thr-Ala-Tyr, the retro modified form is Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-inverso form is tyr-ala-thr (lower case letters refer to D-amino acids). Compared to the parent peptide, a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide. See Goodman et al. "Perspectives in Peptide Chemistry" pp. 283-294 (1981). See also U.S. Pat. No. 4,522,752 by Sisto for further description of "retro-inverso" peptides.

Other derivatives of the modulator compounds of the invention include C-terminal hydroxymethyl derivatives, O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides and compounds in which a C-terminal phenylalanine residue is replaced with a phenethylamide analogue (e.g., Val-Phe-phenethylamide as an analogue of the tripeptide Val-Phe-Phe).

In a preferred embodiment, the ACD of the modulator is modeled after the subregion of .beta.-AP encompassing amino acid positions 17-20 (i.e., Leu-Val-Phe-Phe; SEQ ID NO: 12). As described further in Examples 7, 8 and 9, peptide subregions of A.beta..sub.1-40 were prepared, amino-terminally modified and evaluated for their ability to modulate aggregation of natural .beta.-amyloid peptides. One subregion that was effective at inhibiting aggregation was A.beta..sub.6-20 (i.e., amino acid residues 6-20 of the natural A.beta..sub.1-40 peptide, the amino acid sequence of which is shown in SEQ ID NO: 4). Amino acid residues were serially deleted from the amino-terminus or carboxy terminus of this subregion to further delineate a minimal subregion that was sufficient for aggregation inhibitory activity. This process defined A.beta..sub.17-20 (i.e., amino acid residues 17-20 of the natural A.beta..sub.1-40 peptide) as a minimal subregion that, when appropriately modified, is sufficient for aggregation inhibitory activity. Accordingly, an "A.beta. aggregation core domain" within a modulator compound of the invention can be modeled after A.beta..sub.17-20. In one embodiment, the A.beta. aggregation core domain comprises A.beta..sub.17-20 itself (i.e., a peptide comprising the amino acid sequence leucine-valine-phenylalanine-phenylalanine; SEQ ID NO: 12). In other embodiments, the structure of A.beta..sub.17-20 is used as a model to design an A.beta. aggregation core domain having similar structure and function to A.beta..sub.17-20. For example, peptidomimetics, derivatives or analogues of A.beta..sub.17-20 (as described above) can be used as an A.beta. aggregation core domain. In addition to A.beta..sub.17-20, the natural A.beta. peptide is likely to contain other minimal subregions that are sufficient for aggregation inhibitory activity. Such additional minimal subregions can be identified by the processes described in Examples 7, 8 and 9, wherein a 15mer subregion of A.beta..sub.1-40 is serially deleted from the amino-terminus or carboxy terminus, the deleted peptides are appropriately modified and then evaluated for aggregation inhibitory activity.

One form of the .beta.-amyloid modulator compound comprising an A.beta. aggregation core domain modeled after A.beta..sub.17-20 coupled directly or indirectly to at least one modifying group has the formula -- see Original Patent.

Preferably, a modulator compound of the above formula inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides and/or inhibits A.beta. neurotoxicity. Alternatively, the modulator compound can promote aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. The type and number of modifying groups ("A") coupled to the modulator are selected such that the compound alters (and preferably inhibits) aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. A single modifying group can be coupled to the modulator (i.e., n=1 in the above formula) or, alternatively, multiple modifying groups can be coupled to the modulator. In various embodiments, n is an integer between 1 and 60, between 1 and 30, between 1 and 10, between 1 and 5 or between 1 and 3. Suitable types of modifying groups are described further in subsection II below.

As demonstrated in Example 9, amino acid positions 18 (Val.sub.18) and 20 (Phe.sub.20) of A.beta..sub.17-20 (corresponding to Xaa.sub.2 and Xaa.sub.4) are particularly important within the core domain for inhibitory activity of the modulator compound. Accordingly, these positions are conserved within the core domain in the formula shown above. The terms "valine structure" and "phenylalanine structure" as used in the above formula are intended to include the natural amino acids, as well as non-naturally-occurring analogues, derivatives and mimetics of valine and phenylalanine, respectively, (including D-amino acids) which maintain the functional activity of the compound. Moreover, although Val.sub.18 and Phe.sub.20 have an important functional role, it is possible that Xaa.sub.2 and/or Xaa.sub.4 can be substituted with other naturally-occurring amino acids that are structurally related to valine or phenylalanine, respectively, while still maintaining the activity of the compound. Thus, the terms "valine structure" is intended to include conservative amino acid substitutions that retain the activity of valine at Xaa.sub.2, and the term "phenylalanine structure" is intended to include conservative amino acid substitutions that retain the activity of phenylalanine at Xaa.sub.4. However, the term "valine structure" is not intended to include threonine.

In contrast to positions 18 and 20 of A.beta.17-20, a Phe to Ala substitution at position 19 (corresponding to Xaa.sub.3) did not abolish the activity of the modulator, indicating position 19 may be more amenable to amino acid substitution. In various embodiments of the above formula, positions Xaa.sub.1 and Xaa.sub.3 are any amino acid structure. The term "amino acid structure" is intended to include natural and non-natural amino acids as well as analogues, derivatives and mimetics thereof, including D-amino acids. In a preferred embodiment of the above formula, Xaa.sub.1 is a leucine structure and Xaa.sub.3 is a phenylalanine structure (i.e., modeled after Leu.sub.17 and Phe.sub.19, respectively, in the natural A.beta. peptide sequence). The term "leucine structure" is used in the same manner as valine structure and phenylalanine structure described above. Alternatively, an another embodiment, Xaa.sub.3 is an alanine structure.

The four amino acid structure ACD of the modulator of the above formula can be flanked at the amino-terminal side, carboxy-terminal side, or both, by peptidic structures derived either from the natural A.beta. peptide sequence or from non-A.beta. sequences. The term "peptidic structure" is intended to include peptide analogues, derivatives and mimetics thereof, as described above. The peptidic structure is composed of one or more linked amino acid structures, the type and number of which in the above formula are variable. For example, in one embodiment, no additional amino acid structures flank the Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 core sequence (i.e., Y and Z are absent in the above formula). In another embodiment, one or more additional amino acid structures flank only the amino-terminus of the core sequences (i.e., Y is present but Z is absent in the above formula). In yet another embodiment, one or more additional amino acid structures flank only the carboxy-terminus of the core sequences (i.e., Z is present but Y is absent in the above formula). The length of flanking Z or Y sequences also is variable. For example, in one embodiment, a and b are integers from 1 to 15. More preferably, a and b are integers between 1 and 10. Even more preferably, a and b are integers between 1 and 5. Most preferably, a and b are integers between 1 and 3.

One form of the .beta.-amyloid modulator compound comprising an A.beta. aggregation core domain modeled after A.beta..sub.17-20 coupled directly or indirectly to at least one modifying group has the formula: A--(Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4--(Z)--B wherein Xaa.sub.1 and Xaa.sub.3 are amino acids or amino acid mimetics; Xaa.sub.2 is valine or a valine mimetic Xaa.sub.4 is phenylalanine or a phenylalanine mimetic; Y, which may or may not be present, is a peptide or peptidomimetic having the formula (Xaa).sub.a, wherein Xaa is any amino acid or amino acid mimetic and a is an integer from 1 to 15; Z, which may or may not be present, is a peptide or peptidomimetic having the formula (Xaa).sub.b, wherein Xaa is any amino acid or amino acid mimetic and b is an integer from 1 to 15; and A and B, at least one of which is present, are modifying groups attached directly or indirectly to the amino terminus and carboxy terminus, respectively, of the compound; Xaa.sub.1, Xaa.sub.3, Y, Z, A and B being selected such that the compound modulates the aggregation or inhibits the neurotoxicity of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides.

In this embodiment, the modulator compound is specifically modified at either its amino-terminus, its carboxy-terminus, or both. The terminology used in this formula is the same as described above. Suitable modifying groups are described in subsection II below. In one embodiment, the compound is modified only at its amino terminus (i.e., B is absent and the compound comprises the formula: A--(Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4--(Z)). In another embodiment, the compound is modified only at its carboxy-terminus (i.e., A is absent and the compound comprises the formula: (Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4--(Z)--B). In yet another embodiment, the compound is modified at both its amino- and carboxy termini (i.e., the compound comprises the formula: A--(Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4--(Z)--B and both A and B are present). As described above, the type and number of amino acid structures which flank the Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 core sequences in the above formula is variable. For example, in one embodiment, a and b are integers from 1 to 15. More preferably, a and b are integers between 1 and 10. Even more preferably, a and b are integers between 1 and 5. Most preferably, a and b are integers between 1 and 3.

As demonstrated in Examples 7, 8 and 9, preferred A.beta. modulator compounds of the invention comprise modified forms of A.beta..sub.14-21 (His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5), or amino-terminal or carboxy-terminal deletions thereof, with a preferred "minimal core region" comprising A.beta..sub.17-20. Accordingly, in specific embodiments, the invention provides compounds comprising the formula: A--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-- Xaa.sub.8--B wherein Xaa1 is a histidine structure; Xaa2 is a glutamine structure; Xaa3 is a lysine structure; Xaa4 is a leucine structure; Xaa5 is a valine structure; Xaa6 is a phenylalanine structure; Xaa7 is a phenylalanine structure; Xaa8 is an alanine structure; A and B are modifying groups attached directly or indirectly to the amino terminus and carboxy terminus, respectively, of the compound; and wherein Xaa.sub.1-Xaa.sub.2-Xaa.sub.3, Xaa.sub.1-Xaa.sub.2 or Xaa.sub.1 may or may not be present; Xaa.sub.8 may or may not be present; and at least one of A and B is present.

In one specific embodiment, the compound comprises the formula: A--Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7--B (e.g, a modified form of A.beta..sub.17-20, comprising an amino acid sequence Leu-Val-Phe-Phe; SEQ ID NO: 12).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8--B (e.g, a modified form of A.beta..sub.17-21, comprising an amino acid sequence Leu-Val-Phe-Phe-Ala; SEQ ID NO: 11).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7--B (e.g., a modified form of A.beta..sub.16-20, comprising an amino acid sequence Lys-Leu-Val-Phe-Phe; SEQ ID NO: 10).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8--B (e.g., a modified form of A.beta..sub.16-21, comprising an amino acid sequence Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 9).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7--B (e.g., a modified form of A.beta..sub.15-20, comprising an amino acid sequence Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 8).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-- -B (e.g., a modified form of A.beta..sub.15-21, comprising an amino acid sequence Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 7).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-- -B (e.g., a modified form of A.beta..sub.14-20, comprising an amino acid sequence His-Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 6).

In another specific embodiment, the compound comprises the formula: A--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-- Xaa.sub.8--B (e.g., a modified form of A.beta..sub.14-21, comprising an amino acid sequence His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5).

In preferred embodiments of the aforementioned specific embodiments, A or B is a cholanoyl structure or a biotin-containing structure (described further in subsection II below).

In further experiments to delineate subregions of A.beta. upon which an A.beta. aggregation core domain can be modeled (the results of which are described in Example 11), it was demonstrated that a modulator compound having inhibitory activity can comprise as few as three A.beta. amino acids residues (e.g., Val-Phe-Phe, which corresponds to A.beta..sub.18-20 or Phe-Phe-Ala, which corresponds to A.beta..sub.19-21). The results also demonstrated that a modulator compound having a modulating group at its carboxy-terminus is effective at inhibiting A.beta. aggregation. Still further, the results demonstrated that the cholyl group, as a modulating group, can be manipulated while maintaining the inhibitory activity of the compounds and that an iodotyrosyl can be substituted for phenylalanine (e.g., at position 19 or 20 of the A.beta. sequence) while maintaining the ability of the compound to inhibit A.beta. aggregation.

Still further, the results demonstrated that compounds with inhibitory activity can be created using amino acids residues that are derived from the A.beta. sequence in the region of about positions 17-21 but wherein the amino acid sequence is rearranged or has a substitution with a non-A.beta.-derived amino acid. Examples of such compounds include PPI-426, in which the sequence of A.beta..sub.17-21 (LVFFA SEQ ID NO: 11) has been rearranged (FFVLA SEQ ID NO: 21), PPI-372, in which the sequence of A.beta..sub.16-20 (KLVFF SEQ ID NO: 10) has been rearranged (FKFVL SEQ ID NO: 29), and PPI-388, -389 and -390, in which the sequence of A.beta..sub.17-21 (LVFFA SEQ ID NO: 11) has been substituted at position 17, 18 or 19, respectively, with an alanine residue (AVFFA (SEQ ID NO: 25) for PPI-388, LAFFA (SEQ ID NO: 13) for PPI-389 and LVAFA (SEQ ID NO: 33) for PPI-390). The inhibitory activity of these compounds indicate that the presence in the compound of an amino acid sequence directly corresponding to a portion of A.beta. is not essential for inhibitory activity, but rather suggests that maintenance of the hydrophobic nature of this core region, by inclusion of amino acid residues such as phenylalanine, valine, leucine, regardless of their precise order, can be sufficient for inhibition of A.beta. aggregation. Accordingly, an A.beta. aggregation core domain can be designed based on the direct A.beta. amino acid sequence or can be designed based on a rearranged A.beta. sequence which maintains the hydrophobicity of the A.beta. subregion, e.g., the region around positions 17-20. This region of A.beta. contains the amino acid residues Leu, Val and Phe. Accordingly, preferred A.beta. aggregation core domains are composed of at least three amino acid structures (as that term is defined hereinbefore, including amino acid derivatives, analogues and mimetics), wherein at least two of the amino acid structures are, independently, either a leucine structure, a valine structure or a phenylalanine structure (as those terms are defined hereinbefore, including derivatives, analogues and mimetics).

Thus, in another embodiment, the invention provides a .beta.-amyloid modulator compound comprising a formula -- see Original Patent.

Preferably, the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. In preferred embodiments, Xaa.sub.1 and Xaa.sub.2 are each phenylalanine structures or Xaa.sub.2 and Xaa.sub.3 are each phenylalanine structures. "n" can be, for example, an integer between 1 and 5, whereas "a" and "b" can be, for example, integers between 1 and 5. The modifying group "A" preferably comprises a cyclic, heterocyclic or polycyclic group. More preferably, A contains a cis-decalin group, such as cholanoyl structure or a cholyl group In other embodiments, A can comprise a biotin-containing group, a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a fluorescein-containing group or an N-acetylneuraminyl group. In yet other embodiments, the compound may promotes aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides, may be further modified to alter a pharmacokinetic property of the compound or may be further modified to label the compound with a detectable substance.

In another embodiment, the invention provides a .beta.-amyloid modulator compound comprising a formula: A--(Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3--(Z)--B wherein Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3 are each amino acid structures and at least two of Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3 are, independently, selected from the group consisting of a leucine structure, a phenylalanine structure and a valine structure; Y, which may or may not be present, is a peptidic structure having the formula (Xaa).sub.a, wherein Xaa is any amino acid structure and a is an integer from 1 to 15; Z, which may or may not be present, is a peptidic structure having the formula (Xaa).sub.b, wherein Xaa is any amino acid structure and b is an integer from 1 to 15; and A and B, at least one of which is present, are modifying groups attached directly or indirectly to the amino terminus and carboxy terminus, respectively, of the compound; Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, Y, Z, A and B being selected such that the compound modulates the aggregation or inhibits the neurotoxicity of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides.

Preferably, the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. In preferred embodiments, Xaa.sub.1 and Xaa.sub.2 are each phenylalanine structures or Xaa.sub.2 and Xaa.sub.3 are each phenylalanine structures. In one subembodiment, the compound comprises the formula: A--(Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3--(Z) In another subembodiment, the compound comprises the formula: (Y)--Xaa.sub.1-Xaa.sub.2-Xaa.sub.3--(Z)--B "n" can be, for example, an integer between 1 and 5, whereas "a" and "b" can be, for example, integers between 1 and 5. The modifying group "A" preferably comprises a cyclic, heterocyclic or polycyclic group. More preferably, A contains a cis-decalin group, such as cholanoyl structure or a cholyl group In other embodiments, A can comprise a biotin-containing group, a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a fluorescein-containing group or an N-acetylneuraminyl group. In yet other embodiments, the compound may promote aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides, may be further modified to alter a pharmacokinetic property of the compound or may be further modified to label the compound with a detectable substance.

In preferred specific embodiments, the invention provides a .beta.-amyloid modulator compound comprising a modifying group attached directly or indirectly to a peptidic structure, wherein the peptidic structure comprises amino acid structures having an amino acid sequence selected from the group consisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 5), His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6), Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9), Lys-Leu-Val-Phe-Phe (SEQ ID NO: 10), Leu-Val-Phe-Phe-Ala (SEQ ID NO: 11), Leu-Val-Phe-Phe (SEQ ID NO: 12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19), Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala (SEQ ID NO: 21), Leu-Val-Phe-Phe-Lys (SEQ ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala (SEQ ID NO: 23), Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ ID NO: 25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26), Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO: 28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ ID NO: 30), Lys-Leu-Val-Phe-Phe-.beta.Ala (SEQ ID NO: 31) and Leu-Val-Phe-Phe-DAla (SEQ ID NO: 32).

These specific compounds can be further modified to alter a pharmacokinetic property of the compound and/or further modified to label the compound with a detectable substance.

The modulator compounds of the invention can be incorporated into pharmaceutical compositions (described further in subsection V below) and can be used in detection and treatment methods as described further in subsection VI below.

II. Modifying Groups

Within a modulator compound of the invention, a peptidic structure (such as an A.beta. derived peptide, or an A.beta. aggregation core domain, or an amino acid sequence corresponding to a rearranged A.beta. aggregation core domain) is coupled directly or indirectly to at least one modifying group (abbreviated as MG). In one embodiment, a modulator compounds of the invention comprising an aggregation core domain coupled to a modifying group, the compound can be illustrated schematically as MG-ACD. The term "modifying group" is intended to include structures that are directly attached to the peptidic structure (e.g., by covalent coupling), as well as those that are indirectly attached to the peptidic structure (e.g., by a stable non-covalent association or by covalent coupling to additional amino acid residues, or mimetics, analogues or derivatives thereof, which may flank the A.beta.-derived peptidic structure). For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of an A.beta.-derived peptidic structure, or to a peptidic or peptidomimetic region flanking the core domain. Alternatively, the modifying group can be coupled to a side chain of at least one amino acid residue of an A.beta.-derived peptidic structure, or to a peptidic or peptidomimetic region flanking the core domain (e.g., through the epsilon amino group of a lysyl residue(s), through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s) or other suitable reactive group on an amino acid side chain). Modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.

The term "modifying group" is intended to include groups that are not naturally coupled to natural A.beta. peptides in their native form. Accordingly, the term "modifying group" is not intended to include hydrogen. The modifying group(s) is selected such that the modulator compound alters, and preferably inhibits, aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides or inhibits the neurotoxicity of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides. Although not intending to be limited by mechanism, the modifying group(s) of the modulator compounds of the invention is thought to function as a key pharmacophore which is important for conferring on the modulator the ability to disrupt A.beta. polymerization.

In a preferred embodiment, the modifying group(s) comprises a cyclic, heterocyclic or polycyclic group. The term "cyclic group", as used herein, is intended to include cyclic saturated or unsaturated (i.e., aromatic) group having from about 3 to 10, preferably about 4 to 8, and more preferably about 5 to 7, carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted or substituted at one or more ring positions. Thus, a cyclic group may be substituted with, e.g., halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, --CF.sub.3, --CN, or the like.

The term "heterocyclic group" is intended to include cyclic saturated or unsaturated (i.e., aromatic) group having from about 3 to 10, preferably about 4 to 8, and more preferably about 5 to 7, carbon atoms, wherein the ring structure includes about one to four heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring can be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, --CF.sub.3, --CN, or the like. Heterocycles may also be bridged or fused to other cyclic groups as described below.

The term "polycyclic group" as used herein is intended to refer to two or more saturated or unsaturated (i.e., aromatic) cyclic rings in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic group can be substituted with such substituents as described above, as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, --CF.sub.3, --CN, or the like.

A preferred polycyclic group is a group containing a cis-decalin structure. Although not intending to be limited by mechanism, it is thought that the "bent" conformation conferred on a modifying group by the presence of a cis-decalin structure contributes to the efficacy of the modifying group in disrupting A.beta. polymerization. Accordingly, other structures which mimic the "bent" configuration of the cis-decalin structure can also be used as modifying groups. An example of a cis-decalin containing structure that can be used as a modifying group is a cholanoyl structure, such as a cholyl group. For example, a modulator compound can be modified at its amino terminus with a cholyl group by reacting the aggregation core domain with cholic acid, a bile acid, as described in Example 4 (the structure of cholic acid is illustrated in FIG. 2). Moreover, a modulator compound can be modified at its carboxy terminus with a cholyl group according to methods known in the art (see e.g., Wess, G. et al. (1993) Tetrahedron Letters, 34:817-822; Wess, G. et al. (1992) Tetrahedron Letters 33:195-198; and Kramer, W. et al. (1992) J. Biol. Chem. 267:18598-18604). Cholyl derivatives and analogues can also be used as modifying groups. For example, a preferred cholyl derivative is Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can be used to further modify the modulator compound (e.g., a chelation group for .sup.99mTc can be introduced through the free amino group of Aic). As used herein, the term "cholanoyl structure" is intended to include the cholyl group and derivatives and analogues thereof, in particular those which retain a four-ring cis-decalin configuration. Examples of cholanoyl structures include groups derived from other bile acids, such as deoxycholic acid, lithocholic acid, ursodeoxycholic acid, chenodeoxycholic acid and hyodeoxycholic acid, as well as other related structures such as cholanic acid, bufalin and resibufogenin (although the latter two compounds are not preferred for use as a modifying group). Another example of a cis-decalin containing compound is 5.beta.-cholestan-3.alpha.-ol (the cis-decalin isomer of (+)-dihydrocholesterol). For further description of bile acid and steroid structure and nomenclature, see Nes, W. R. and McKean, M. L. Biochemistry of Steroids and Other Isopentanoids, University Park Press, Baltimore, Md., Chapter 2.

In addition to cis-decalin containing groups, other polycyclic groups may be used as modifying groups. For example, modifying groups derived from steroids or .beta.-lactams may be suitable modifying groups. Moreover, non-limiting examples of some additional cyclic, heterocyclic or polycyclic compounds which can be used to modify an A.beta.-derived peptidic structure are shown schematically in FIG. 2. In one embodiment, the modifying group is a "biotinyl structure", which includes biotinyl groups and analogues and derivatives thereof (such as a 2-iminobiotinyl group). In another embodiment, the modifying group can comprise a "fluorescein-containing group", such as a group derived from reacting an A.beta.-derived peptidic structure with 5-(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. In various other embodiments, the modifying group(s) can comprise an N-acetylneuraminyl group, a trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl group, an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl group, a 2-norbornaneacetyl group, a .gamma.-oxo-5-acenaphthenebutyryl, a (-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a 2-thiophenesulfonyl group.

Preferred modifying groups include groups comprising cholyl structures, biotinyl structures, fluorescein-containing groups, a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, and a N-acetylneuraminyl group. More preferred modifying groups those comprising a cholyl structure or an iminiobiotinyl group.

In addition to the cyclic, heterocyclic and polycyclic groups discussed above, other types of modifying groups can be used in a modulator of the invention. For example, small hydrophobic groups may be suitable modifying groups. An example of a suitable non-cyclic modifying group is an acetyl group.

Yet another type of modifying group is a compound that contains a non-natural amino acid that acts as a beta-turn mimetic, such as a dibenzofuran-based amino acid described in Tsang, K. Y. et al. (1994) J. Am. Chem. Soc. 116:3988-4005; Diaz, H and Kelly, J. W. (1991) Tetrahedron Letters 41:5725-5728; and Diaz, H. et al. (1992) J. Am. Chem. Soc. 114:8316-8318. An example of such a modifying group is a peptide-aminoethyldibenzofuranyl-proprionic acid (Adp) group (e.g., DDIIL-Adp (SEQ ID NO: 34)). This type of modifying group further can comprise one or more N-methyl peptide bonds to introduce additional steric hindrance to the aggregation of natural .beta.-AP when compounds of this type interact with natural .beta.-AP.

III. Additional Chemical Modifications of A.beta. Modulators

A .beta.-amyloid modulator compound of the invention can be further modified to alter the specific properties of the compound while retaining the ability of the compound to alter A.beta. aggregation and inhibit A.beta. neurotoxicity. For example, in one embodiment, the compound is further modified to alter a pharmacokinetic property of the compound, such as in vivo stability or half-life. In another embodiment, the compound is further modified to label the compound with a detectable substance. In yet another embodiment, the compound is further modified to couple the compound to an additional therapeutic moiety. Schematically, a modulator of the invention comprising an A.beta. aggregation core domain coupled directly or indirectly to at least one modifying group can be illustrated as MG-ACD, whereas this compound which has been further modified to alter the properties of the modulator can be illustrated as MG-ACD-CM, wherein CM represents an additional chemical modification.

To further chemically modify the compound, such as to alter the pharmacokinetic properties of the compound, reactive groups can be derivatized. For example, when the modifying group is attached to the amino-terminal end of the aggregation core domain, the carboxy-terminal end of the compound can be further modified. Preferred C-terminal modifications include those which reduce the ability of the compound to act as a substrate for carboxypeptidases. Examples of preferred C-terminal modifiers include an amide group, an ethylamide group and various non-natural amino acids, such as D-amino acids and .beta.-alanine. Alternatively, when the modifying group is attached to the carboxy-terminal end of the aggregation core domain, the amino-terminal end of the compound can be further modified, for example, to reduce the ability of the compound to act as a substrate for aminopeptidases.

A modulator compound can be further modified to label the compound by reacting the compound with a detectable substance. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include .sup.14C, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.99mTc, .sup.35S or .sup.3H. In a preferred embodiment, a modulator compound is radioactively labeled with .sup.14C, either by incorporation of .sup.14C into the modifying group or one or more amino acid structures in the modulator compound. Labeled modulator compounds can be used to assess the in vivo pharmacokinetics of the compounds, as well as to detect A.beta. aggregation, for example for diagnostic purposes. A.beta. aggregation can be detected using a labeled modulator compound either in vivo or in an in vitro sample derived from a subject.

Preferably, for use as an in vivo diagnostic agent, a modulator compound of the invention is labeled with radioactive technetium or iodine. Accordingly, in one embodiment, the invention provides a modulator compound labeled with technetium, preferably .sup.99mTc. Methods for labeling peptide compounds with technetium are known in the art (see e.g., U.S. Pat. Nos. 5,443,815, 5,225,180 and 5,405,597, all by Dean et al.; Stepniak-Biniakiewicz, D., et al. (1992) J. Med. Chem. 35:274-279; Fritzberg, A. R., et al. (1988) Proc. Natl. Acad. Sci. USA 85:4025-4029; Baidoo, K. E., et al. (1990) Cancer Res. Suppl. 50:799s-803s; and Regan, L. and Smith, C. K. (1995) Science 270:980-982). A modifying group can be chosen that provides a site at which a chelation group for .sup.99mTc can be introduced, such as the Aic derivative of cholic acid, which has a free amino group (see Example 11). In another embodiment, the invention provides a modulator compound labeled with radioactive iodine. For example, a phenylalanine residue within the A.beta. sequence (such as Phe.sub.19 or Phe.sub.20) can be substituted with radioactive iodotyrosyl (see Example 11). Any of the various isotopes of radioactive iodine can be incorporated to create a diagnostic agent. Preferably, .sup.123I (half-life=13.2 hours) is used for whole body scintigraphy, .sup.124I (half life=4 days) is used for positron emission tomography (PET), .sup.125I (half life=60 days) is used for metabolic turnover studies and .sup.131I (half life=8 days) is used for whole body counting and delayed low resolution imaging studies.

Furthermore, an additional modification of a modulator compound of the invention can serve to confer an additional therapeutic property on the compound. That is, the additional chemical modification can comprise an additional functional moiety. For example, a functional moiety which serves to break down or dissolve amyloid plaques can be coupled to the modulator compound. In this form, the MG-ACD portion of the modulator serves to target the compound to A.beta. peptides and disrupt the polymerization of the A.beta. peptides, whereas the additional functional moiety serves to break down or dissolve amyloid plaques after the compound has been targeted to these sites.

In an alternative chemical modification, a .beta.-amyloid compound of the invention is prepared in a "prodrug" form, wherein the compound itself does not modulate A.beta. aggregation, but rather is capable of being transformed, upon metabolism in vivo, into a .beta.-amyloid modulator compound as defined herein. For example, in this type of compound, the modulating group can be present in a prodrug form that is capable of being converted upon metabolism into the form of an active modulating group. Such a prodrug form of a modifying group is referred to herein as a "secondary modifying group." A variety of strategies are known in the art for preparing peptide prodrugs that limit metabolism in order to optimize delivery of the active form of the peptide-based drug (see e.g., Moss, J. (1995) in Peptide-Based Drug Design: Controlling Transport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter 18. Additionally strategies have been specifically tailored to achieving CNS delivery based on "sequential metabolism" (see e.g., Bodor, N., et al. (1992) Science 257:1698-1700; Prokai, L., et al. (1994) J. Am. Chem. Soc. 116:2643-2644; Bodor, N. and Prokai, L. (1995) in Peptide-Based Drug Design: Controlling Transport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter 14. In one embodiment of a prodrug form of a modulator of the invention, the modifying group comprises an alkyl ester to facilitate blood-brain barrier permeability.

Modulator compounds of the invention can be prepared by standard techniques known in the art. The peptide component of a modulator composed, at least in part, of a peptide, can be synthesized using standard techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant, G. A (ed.). Synthetic Peptides: A User's Guide, W.H. Freeman and Company, New York (1992). Automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Additionally, one or more modulating groups can be attached to the A.beta.-derived peptidic component (e.g., an A.beta. aggregation core domain) by standard methods, for example using methods for reaction through an amino group (e.g., the alpha-amino group at the amino-terminus of a peptide), a carboxyl group (e.g., at the carboxy terminus of a peptide), a hydroxyl group (e.g., on a tyrosine, seine or threonine residue) or other suitable reactive group on an amino acid side chain (see e.g., Greene, T. W and Wuts, P. G. M. Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York (1991)). Exemplary syntheses of preferred .beta. amyloid modulators is described further in Examples 1, 4 and 11.

Claim 1 of 20 Claims

1. An amyloid modulator compound having the structure -- see Original Patent.

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