AU2006228064A1 - Method for modulating macrophage activation - Google Patents

Description

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N AUSTRALIA O PATENTS ACT 1990 COMPLETE SPECIFICATION o0 FOR A STANDARD PATENT

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SORIGINAL

Name of Applicant/s: Neurochem (International) Limited Actual Inventor/s: Celine Morrissette and Francine Gervais Address for Service is: SHELSTON IP Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: METHOD FOR MODULATING MACROPHAGE ACTIVATION Details of Original Application No. 2002301959 dated 08 Nov 2002 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 29084AUP02 500996103_1.DOC5844 lac, METHOD FOR MODULATING MACROPHAGEACTIVATION c)

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This application is a divisional application derived from Australian Patent Application No. 24378/99, the entire contents of which are incorporated herein by reference.

00 Background of the Invention Amyloidogenic proteins are a group of proteins which are capable of organizing I into extracellular fibrillary protein deposits. These proteins, although different in nature, have a unique set of structural properties: they bind to Congo Red Staining and display an apple green birefringence when observed under polarized light.

Extracellular deposition of AP protein in specific regions of the brain is one of the hallmarks of Alzheimer's Disease. Ap protein is derived from an abnormal proteolytic cleavage of the precursor protein, the PAPP. Ohce deposited into the brain, it forms senile plaques which have been found in greater numbers in the brains of patients with Alzheimer's Disease. It has also been shown to infiltrate cerebrovascular walls and cause angiopathy. A progressive neuronal cell loss accompanies the deposition of AP amyloid fibrils in senile plaques. In vitro, AP has been shown by several groups to be highly toxic to neurons. La Ferla et al. have recently shown that neuronal cells when exposed in vitro to soluble AP can become apoptotic. Once internalized, AP protein is stabilized and induces DNA fragmentation, which is characteristic ofapoptosis. It has been shown that the 25-35 domain of the AP protein is responsible for such an excitotoxic activity. These results have brought scientists to consider that not only the organization of A3 fibrils into senile plaques, which.are observed late in the disease, would be detrimental to the host but that even soluble AP protein can induce neuronal cell loss earlier in the disease process.

.Activated. microgliaells have also. been observed in brains of patients -with Alzheimer's Disease. The activation process of these brain macrophages are thought:to beiresponsible for the presence of inflammatory mediators in brain extracts: These mediators, inflammatory cytokines,. nitric oxide and reactive oxygen intermediates, could play a major role in inducing-neuronal.celltoxicity. Soluble AP protein has recently been shown to be capable bfgetting-interalized by microglial cellsand to: induce an activation process as determined by production.of inflammatory.mediators such as NO (Barger et Giulian et al have also shown that this activation process is due to a specific domain of A3- the.domain of residues 10-16. It is possible that.this activation process is due to adherence of the protein (in particular the 10-16 domain of the Ap protein) to the macrophage cell surface.

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O International publication number WO 94/22437 decribes therapeutic compounds and methods for inhibiting amyloid deposition in a subject The amyloid O deposition is inhibited by the administration of an effective amount of a therapeutic compound comprising an anionic group and a carrier molecule, or a pharmaceutically acceptable salt thereof, such that an interaction between an amyloidogenic protein and a basement menbrane constituent is inhibited.

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0 U.S. Patent No. 5,276,059 discloses a method for treating a mammal having a 00 C-i condition associated with deposition of amyloidogenic protein in plaques.

I The above references relate to methods of inhibiting amyloid deposition, as does Kisilevsky et al. (Nature Medicine, Vol. 1(2) Feb. 1995, 143-148). Kisilevsky et al. uses, anionic sulfonate or sulfate compounds to substantially reduce murine splenic aa amyloid progression by interfering with Heparan sulfate-stimulated Bpeptide fibril aggregation.

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-2- SSummary of the Invention 0 It has now been discovered that certain anionic compounds, including sulfonates, C are capable of blocking AP-induced macrophage activation (or macrophage activation induced by other amyloidogenic proteins or peptides). By interfering with the ability of A3 to activate macrophages, such compounds can inhibit the inflammatory process, e.g., ND in the brain of a subject suffering from a disease characterized by AP deposition, such as 00 Alzheimer's disease.

Thus, in one aspect, the invention provides a method for inhibiting macrophage IN activation by an amyloidogenic protein or peptide. The method comprises contacting a macrophage in the presence of an amyloidogenic protein or peptide with an anionic compound, an anionic compound of Formulae I or II, such that macrophage activation is inhibited.

In one aspect, the invention provides a method for inhibiting macrophage activation by an amyloidogenic protein or peptide. The method comprises contacting a macrophage in the presence of an amyloidogenic protein or peptide with an anionic compound, an anionic compound of Formulae I or II), such that macrophage activation is inhibited. In one embodiment, the therapeutic compound can have the following formula (Formula I): wherein Y- is an anionic group at physiological pH; Q is a carrier molecule; X is a cationic group; and n is an integer. The number of anionic groups is selected such that the biodistribution of the compound for an intended target site is not prevented while maintaining activity of the compound. For example, the number of anionic groups is not so great as to inhibit traversal of an anatomical barrier, such as i cell membrane, or entry across a'physiological bariier, such as the blood-brain barrier, in situations where such properties are desired. In one embodiment, n is an integer between 1 and 10. In another embodimnt, n is an-integer between -3and 8.

In another emb6diment,the compound can be represented by the followingformula (Fdriiila II):

X

II

-P-(CYly 2 )nC(X)XR 3

RIXI

Z

wherein Z is XR 2 or R 4

R

1 and R 2 are each independently hydrogen, a substituted or unsubstituted aliphatic group, an aryl group, a heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a salt-forming cation; R 4 is hydrogen, lower alkyl, aryl or amino; X is, independently for each occurrence, 0 or S; yl and Y 2 are O -3each independently hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n O is an integer from 0 to 12.

Preferred compounds include sulfates, sulfonates, phosphates, carboxylates, and compounds which include combinations of these functional groups. Particularly preferred compounds include substituted and unsubstituted lower alkyl sulfates and IDsulfonates (including without limitation, 1,4-butanediol disulfate, sodium 00 pentanedisulfonate, taurine (sodium 2-amino-ethanesulfonate), and homotaurine (3aminopropanesulfonic acid). Other preferred compounds include 3-(cyclohexylamino)- D -propane sulfonate, 4-(2-hydroxyethyl)- 1 -piperazine-ethanesulfonate, 3-(N- O 10 morpholino)propanesulfonic acid, sodium tetrahydrothiophene- 1,1 -dioxide-3,4-disulfate trihydrate, sodium 4-hydroxybutane-1-sulfonate, sodium 1,3,5-pentanetriol trisulfate, 2aminoethyl hydrogen sulfate, phosphonoformic acid, phosphonoacetic acid, or indigo carmine. A preferred compound is 3-aminopropanesulfonic acid, or a salt thereof (see Example, infra).

In another aspect, the invention provides a method for inhibiting an inflammatory process an inflammatory process due to the presence of, or activation of macrophages by, an amyloidogenic protein or peptide). The method comprises administering to a subject in need thereof a subject having amyloid deposition) an effective therapeutic amount of an anionic compound, such that the inflammatory process is inhibited, by-inhibition of macrophage activation by an-amyloidogenic protein or peptide, suich as Ap. In a preferred embodiment, the subject is a subject suffering from Alzheimer's disease.

In certain embodiments, the anionic compoundis acompound represented by Formulas I or II.-Preferred compounds include sulfatessulfonates, phosphates, carboxylates, and compounds which include combinations-of these functional groups.

Particularly preferred compounds include substituted add-unsubstituted lower alkyl sulfates and sulfonates.(including without limitation, 1.,4biitanediol disulfate, sodium taurine (sodium 2-amino-ethanesulfonate);,-and homotaurine (3aminopropanesuilfonic acid). Other preferred compounds. iilud3-(cyclohexylamino)- 1-propane sulfonate, 4-(2-hydroxyethyl)- 1 -piperazine-ethanesulfonate, 3-(Nmorpholino)propanesulfonic acid, sodium tetrahydrothiophene-1 ,1-dioxide-3,4-disulfate trihydrate, sodium 4-hydroxybutane-1-sulfonate, sodium 1,3,5-pentanetriol trisulfate, 2aminoethyl hydrogen sulfate, phosphonoformic acid, phosphonoacetic acid, or indigo carmine. A preferred compound is 3-aminopropanesulfonic acid, or a salt thereof.

-4- Brief Description of the Drawings 0 Figure 1 is a bar graph showing the effect of various conditions on nitric oxide C(NO) production by macrophages in cell culture.

Figure 2 is a bar graph showing the effect of various conditions on Ap-induced TNFa production by macrophages in cell culture.

SFigure 3 is a graph showing the ability of a compound of the invention, 3- Saminopropanesulfonic acid, to block or inhibit macrophage activation.

00 N Detailed Description of the Invention The methods of the invention provide therapeutic treatments for subjects Ssuffering from conditions, including inflammation and neuronal cell death, subjects suffering from Alzheimer's disease or other diseases in which amyloidogenic proteins or peptides are present. By inhibiting the ability of AP to induce the activation process of macrophages, neuronal cell loss due to the brain inflammatory status can be slowed or prevented.

As used herein, the terms "inhibiting macrophage activation" or "inhibiting an inflammatory process" refer to decreasing, inhibiting, slowing, ameliorating, or reversing the course or degree ofmacrophage activation or inflammation, respectively, in vitro or in a subject; In one aspect, the invention provides a method for inhibiting macrophage activation by an amyloidogenic protein or peptide. The method comprises contacting a macrophage in the presence of an amyloidogenic protein or peptide with an anionic compound, an anionic compound of Formulae I or II, such that macrophage activation is inhibited. In one embodiment, the compound can have the following forinula.(Formula I): Q--4-Y-X]n wherein Y- is an anionic group at physiological pH; Q is acarrier molecule; X is a' cationic group; and n is an integer. The numer: of anionic groups is selected such that the biodistribution of the compoud foran intended target site is not preventeidwhile maintaining activity of the compound. For example, the number of anionic groups is not so great as to inhibit traversal of an anatomical barrier, such as a cell membrane, or entry across a physiological barrier, such as the blood-brain barrier, in situations where such properties are desired. In one embodiment, n is an integer between 1 and 10. In another embodiment, n is an integer between 3 and 8.

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0 0 An anionic group of a therapeutic compound of the invention is a negatively O charged moiety that, when attached to a carrier molecule, can inhibit macrophage Cn activation by an amyloidogenic protein or peptide, or fragment thereof AP or a fragment thereof). For purposes of this invention, the anionic group is negatively charged at physiological pH. Preferably, the anionic therapeutic compound mimics the s structure of a sulfated proteoglycan, is a sulfated compound or a functional 00 equivalent thereof. "Functional equivalents" of sulfates are intended to include compounds such as sulfamates as well as bioisosteres. Bioisosteres encompass both O classical bioisosteric equivalents and non-classical bioisosteric equivalents. Classical 0 10 and non-classical bioisosteres of sulfate groups are known in the art (see, e.g., Silverman, R.B. The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc.: San Diego, CA, 1992, pp. 19-23). Accordingly, a therapeutic compound of the invention can comprise at least one anionic group including sulfonates, sulfates, sulfamates, phosphonates, phosphates, carboxylates, and heterocyclic groups of the following formulae: 0-I

O

Depending on the carrier molecule, more than one anionic group can be attached thereto.

When more than one anionic group is attached to a carrier molecule, the multiple anionic groups can be the same structural group (e.g.,-all sulfonates) or, alternatively, a combination of different anionic groups can be used sulfonates and-sulfates, etc.).

It will be understood that the term "anionic group" includes salts, such as ._pharmaceutically acceptable salts, of an anionic group. For examples of useful compounds having anionic groups in the invention, see, U. S. Patent No. 5,643,562 (incorporated herein-by reference). In another.embodiment, the compound can be represented .by thefollowingformula (Formula II):

X

II

,P-(CYlY2)nC(X)XR 3 RIX I z wherein Z is XR 2 or R 4

R

1 and R 2 are each independently hydrogen, a substituted or unsubstituted aliphatic group (preferably a branched or straight-chain aliphatic moiety having from 1 to 24 carbon atoms in the chain; or an unsubstituted or substituted cyclic S-6aliphatic moiety having from 4 to 7 carbon atoms in the aliphatic ring; preferred 0 aliphatic and cyclic aliphatic groups are alkyl groups, more preferably lower alkyl), an aryl group, a heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a salt-forming cation; R 4 is hydrogen, lower alkyl, aryl or amino (including alkylamino, dialkylamino (including cyclic amino moieties), arylamino, diarylamino, and alkylarylamino); X is, independently for each occurrence, O or S; yl and Y 2 are each independently hydrogen, halogen F, Cl, Br, or alkyl (preferably lower OC alkyl), amino, hydroxy, alkoxy, or aryloxy; and n is an integer from 0 to 12 (more N preferably 0 to 6, more preferably 0 or 1).

An anionic compound of the invention typically further comprises a counter N cation X in the general formula: Cationic groups include positively charged atoms and moieties. Cationic groups include positively charged atoms and moieties. If the cationic group is hydrogen, then the compound is considered an acid, ethanesulfonic acid. If hydrogen is replaced by a metal or its equivalent, the compound is a salt of the acid. Pharmaceutically acceptable salts of the anionic compound are within the scope of the invention. For example, X can be a pharmaceutically acceptable alkali metal, alkaline earth, higher valency cation aluminum salt), polycationic counter ion or ammonium. A preferred pharmaceutically acceptable salt is a sodium salt but other salts are also contemplated within their pharmaceutically acceptable range.., Within the anionic compound, the anionic group(s) is covalently attached to a carrier molecule. Suitable carrier molecules include carbohydrates, polymers, peptides, peptide derivatives;: aliphatic groups, alicyclic groups, heterocyclic groups, aromatic groups or combinations thereof. A carrier molecule can be substituted, e.g. with one or more amino, nitro, halogen, thiol or hydroxy groups. As used herein, the term "carbohydrate" is intended.toinclude substituted and unsubstituted mono-,.oligo-, and polysaccharides. Monosaccharides are simple sugars usually of the formula-.C- 6

H

2 0 6 that can be combined toform.pligosadcharides or polysaccharides.'-Monosaccharides include enantiomers-and-both the D and L stereoisomers of monosaccharides. Carbohydrates can have multiple anionic groups attached to each monosaccharide moiety. For example, in sucrose octasulfate, four sulfate groups are attached to each of the two monosaccharide moieties.

As used herein, the term "polymer" is intended to include molecules formed by the chemical union of two or more combining subunits called monomers. Monomers are molecules or compounds which usually contain carbon and are of relatively low molecular weight and simple structure. A monomer can be converted to a polymer by

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S-7combination with itself or other similar molecules or compounds. A polymer may be O composed of a single identical repeating subunit or multiple different repeating subunits c (copolymers). Polymers within the scope of this invention include substituted and unsubstituted vinyl, acryl, styrene and carbohydrate-derived polymers and copolymers and salts thereof. In one embodiment, the polymer has a molecular weight of approximately 800-1000 Daltons. Examples of polymers with suitable covalently 00 attached anionic groups sulfonates or sulfates) include poly(2-acrylamido- 2 methyl-I-propanesulfonic acid); poly(2-acrylamido-2-methyl-l-propanesulfonic acid-co- ND acrylonitrile); poly(2-acrylamido-2-methyl-l-propanesulfonic acid-co-styrene); S 10 poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); and sulfates and sulfonates derived from: poly(acrylic acid); poly(methyl acrylate); poly(methyl methacrylate); and poly(vinyl alcohol); and pharmaceutically acceptable salts thereof.

Examples of carbohydrate-derived polymers with suitable covalently attached anionic groups include those of the formula:

CH

2 R CH 2 R CH 2 R CH 2

R

RCH

2 0 0

CH

2

R

CH

2

R

wherein R is SO 3 or OSO3-; and pharmaceutically acceptable salts thereof.

Peptides and peptide derivatives can also act as carrier molecules. The term "peptide" includes two or more amino acids covalently attached through a peptide bond.

Amino acids which can be used in peptide carrier molecules include those naturally occurring amino acids found in proteins such as glycine, alanine, valine, cysteine, leucine, isoleucine, serine,-threonine, methionine, glutamic acid, aspartic acid, glitamine, asparagine, lysine-arginine, proline, histidine, phenylalanine, tyrosine, and 25 tryptophan. The term amino-acid further includes- analogs;-derivatives and congeners of naturally occurring amino acids, one;or more of which can be present in apeptide- derivative. .For example, amino acid analogs can niave lengthened-or-shortened side chains or variant side chains with appropriate functional groups. Also.included are the D Sand L stereoisomers of an amino acid when the structure of the amino. acid admits of-; stereoisomeric forms. The term "peptide derivative" further includes compounds which contain molecules which mimic a peptide backbone but are not amino acids (so-called peptidomimetics), such as benzodiazepine molecules (see e.g. James, G. L. et al. (1993) Science 260:1937-1942). The anionic groups can be attached to a peptide or peptide derivative through a functional group on the side chain of certain amino acids or other 0-8suitable functional group. For example, a sulfate or sulfonate group can be attached O through the hydroxyl side chain of a serine residue. A peptide can be designed to Sinteract with a binding site for a basement membrane constituent a GAG) in an Abpeptide (as described above). Accordingly, in one embodiment, the peptide comprises four amino acids and anionic groups sulfonates) are attached to the first, second and fourth amino acid. For example, the peptide can be Ser-Ser-Y-Ser, wherein an 00 anionic group is attached to the side chain of each serine residue and Y is any amino Sacid. In addition to peptides and peptide derivatives, single amino acids can be used as ND carriers in the anionic compound of the invention. For example, cysteic acid, the sulfonate derivative of cysteine, can be used.

The term "aliphatic group" is intended to include organic compounds characterized by straight or branched chains, typically having between 1 and 22 carbon atoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynyl groups. In complex structures, the chains can be branched or cross-linked. Alkyl groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups and branched-chain alkyl groups.. Such hydrocarbon moieties may be substituted on one or more carbons with, for example, a halogen, a hydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the number of carbons is otherwise specified, "lower aliphatic" as used herein means an aliphatic group, as defined above lower alkyl, lower alkenyl, lower alkynyl), but having from one to six carbon atoms. Representative of such lower aliphatic groups, lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2aminobutyl, isobutyl, tert-butyl; 3-thioiSentyl, and the like. As used -herein, the term "amino" means -NH 2 the term "nitro" means -NO 2 the term "halogen" designates Cl, -Br or the term "thiol" means-SH; and the term "hydroxyl";means -OH. Thus, the term "alkylamino" as used herein-means an alkyl group, as defined above, having an amino group attached thereto. The term "alkylthio" refers to an alkyl-group, as defined above, having a sulfhydryl group attached thereto. The term "alkylcairbxyl"'as used herein means an alkyl group; as defined-above, having a carboxyl grioup attached thereto.

The term "alkoxy" as used herein means an alkyl group, as defined above,-having an oxygen atom, attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple bond respectively.

-9- The term "alicyclic group" is intended to include closed ring structures of three O or more carbon atoms. Alicyclic groups include cycloparaffins or naphthenes which are saturated cyclic hydrocarbons, cycloolefins which are unsaturated with two or more double bonds, and cycloacetylenes which have a triple bond. They do not include aromatic groups. Examples of cycloparaffins include cyclopropane, cyclohexane, and cyclopentane. Examples of cycloolefins include cyclopentadiene and cyclooctatetraene.

SAlicyclic groups also include fused ring structures and substituted alicyclic groups such 00 C as alkyl substituted alicyclic groups. In the instance of the alicyclics such substituents O can further comprise a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, -CF 3 -CN, or the like.

"1 The term "heterocyclic group" is intended to include closed ring structures in which one or more of the atoms in the ring is an element other than carbon, for example, nitrogen, or oxygen. Heterocyclic groups can be saturated or unsaturated and heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, -CF 3 -CN, or the like; i The term "aromatic group" is intended to include unsaturated cyclic hydrocarbons containing one or more rings. Aromatic groups include 5- and 6membered single-ring groups which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,-pyrazine, pyridazine and pyrimidine, and the like.' The aromatic ring may be substituted at one or more ring positions with, for example, a halogen, a lower alkyl, a lower-alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylainino, a lower alkylcarboxyl, a nitro, a hydroxyl, -CF 3 or the like. Int preferred eibodiment of the method of.the invention, the aiiibnic compound administered to the'subject is comprised of at least one sulfonafi'group covalently.

attached to a carrier molecule, or a pharmaceutically acceptable salt thereof.

Accordingly, an anionic compound can have the structure: Q--S0 3 -Xln wherein Q is a carrier molecule; X is a cationic group; and n is an integer. Suitable carrier molecules and cationic groups are those described hereinbefore. The number of ID sulfonate groups is selected such that the biodistribution of the compound for an Sintended target site is not prevented while maintaining activity of the compound as discussed earlier. In one embodiment, n is an integer between 1 and 10. In another embodiment, n is an integer between 3 and 8. As described earlier, an anionic compound with multiple sulfonate groups can have the sulfonate groups spaced such that the compound interacts optimally with an HSPG binding site within the Ab peptide.

O In preferred embodiments, the carrier molecule for a sulfonate(s) is a lower 00 Saliphatic group a lower alkyl, lower alkenyl or lower alkynyl), a heterocyclic Sgroup, a disaccharide, a polymer or a peptide or peptide derivative. Furthermore, the carrier can be substituted, e.g. with one or more amino, nitro, halogen, thiol or hydroxy C groups. In certain embodiments, the carrier molecule for a sulfonate(s) is an aromatic group.

Examples of suitable sulfonated polymeric anionic compounds include poly(2acrylamido-2-methyl-l-propanesulfonic acid); poly(2-acrylamido-2-methyl-1propanesulfonic acid-co-acrylonitrile); poly(2-acrylamido-2-methyl-l-propanesulfonic acid-co-styrene); poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); a sulfonic acid derivative of poly(acrylic acid); a sulfonic acid derivative of poly(methyl acrylate); a sulfonic acid derivative of poly(methyl methacrylate); and a sulfonate derivative of poly(vinyl alcohol); and pharmaceutically acceptable salts thereof. A preferred sulfonated polymer is poly(vinylsulfonic acid) (PVS) or a., pharmaceutically acceptable salt thereof, preferably the sodium salt thereof. In one embodiment, PVS having a molecular weight of about 800-1000-Daltons is used. PVS may be used as a mixture of stereoisomers or as a-singleactive isomer.

A preferred sulfonated disaccharide is afully- or partially sulfonated sucrose, or pharmaceutically acceptable salt thereof, such as sucrose octasulfonate. Other sulfonated saccharides include 5-deoxy-l,2--O-isopropylidene-a-D-xylofuranose-5sulfonic acid (XXIII, shown as the sodium, st) Preferred lower aliphatic sulfonated anionic-compounds include ethanesulfonic acid; 2 -aminoethanesulfonic acid (taurine); cysteic acid,(3-sulfoalanine or a-amin-bsulfopropionic acid); 1-propanesulfonic acid; 1,2-ethanedisulfonic acid; 1,3propanedisulfonic acid; 1,4-butanedisulfonic acid; 1,5-pentanedisulfonic acid; and 4hydroxybutane-1-sulfonic acid; and pharmaceutically acceptable salts thereof. Other aliphatic sulfonated anionic compounds include 1-butanesulfonic acid, 2propanesulfonic acid, 3-pentanesulfonic acid, 4-heptanesulfonic acid, 1-decanesulfonic acid; and pharmaceutically acceptable salts thereof. Sulfonated substituted aliphatic anionic compounds include 3-amino-l-propanesulfonic acid, 3-hydroxypropanesulfonic S-11acid sulfate, 1,7-dihydroxy-4-heptanesulfonic acid; and pharmaceutically acceptable 0 salts thereof. Yet other sulfonated compounds include ^c pyridinyl)amido]ethanesulfonic acid, and pharmaceutically acceptable salts thereof.

Preferred heterocyclic sulfonated anionic compounds include 3-(Nmorpholino)propanesulfonic acid; and tetrahydrothiophene- l,l-dioxide-3,4-disulfonic acid; and pharmaceutically acceptable salts thereof.

0 Aromatic sulfonated anionic compounds include 1,3-benzenedisulfonic acid, dimethoxy-1,4-benzenedisulfonic acid, 4-amino-3-hydroxy-l-naphthalenesulfonic acid, S 3,4-diamino- -naphthalenesulfonic acid; and pharmaceutically acceptable salts thereof.

In another embodiment of the method of the invention, the anionic compound

C

administered to the subject is comprised of at least one sulfate group covalently attached to a carrier molecule, or a pharmaceutically acceptable salt thereof. Accordingly, the anionic compound can have the structure: Q----OSO3-X+]n wherein Q is a carrier molecule; X is a cationic group; and n is an integer. Suitable carrier molecules and cationic groups are those described hereinbefore. The number of sulfate groups is selected such that the biodistribution of the compound for an intended target site is not prevented while maintaining activity of the anionic compound as discussed earlier. In one embodiment, n is an integer between 1 and 10. In another embodiment, n is an integer between 3 and 8. As described earlier, an anionic compound with multiple sulfate groups can have.the sulfate groups spaced such that the compound interacts optimally-with a GAG binding site within an Ab.peptide.

In preferred embodiments, the carrier molecule for a sulfate(s) is a.lower aliphatic group a lower.alkyl, lower-alkenyl or lower alkynyl), an aromatic group, a disaccharide, a polymer orapeptide or.peptide derivative. Furthermore, the:carriercan be substituted, e.g. with one or more amino,.nitro, halogen, thiol or hydroxy-.groups., Examples of suitable sulfated polymeric anionic compounds include poly( 2 acrylamido-2-methyl-propyl sulfuric acid); poly(2-acrylamido-2-methyl-propyl sulfuric acid-co-acrylonitrile); poly(2-acrylamido-2-methyl-propyl sulfuric acid-co-styrene); poly(vinylsulfuric acid); poly(sodium 4-styrenesulfate); a sulfate derivative of poly(acrylic acid); a sulfate derivative of poly(methyl acrylate); a sulfate derivative of poly(methyl methacrylate); and a sulfate derivative ofpoly(vinyl alcohol); and pharmaceutically acceptable salts thereof.

-12- A preferred sulfated disaccharide is sucrose octasulfate or pharmaceutically o acceptable salt thereof. Other sulfated saccharides include the acid form of methyl-ca-Dglucopyranoside 2,3-disulfate, methyl 4,6-O-benzylidene-a-D-glucopyranoside 2,3disulfate, 2,3,4,3',4'-sucrose pentasulfate, 1,3 :4,6-di-O-benzylidene-D-mannitol disulfate, D-mannitol 2,5-disulfate, 2,5-di-O-benzyl-D-mannitol tetrasulfate; and pharmaceutically acceptable salts thereof.

0 Preferred lower aliphatic sulfated anionic compounds for use in the invention 00 0 include ethyl sulfuric acid; 2-aminoethan-1-ol sulfuric acid; 1-propanol sulfuric acid; 1,2-ethanediol disulfuric acid; 1,3-propanediol disulfuric acid; 1,4-butanediol disulfuric acid; 1,5-pentanediol disulfuric acid; and 1,4-butanediol monosulfuric acid; and CN pharmaceutically acceptable salts thereof. Other sulfated aliphatic anionic compounds contemplated for use in the invention include the acid form of 1,3-cyclohexanediol disulfate, 1,3,5-heptanetriol trisulfate, 2-hydroxymethyl- 1,3-propanediol trisulfate, 2hydroxymethyl-2-methyl-1,3-propanediol trisulfate, 1,3,5,7-heptanetetraol tetrasulfate, 1,3,5,7,9-nonane pentasulfate; and pharmaceutically acceptable salts thereof. Other sulfated anionic compounds contemplated for use in the invention include the acid form of 2-amino-2-hydroxymethyl- 1,3-propanediol trisulfate, 2-benzyloxy-1 ,3-propanediol disulfate, 3-hydroxypropylsulfamic acid sulfate, 2,2'-iminoethanol disulfate, N,N-bis(2hydroxyethyl)sulfamic acid disulfate,; and pharmaceutically acceptable salts thereof.

Preferred heterocyclic sulfated anionic compounds include morpholino)propanesulfuric acid; and tetrahydrothiophene-1,1-dioxide-3,4-diol disulfuric acid; and pharmaceutically acceptable salts thereof.

S The invention further contemplates the use ofprodrugs.which are converted in vivo to the anionic compounds used in the methods of the invention (see, RB.

Silverman, 1992,."'The Organic Chemistry of Drug.Design and Drug Action", Academic Press, Chp. Such prodrugs can be used to alter.thebiodistribution to allow compounds which would not typically cross the bloodrbrain barrier to cross the bloodbrain barrier) or the pharmacokinetics of the anionic-comppund:. For example, an anionic group, a sulfate or sulfonate, can be.esterified, with a methyl group or a phenyl group, to yield a sulfate or sulfonate ester. When the sulfate or sulfonate ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively or hydrolytically, to reveal the anionic group. Such an ester can be cyclic, a cyclic sulfate or sultone, or two or more anionic moieties may be esterified through a linking group. Exemplary cyclic anionic compounds include, for example, 2sulfobenzoic acid, propane sultone, butane sultone, 1,3-butanediol cyclic sulfate, achloro-a-hydroxy-o-toluenesulfonic acid sultone, and 6-nitronaphth-[1,8-cd]-1,2,- -13oxathiole 2,2-dioxide. In a preferred embodiment, the prodrug is a cyclic sulfate or 0 sultone. An anionic group can be esterified with moieties acyloxymethyl esters) Swhich are cleaved to reveal an intermediate anionic compound which subsequently decomposes to yield the active anionic compound. In another embodiment, the prodrug is a reduced form of a sulfate or sulfonate, a thiol, which is oxidized in vivo to the 0 anionic compound. Furthermore, an anionic moiety can be esterified to a group which is 0 actively transported in vivo, or which is selectively taken up by target organs. The ester 0 can be selected to allow specific targeting of the anionic compounds to particular organs, IN as described below for carrier moieties.

0 10 Carrier molecules useful in the anionic compounds-include carrier molecules c previously described, e.g. carbohydrates, polymers, peptides, peptide derivatives, aliphatic groups, alicyclic groups, heterocyclic groups, aromatic groups or combinations thereof. Suitable polymers include substituted and unsubstituted vinyl, acryl, styrene and carbohydrate-derived polymers and copolymers and salts thereof. Preferred carrier molecules include a lower alkyl group, a heterocyclic group, a disaccharide, a polymer or a peptide or peptide derivative.

Carrier molecules useful in the present invention may also include moieties which allow the anionic compound to be selectively delivered to a target organ or organs. For example, if delivery of a tanionic compound to the brain is desired, the carrier molecule may include a moiety capable of targeting the anionic. compound to the brain, by either active or passive transport (a "targeting moiety"). Illustratively, the carrier molecule may include a redox moiety, as described in, for example, U.S. Patents 4,540,564 and 5,389,623, both to Bodor.- These patents disclose drugs linked dihydropyridine moieties which can enter:the brain, where they are oxidized to a charged pyridinium species which is trapped in thebrain. Thus, drug accumulates in the brain.

Exemplary pyridine/dihdropyridine compounds of the invention include sodium l-(3- .sulfopropyl)- 1,4-dihydropyridine, sodium 2(nicotinylamido)-ethanesulfonate, and 1-(3sulfopropyl)-pyridinium betaine. Other carriermoieties include compounds, such as..

amino acids or thyroxine, which can be passively or actively transported in vivo: -An -7 illustrative compound is phenylalanyltaurine, in which a taurine molecule is conjugated to a phenylalanine (a large neutral amino acid). Such a carrier moiety can be metabolically removed in vivo, or can remain intact as part of an active anionic compound. Structural mimics of amino acids (and other actively transported moieties) are also useful in the invention 1-(aminomethyl)-l-(sulfomethyl)-cyclohexane).

Other exemplary amino acid mimetics include p-(sulfomethyl)phenylalanine, p-(1,3disulfoprop-2-yl)phenylalanine, and O-(1,3-disulfoprop-2-yl)tyrosine. Many targeting N -14moieties are known, and include, for example, asialoglycoproteins (see, e.g. Wu, U.S.

Patent 5,166,320) and other ligands which are transported into cells via receptormediated endocytosis (see below for further examples of targeting moieties which may be covalently or non-covalently bound to a carrier molecule). Furthermore, the anionic compounds of the invention may bind to amyloidogenic proteins, Ab peptide, in the circulation and thus be transported to the site of action.

O The targeting and prodrug strategies described above can be combined to 00 produce an anionic compound that can be transported as a prodrug to a desired site of N action and then unmasked to reveal an active anionic compound. For example, the dihydropyrine strategy of Bodor (see supra) can be combined with a cyclic prodrug, as Sfor example in the compound 2 -(1-methyl-1,4-dihydronicotinyl)amidomethylpropanesultone.

In one embodiment, the anionic compound in the pharmaceutical compositions is a sulfonated polymer, for example poly(2-acrylamido-2-methyl-l-propanesulfonic acid); poly( 2 -acrylamido-2-methyl-1 -propanesulfonic acid-co-acrylonitrile); poly(2acrylamido-2-methyl-l-propanesulfonic acid-co-styrene); poly(vinylsulfonic acid); poly(sodium 4 -styrenesulfonic acid); a sulfonate derivative ofpoly(acrylic acid); a sulfonate derivative of poly(methyl acrylate); a sulfonate derivative of poly(methyl methacrylate); and a sulfonate derivative of poly(vinyl alcohol);.and pharmaceutically .acceptable salts thereof.

In another embodiment, the anionic compound in the pharmaceutical compositions is a sulfated polymer, for example poly(2-acrylamido-2-methyl-lpropanesulfuric acid); poly( 2 -acrylamido-2-methyl-l-propanesulfuric acid-coacrylonitrile); poly( 2 -acrylamido-2-methyl- l-propanesulfuric acid-co-styrene); poly(vinylsulfuric acid); poly(sodium 4-styrenesulfate); a sulfate derivative of poly(acrylic acid); a sulfate derivative of poly(methyl acrylate); a sulfate derivative of poly(methyl methacrylate); and a sulfate derivative of Dolv(vinyl alcohol); and pharmaceutically acceptable salts thereof.

The anionic compound can also have the structure:

X

II

P---(CYIY

2 )nC(X)XR 3 RIXA I

Z

in which Z is XR 2 or R 4

R

I and R 2 are each independently hydrogen, a substituted or unsubstituted aliphatic group (preferably a branched or straight-chain aliphatic moiety having from 1 to 24 carbon atoms in the chain; or an unsubstituted or substituted cyclic N aliphatic moiety having from 4 to 7 carbon atoms in the aliphatic ring; preferred Saliphatic and cyclic aliphatic groups are alkyl groups, more preferably lower alkyl), an aryl group, a heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a salt-forming cation; X is, independently for each occurrence, O or S; R 4 is hydrogen, lower alkyl, aryl or amino; YI and Y 2 are each independently hydrogen, halogen F, Cl, Br, or lower alkyl, amino (including alkylamino, dialkylamino, O arylamino, diarylamino, and alkylarylamino), hydroxy, alkoxy, or aryloxy; and n is an

OO

00 integer from 0 to 12 (more preferably 0 to 6, more preferably 0 or These compounds N are described in U.S. Application Serial No. 08/912,574, the contents of which are S 10 incorporated herein by reference.

Ci Preferred anionic compounds for use in the invention include compounds in which both R I and R 2 are pharmaceutically acceptable salt-forming cations. It will be appreciated that the stoichiometry of an anionic compound to a salt-forming counterion (if any) will vary depending on the charge of the anionic portion of the compound (if any) and the charge of the counterion. In a particularly preferred embodiment, R 1

R

2 and R 3 are each independently a sodium, potassium or calcium cation. In certain embodiments in which at least one ofR 1 and R 2 is an aliphatic group, the aliphatic group has between 1 and 10 carbons atoms in the straight or branched chain, and is more preferably a lower alkyl group. In other embodiments in which at least one of Rj and R 2 is an aliphatic group, the aliphatic group has between 10 and 24 carbons atoms in thestraight or branched chain. In certain preferred embodiments, n is 0 or 1; more preferably, n is 0. In certain preferred embodiments of the therapeutic compounds, Y 1 and Y 2 are each hydrogen. In certain preferred embodiments, the anionic.compound of the invention can have the structure:

X

II

,P--(CYIY

2 )nC(0)OR 3 RIX I XR2 in which R 1

R

2

R

3 yl, Y 2 X and n are as defined above. In more preferred embodiments, the anionic compound of the invention can have the structure:

X

II

P--(CYIY2)nCH(NRaRb)C()OR3 RIO I OR2 in which R 1

R

2 R3, Yl, Y 2 and X are as defined above, Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with -16the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 0 atoms in the ring, and n is an integer from 0 to 6. In certain preferred embodiments, Ra C and Rb are each hydrogen. In certain preferred embodiments, a compound of the invention comprises an a-amino acid (or ca-amino acid ester), more preferably a L-aamino acid or ester.

The Z, Q, R

I

R

2

R

3 yl, Y 2 and X groups are each independently selected such O that the biodistribution of the anionic compound for an intended target site is not

OO

prevented while maintaining activity of the anionic compound. For example, the N number of anionic groups (and the overall charge on the therapeutic compound) should not be so great as to inhibit traversal of an anatomical barrier, such as a cell membrane, C or entry across a physiological barrier, such as the blood-brain barrier, in situations where such properties are desired. For example, it has been reported that esters of phosphonoformate have biodistribution properties different from, and in some cases superior to, the biodistribution properties of phosphonoformate (see, U.S. Patent Nos. 4,386,081 and 4,591583 to Helgstrand et al., and U.S. Patent Nos. 5,194,654 and 5,463,092 to Hostetler et Thus, in certain embodiments, at least one of RI and R 2 is an aliphatic group (more preferably an alkyl group), in which the aliphatic group has between 10 and 24 carbons atoms in the straight or branched chain. The number, length, and degree of branching of the aliphatic chains can-be selected to provide a desired characteristic, lipophilicity. In other embodiments, at least one of R 1 and

R

2 is an aliphatic group (more preferably an alkyl group); in which the aliphatic group has between 1 and 10 carbons atoms in the straight or branched chain. Again, the number, length, and degree of branching of the aliphatic chains can be selected to provide a desired characteristic, lipophilicity or ease of ester cleavage by enzymes.

In certain embodiments, a preferred aliphatic group is an ethyl group.

In another embodiment, the anionic compound of the invention can have the structure: 00 II II

)-C-P-O-L

I ~\0

G

in which G represents hydrogen or one or more substituents on the aryl ring alkyl, aryl, halogen, amino, and the like) and L is a substituted alkyl group (in certain embodiments, preferably a lower alkyl), more preferably a hydroxy-substituted alkyl or an alkyl substituted with a nucleoside base. In certain embodiments, G is hydrogen or an electron-donating group. In embodiments in which G is an electron-withdrawing

O

-17- Sgroup, G is preferably an electron withdrawing group at the meta position. The term 0 "electron-withdrawing group" is known in the art, and, as used herein, refers to a group m which has a greater electron-withdrawing than hydrogen. A variety of electronwithdrawing groups are known, and include halogens fluoro, chloro, bromo, and iodo groups), nitro, cyano, and the like. Similarly, the term "electron-donating group", 1 as used herein, refers to a group which is less electron-withdrawing than hydrogen. In 0 embodiments in which G is an electron donating group, G can be in the ortho, meta or Spara position.

0 In certain preferred embodiments, L is a moiety selected from the group 0 10 consisting of: OHH -OH

-OH

IVa OC(O)C H 23

SC(O)C

11

H

2 3

(O)C

7 His IVb IVc IVd

NH

2 NH 2 N N N OH

OH

N N N N OH OH IVe IVf IVg STable 1 lists data pertinent to the characterizatin of these compounds using artrecognized techniques.

-18- Table 1

COMPOUND

IVa lVb 11iP NNM -6.33(DMSO-d6) -6.41 (DMSO-d 6 lic NMR FAB-MS(-) 60.97 CHiOH(d, J=6Hz) 66.76 CHOH(d, J=7.8Hz) 121.65, 121.78, 121.99, 125.71, 129.48, 129.57, 126.43 Aromatic CH 134.38 Aniline C-N 150.39 Phenyl C-O(d, J=7-z) 171.57 P-C=O(d, J=234H1z) 13.94 CH 3 22.11, 24.40, 28.56, 28.72, 28.99, 29.00, 31.30, 33.43, -(CH 2 1 0o 65.03, CH 2

-OC(O)

66.60 CH 2 -OP(d, J=5.6Hz) 67.71 CH2-OH(d, J=6 Hz) 121.73, 121.10, 125.64, 126.57, 129.40, 129.95, Aromatic CH 134.04 Aniline C-N 150.31 Phenyl C-0 171,44PR-C0(d, J=6.7 Hz) 245.2 456 172.83 O-C~iO LVc 6.46(DMSO-d 6 13.94 CHI- 22.11, 25! 10, 28.68, 28.72, 28.85, 20'00, 30.76, 31.31, 32.10, 43.36 C H 2

-S

68,43 !CH 2 -OH 68.43 CH-OH(d, J=6.3 Hz) 68.76 P-O-CH 2 -9d, J--5.8 Hz) 121.75, 122-03, 125.62, 126.37, 129.30, 129.53, Aromatic CH 134-23 Aniline C-N 150.37 Phenyl C-O(d, J=6.7 Hz) 171.47 P.C=O(d, J=234.0 Hz) 198.47 S-C'=O 471 -19-

COMPOUND

IVd 2-1P NMR -6.6 1 (DMSO-d 6 13C NMR 13.94 CH 3 22.06, 25.14, 28.24, 28.35, 31.09, 32.14

FAB-MS(-)

416

-CH

2 43.40 CH 2

-S

68.50 P-O-CH 2 J=5.8 Hz) 68.77 CH-OH4(d, 6.4 Hz) 121.78, 122.59, 125.69, 127.06, .129.43, 129.59 Aromatic CH 133.39 Aniline C-N 150.38 Phenyl C-O(d, J=6.7 Hz) 171.47 P-C0O(d, J=234.4 Hz) 198.54 S-C=O -5.76(D 2 0)

N/A

IVf lVg -7.00(DMSO-d 6 -6.60(DMSO-D6)

N/A

70.84 CH2-OH 72.17 CH-OH 121.68, 12r:79, 121.85, 125.71 127.10, 127.92, 129.36, 129.50, 129.59 Aromatic CH 134.51 Aniline C-N 142.34 Aromatic C-CR 150.37 Phenyl C-O(d, 3z=6.2 Hz) 171.59 P-C0O(d, J=232.6 Hz)

N/A

N/A

321 It will be noted that the structure of some of thae anionic compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers enantiomers and diastereomers) arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure

IO

Sform by classical separation techniques and by sterically controlled synthesis. For the O purposes of this application, unless expressly noted to the contrary, an anionic C compound shall be construed to include both the R or S stereoisomers at each chiral center.

In certain embodiments, an anionic compound of the invention comprises a cation in certain embodiments, at least one of R 1

R

2 or R 3 is a cation). If the 00 cationic group is hydrogen, then the anionic compound is considered an acid, e.g., phosphonoformic acid. If hydrogen is replaced by a metal ion or its equivalent, the D anionic compound is a salt of the acid. Pharmaceutically acceptable salts of the anionic O 10 compound are within the scope of the invention. For exarfiple, at least one of R 1

R

2 or

R

3 can be a pharmaceutically acceptable alkali metal Li, Na, or ammonium cation, alkaline earth cation Ca 2 Ba 2 Mg 2 higher valency cation, or polycationic counter ion a polyammonium cation). (See, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). It will be appreciated that the stoichiometry of an anionic compound to a salt-forming counterion (if any) will vary depending on the charge of the anionic portion of the compound (if any) and the charge of the counterion. Preferred pharmaceutically acceptable salts include a sodium, potassium or calcium salt, but other salts are also contemplated within their pharmaceutically acceptable range.

The term "pharmaceutically acceptable esters" refers to the relatively non-toxic, esterified products of the anionic compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the anionic compounds or by separately reacting the purified anionic compound in its free acid-form or hydroxyl with a suitable esterifying agent;. either of which are methods known to those skilled in the art. Carboxylic acids and phosphonic-acidscan be converted into esters according to methods well known to one of.ordinary skill in the art, via treatment with an-alcohol in the presence of a catalyst, A preferred ester group when R 3 is lower alkyl) is an ethyl ester group.

The term "alkyl" refers to the saturated aliphatic-groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone CI-C 3 0 for straight chain, C 3

-C

3 0 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 4-7 carbon atoms in the ring structure.

\0 -21- SThe term "lower alkyl" refers to alkyl groups having from 1 to 6 carbons in the chain, O and to cycloalkyls having from 3 to 6 carbons in the ring structure.

Moreover, the term "alkyl" (including "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents Sreplacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, IN alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, N diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, with the substituents described above. An "aralkyl" moiety is an alkyl substituted with an aryl phenylmethyl (benzyl)).

The term "alkoxy", as used herein, refers to a moiety having the structure -0alkyl, in which the alkyl moiety is described above.

The term "aryl" as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like..Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl; and the like. The aromatic ring can be substituted at one or more ring positions with such substituents, e.g.,-as described above for alkyl groups. Preferred-a-yL groups include unsubstituted and substituted phenyl groups.

The term "aryloxy", as used herein, refers to a group having the structure -O-aryl, in which the aryl moiety is as defined above.

The term "amino," as used herein, refers to an unsubstituted or substituted moiety of the formula -NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term "amino" is intended to include cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated. An "amino-substituted amino group" refers to an o -22amino group in which at least one of Ra and Rb, is further substituted with an amino 0 group.

In a preferred embodiment, R 1 or R 2 can be (for at least one occurrence) a longchain aliphatic moiety. The term "long-chain aliphatic moiety" as used herein, refers to a moiety having a straight or branched chain aliphatic moiety an alkyl or alkenyl Smoiety) having from 10 to 24 carbons in the aliphatic chain, the long-chain aliphatic moiety is an aliphatic chain of a fatty acid (preferably a naturally-occurring fatty acid).

SRepresentative long-chain aliphatic moieties include the aliphatic chains of stearic acid, N oleic acid, linolenic acid, and the like.

In certain embodiments, the anionic compound of the invention can have the C structure: 0

II

SP-(CYIY

2 )nCOOR 3 RIO I

OR

2 in which R I and R 2 are each independently hydrogen, an aliphatic group (preferably a branched or straight-chain aliphatic moiety having from 1 to 24 carbon atoms, more preferably 10-24 carbon atoms, in the chain; or an unsubstituted or substituted cyclic aliphatic moiety having from 4 to 7 carbon atoms in the aliphatic ring), an aryl group, a.

heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a saltforming cation; yl and Y 2 are each independently hydrogen, halogen F, Cl, Br,.or lower alkyl, hydroxy, alkoxy, or aryloxy; and n is an integer from 0 to 12. Preferred anionic compounds for use in the invention include compounds in which both R 1 and R 2 are pharmaceutically acceptable salt-forming cations. In a particularly preferred embodiment, R 1

R

2 and R 3 are each independently a sodium, potassium or calcium catioh, and n is 0. In certain preferred embodiments of he therapeutic compounds; Y 1 aid Y 2 are each hydrogen. Particularly preferred aionie compounds are salts of phosphonoformate. Trisodium phosphonoformate (foscamet sodium or Foscavir®) is commercially available from Astra), and its clinical pharmacology has been investigated (see, "Physician's Desk Reference", 51st Ed., pp. 541-545 (1997)).

In another embodiment, the anionic compound used in the invention can be an aminophosphonate, a biphosphonate, a phosphonocarboxylate derivative, a phosphonate derivative, or a phosphono carbohydrate.

IO

-23- Pharmaceutically Acceptable Formulations O In the method of the invention, the anionic compound can be administered in a c pharmaceutically acceptable formulation. The present invention pertains to any pharmaceutically acceptable formulations, such as synthetic or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres, or beads, and lipid- Sbased formulations including oil-in-water emulsions, micelles, mixed micelles, synthetic 00 membrane vesicles, and resealed erythrocytes.

In one embodiment, the pharmaceutically acceptable formulations comprise a Spolymeric matrix.

The terms "polymer" or "polymeric" are art-recognized and include a structural framework comprised of repeating monomer units which is capable of delivering an anionic compound, such that treatment of a targeted condition occurs. The terms also include co-polymers and homopolymers, synthetic or naturally occurring. Linear polymers, branched polymers, and cross-linked polymers are also meant to be included.

For example, polymeric materials suitable for forming the pharmaceutically acceptable formulation employed in the present invention, include naturally derived polymers such as albumin, alginate, cellulose derivatives, collagen, fibrin, gelatin, and polysaccharides, as well as synthetic polymers such as polyesters (PLA, PLGA), -polyethylene glycol, poloxomers, polyanhydrides, and pluronics. These polymers are biocompatible-with the nervous system, including the central nervous system, they are biodegradable within the central nervous system without producing any toxic byproducts of degradation, and they possess the ability to modify the manner and duration of anionic compound release by manipulating the polymer's kinetic characteristics. As used herein, the term "biodegradable" means that the polymer will degrade overtime by, the action of enzymes, by hydrolytic action and/or by other similar mechanisms in thebody of the subject As used herein, the term "biocompatible" means that the polymer is compatible with a living tissue or a living organism by not being toxic or injurious and by not causing an immunological rejection.

SPolymers can be prepared using methods known in the art (Sandier, S. R.;Karo, W. Polymer Syntheses; Harcourt Brace: Boston, 1994; Shalaby, Ikada, Langer, LR; Williams, J. Polymers of Biological and Biomedical Significance (ACS Symposium Series 540; American Chemical Society: Washington, DC, 1994). Polymers can be designed to be flexible; the distance between the bioactive side-chains and the length of a linker between the polymer backbone and the group can be controlled. Other suitable polymers and methods for their preparation are described in U.S. Patent Nos. 5,455,044 and 5,576,018, the contents of which are incorporated herein by reference.

IO

-24- The polymeric formulations are preferably formed by dispersion of the anionic O compound within liquefied polymer, as described in U.S. Pat. No. 4,883,666, the Steachings of which are incorporated herein by reference, or by such methods as bulk polymerization, interfacial polymerization, solution polymerization and ring polymerization as described in Odian Principles of Polymerization and ring opening Spolymerization, 2nd ed., John Wiley Sons, New York, 1981, the contents of which are incorporated herein by reference. The properties and characteristics of the formulations are controlled by varying such parameters as the reaction temperature, concentrations of 0 polymer and anionic compound, types of solvent used, and reaction times.

8 10 In addition to the anionic compound and the pharmaceutically acceptable C polymer, the pharmaceutically acceptable formulation used in the method of the invention can comprise additional pharmaceutically acceptable carriers and/or excipients. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and anti fungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. For example, the carrier can be suitable for injection into the cerebrospinal fluid. Excipients include pharmaceutically acceptable stabilizers and disintegrants.

The anionic compound can be encapsulated in one or more pharmaceutically Sacceptable polymers, to form a microcapsule, microsphere, or microparticle, terms used .herein interchangeably. Microcapsules, microspheres, and microparticles are conventionally free-flowing powders consisting of spherical particles of 2 millimeters or less in diameter, usually 500 microns or less in diameter. Particles less than 1 micron are-conventionally referred to as nanocapsules, nanoparticles or nanospheres. For the most part, the difference between a microcapsule and a nariocapsule, a microsphere and a nanosphere, or microparticle and nanoparticle is size; generally there is little, if any, difference between the internal structure of the two. In one-aspect of the present invention, the mean average diameter is less than-about 45 pm,,preferably less than 20 m, and more preferably between about 0.1 and 10 pm. 2 In another embodiment, the pharmaceutically acceptable formulations comprise lipid-based formulations. Any of the known lipid-based drug delivery systems can be used in the practice of the invention. For instance, multivesicular liposomes (MVL), multilamellar liposomes (also known as multilamellar vesicles or unilamellar liposomes, including small unilamellar liposomes (also known as unilamellar vesicles or "SUV") and large unilamellar liposomes (also known as large unilamellar vesicles or can all be used so long as a sustained release rate of the encapsulated anionic compound can be established. In one embodiment, the lipid-based formulation can be a S- multivesicular liposome system. Methods of making controlled release multivesicular O liposome drug delivery systems is described in PCT Application Serial Nos.

c US96/11642, US94/12957 and US94/04490, the contents of which are incorporated herein by reference.

The composition of the synthetic membrane vesicle is usually a combination of 0 phospholipids, usually in combination with steroids, especially cholesterol. Other 0 phospholipids or other lipids may also be used.

Examples of lipids useful in synthetic membrane vesicle production include phosphatidylglycerols, phosphatidylcholines, phosphatidylserines, 0 10 phosphatidylethanolamines, sphingolipids, cerebrosides, and gangliosides. Preferably Sphospholipids including egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol are used.

In preparing lipid-based vesicles containing an anionic compound, such variables as the efficiency of anionic compound encapsulation, lability of the anionic compound, homogeneity and size of the resulting population of vesicles, anionic compound-to-lipid ratio, permeability, instability of the preparation, and pharmaceutical acceptability of the formulation should be considered (see Szoka, et al., Annual Reviews of Biophysics and Bioengineering, 9:467, 1980; Deamer, et al., in Liposomes, Marcel Dekker, New York, 1983, 27; and Hope, et al., Chem. Phys. Lipids, 40:89, 1986, the contents of which are incorporated herein by reference).

Administration of the Pharmaceutically Acceptable Formulation In one embodiment, the anionic compound is administered by introduction into the central nervous system of the subject, into the cerebrospinal fluid of the-subject.

In certain aspects of the invention, the anionic compound is introduced intrathecally;: into a cerebral ventricle, the lumbar area, or the cisterna magna.

The pharmaceutically acceptable formulations can easily be suspended in aqueous vehicles and introduced through conventional hypodermic needles or usinginfusion pumps. Prior to introduction, the formulations can be sterilized with, preferably, gamma radiation or electron beam sterilization, described in US patent no.

436,742 the contents of which are incorporated herein by reference.

In another embodiment of the invention, the anionic compound formulation is administered into a subject intrathecally. As used herein, the term "intrathecal administration" is intended to include delivering an anionic compound formulation directly into the cerebrospinal fluid of a subject, by techniques including lateral

\D

S- 26cerebroventricular injection through a burrhole or cistemal or lumbar puncture or the like O (described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Cr< Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of which are incorporated herein by reference). The term "lumbar region" is intended to include the area between the third and fourth lumbar (lower back) vertebrae.

\The term "cisterna magna" is intended to include the area where the skull ends and the 00 spinal cord begins at the back of the head. The term "cerebral ventricle" is intended to Sinclude the cavities in the brain that are continuous with the central canal of the spinal N0 cord. Administration of an anionic compound to any of the above mentioned sites can be achieved by direct injection of the anionic compound formulation or by the use of infusion pumps. For injection, the anionic compound formulation of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the anionic compound formulation may be formulated in solid form and re-dissolved or suspended immediately prior to use.

Lyophilized forms are also included. The injection can be, for example, in the form of a bolus injection or continuous infusion using infusion pumps) of the anionic compound formulation.

Duration and Levels of Administration In another embodiment of the method of the invention, the pharmaceutically -acceptable formulation provides sustained delivery, "slow release" of the anionic -compound to a subject for at least one, two, three, or four weeks after the pharmaceutically acceptable formulation is administered to the subject.

As used herein, the term "sustained delivery" is intended to include continual -delivery of an anionic compound in vivo over a period of time following administration, preferably at least several days, a week or several weeks. Sustained delivery of the anionic compound.can be demonstrated by, for example, the continued therapeutic.effect Sof the.anionic compound over time sustained delivery of the anionic compound can- Sbe demonstrated-by continued-inhibition of neuronal cell death over time).

Alternatively, sustained delivery of the anionic compound may be demonstrated by detecting the presence of the anionic compound in vivo over time.

In one embodiment, the pharmaceutically acceptable formulation provides sustained delivery of the anionic compound to a subject for less than 30 days after the anionic compound is administered to the subject For example, the pharmaceutically acceptable formulation, "slow release" formulation, can provide sustained delivery of the anionic compound to a subject for one, two, three or four weeks after the anionic

IN

-27compound is administered to the subject. Alternatively, the pharmaceutically acceptable O formulation may provide sustained delivery of the anionic compound to a subject for C€ more than 30 days after the anionic compound is administered to the subject.

The pharmaceutical formulation, used in the method of the invention, contains a therapeutically effective amount of the anionic compound. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to 0 achieve the desired result. A therapeutically effective amount of the anionic compound may vary according to factors such as the disease state, age, and weight of the subject, IN and the ability of the anionic compound (alone or in combination with one or more other agents) to elicit a desired response in the subject. Dosage regimens may be adjusted to

C

provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anionic compound are outweighed by the therapeutically beneficial effects. A non-limiting range for a therapeutically effective concentration of an anionic compound is 100 mM to 1 mM. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the anionic compound and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

Preferred compounds of the invention include sulfates, sulfonates, phosphates, carboxylates, and compounds which include combinations of these functional groups.

Particularly preferred compounds include substituted and unsubstituted lower alkyl sulfates and sulfonates (including without limitation, 1,4-butanediol disulfate, sodium.

taurine (sodium 2-amino-ethanesulfonate), and homotaurine aminopropanesulfonic acid). Other preferred compounds include 3-(cyclohexylamino)- 1-propane sulfonate, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonate, 3-(Nmorpholino)propanesulfonic acid, sodium tetrahydrothiophene-1,1-dioxide-3,4-disulfate trihydrate, sodium 4-hydroxybutane-1-sulfonate, sodium 1,3,5-pentanetriol trisulfate, 2-.

aminoethyl hydrogen sulfate.-phosphonoformic acid, phosphonoacetic.acid, or indigo carmine. A preferred compound is 3-aminopropanesulfonic acid, or a salt thereof (see Example, infra).

In another aspect, the invention provides a method for inhibiting an inflammatory process an inflammatory process due to the presence of, or activation of macrophages by, an amyloidogenic protein or peptide). The method comprises administering to a subject in need thereof a subject having amyloid deposition) an effect therapeutic amount of an anionic compound, such that the inflammatory process is -28inhibited, by inhibition ofmacrophage activation by an amyloidogenic protein or o peptide, such as Ap. In a preferred embodiment, the subject is a subject suffering from c Alzheimer's disease. In certain embodiments, the anionic compound is a compound represented by Formulas I or II. Preferred therapeutic compounds include sulfates, sulfonates, phosphates, carboxylates, and compounds which include combinations of these functional groups. Particularly preferred compounds include substituted and unsubstiruted lower alkyl sulfates and sulfonates (including without limitation, 1,4- 00 butanediol disulfate, sodium 1,5-pentanedisulfonate, taurine (sodium 2 -amino- N ethanesulfonate), and homotaurine (3-aminopropanesulfonic acid). Other preferred compounds include 3 -(cyclohexylamino)-l-propane sulfonate, 4-(2-hydroxyethyl)-1- N piperazine-ethanesulfonate, 3-(N-morpholino)propanesulfonic acid, sodium tetrahydrothiophene- 1,1-dioxide-3,4-disulfate trihydrate, sodium 4-hydroxybutane- sulfonate, sodium 1,3,5-pentanetriol trisulfate, 2-aminoethyl hydrogen sulfate, phosphonoformic acid, phosphonoacetic acid, or indigo carmine. A preferred compound is 3 -aminopropanesulfonic acid, or a salt thereof.

Example 1 Macrophages (Bone-marrow derived macrophages RAW cells) were incubated in serum free medium with Ai1 -40 fibrils (AI 1-40 is a polypeptide corresponding to residues 1-40 of the Ap protein) (final concentration 2.5 tM) with or without the presence of lipopolysaccharide (LPS) (0.01 pg/ml) as a co-inducer of activation. The macrophages were incubated overnight. Supematants were harvested and inflammatory cytokines TNFa, IL-6, as well as nitric oxide were measured. TNFa, IL-6-were measured by an ELISA, while NO was measured by Griess Reagent.

Negative controls consisted of cells incubated with LPS or AP alone. Positive control consisted of cells incubated with LPS and IFNy at concentrations known to induce an optimal activation of these cells.

As shown in.Figures 1 and 2, 3-aminopropanesulfonic acid (present in solution as the salt form) was -found to block about 60% of the Ap-mlnduced TNFa production, while IL-6 production was not shown to be affected. NO production was also shown to be inhibited by 3 -aminopropanesulfonic acid, while this compound did not appear to have any significant effect on the TNFa and NO produced by RAW cells in presence of LPS and IFNy.

The contents of all references, issued patents, and published patent applications cited throughout this application, including the background, are hereby incorporated by reference.

IO

-29- Example 2 O The following example demonstrates the ability of compounds of the invention to inhibit Ap-induced microglia activation.

Human microglia THP-1 cells were primed with LPS (lipopolysaccharide) (0.25 .g/ml) and then incubated with a 5 uM preparation of fibrillary AP peptide. Activation \0 was determined by measuring the amount of IL-I P released in the cell culture Ssupernatant. The ability of a compound to block/inhibit the activation process was Sdetermined by comparing the amount of cytokine (here, IL- 13) present in the Is0 supernatant when cells were incubated with a compound to that obtained in the supernatant of control cells (incubated with Ap).

When cells were treated with a sulfonated compound, here, 3aminopropanesulfonic acid, a significant decrease (shown in FIG. 3) in the amount of IL-lp was seen at concentration of 10" M to 10-3M, indicating inhibition of microglia.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims (16)

1. 3. The method of claim 1 wherein said anionic compound has the structure: X II ,P-(CYlY 2 )nC(X)XR3 RIX I Z wherein Z is XR 2 or R 4 R 1 and R 2 are each independently hydrogen, a substituted or unsubstituted aliphatic group, an aryl group, a heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a salt-forming cation; R 4 is.hydrogen, lower alkyl, aryl or amino; X is, independently for each occurrence, O or S;-Y l and Y 2 are each independently hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n is an integer from 0 to 12.
2. 4. Themethod of claim 1 wherein said anionic compound is selected from the group consistingof- 1,4-butanediol disulfate, sodium taurine (sodium 2 -amino-ethanesulfonate), homotaurine (3- aminopropanesulfonic acid). 3 -(cyclohexylamino)-1 -propane sulfonate, 4-(2- hydroxyethyl)-1 -piperazine-ethanesulfonate, 3-(N-morpholino)propanesulfonic acid, sodium tetrahydrothiophene-1,1-dioxide-3,4-disulfate trihydrate, sodium 4- hydroxybutane- -sulfonate, sodium 1, 3 ,5-pentanetriol trisulfate, 2-aminoethyl hydrogen sulfate, phosphonoformic acid, phosphonoacetic acid, indigo carmine, pharmaceutically acceptable salts thereof. ID -31- The method of claim 1, wherein the anionic compound is 3- 0 aminopropanesulfonic acid, or a pharmaceutically acceptable salt thereof.
3. 6. A method for inhibiting an inflammatory process, comprising: administering to a subject in need thereof an effect therapeutic amount of INO an anionic compound, such that the inflammatory process is inhibited. 0 OO
4. 7. The method of claim 6 wherein said anionic compound has the structure O 10 wherein Y- is an anionic group at physiological pH,-Q is a carrier molecule; X is a cationic group; and n is an integer selected such that the biodistribution of -the compound for an intended target site is not prevented while maintaining activity of said compound.
5. 8. The method of claim 6 wherein said anionic compound has the structure: X II ,P-(CYlY 2 )nC(X)XR 3 RIX I Z wherein Z is XR 2 or R 4 R I and R 2 are. each independently hydrogen, a substituted or unsubstituted aliphatic group, an aryl group, a heterocyclic group, or a salt-forming cation; R 3 is hydrogen, lower alkyl, aryl, or a salt-forming cation; R 4 is hydrogen, lower alkyl, aryl or amino; X is, independently for each occurrence, O or S; yl and Y 2 are each independently hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n is an integer from 0 to 12. The method of claim 6 wherein said anionic compound is selected from the group consisting of 1,4-butanediol disulfate, sodium taurine (sodium 2-amino-ethanesulfonate), homotaurine (3- aminopropanesulfonic acid). 3-(cyclohexylamino)- -propane sulfonate, 4-(2- hydroxyethyl)-l-piperazine-ethanesulfonate, 3-(N-morpholino)propanesulfonic acid, sodium tetrahydrothiophene-1,1 -dioxide-3,4-disulfate trihydrate, sodium 4- hydroxybutane-1-sulfonate, sodium 1,3,5-pentanetriol trisulfate, 2-aminoethyl hydrogen sulfate, phosphonoformic acid, phosphonoacetic acid, indigo carmine, pharmaceutically acceptable salts thereof. IND -32- The method Of claim 9, wherein the anionic compound is 3- am ifopropanesulfonic acid, or a salt thereof. It1. The method of claim 9, wherein the subject is a subject suffering from Alzheimer's disease.
6. 12. The method of claim 11, wherein the inflammatory process is due to activation of macrophages, by an amyloidogenic protein or peptide, or a fragmeni thereof. N
7. 13. The method of claim 12, wherein the arnyloidogenic protein or peptide is AP.
8. 14. Use of an amyloidogenic protein or peptide with an anionic compound for the manufacture of a medicanment for inhibiting macrophage activation. The use of claim 14, wherein said anionic compound has the structure: wherein Y- is an anionic group at physiological pH-; Q is a carrier molecule; X+ is a cationic group; and a is an integer s~lected such that the biodistribution of the compound for an intended target site is not prevented while maintainiing activity of said compound. -16. The use of claim 14, wherein, said anionic compound has the structure: x IP-(C Y.Y 2 )nC (X)XR 3 wherein Z is XR 2 or R 4 R1 and R 2 are each.independendly hydrogen, a. substituted or unsubstituted aliphatic group, an aryl group, a heterocycl~c group. or a salt-forming cation; R3) is hydrogen, lower alkyl, aryl, or a salt-fornin. cation; R 4 is hydrogen, lower alkyl, aryl- or amino; X is, independently for each: occurrence, 0 or S; Y I and Y? are eacti independently hydrogea, halogeit alkyvl.* amino, hydroxy. alkoxy, or aryloxy; and n is an integer from 0 to 12..
9. 17. The use of claim 14, wherein said anionic compound is selected from the IND -33- group consisting of 1,4-butanediol disulfate, sodium taurine (sodium 2 -amino -ethanesul fonate), homotaurine (3- oaminopropanesuifonic acid). 3-(cyciohe~xvlamnino)-li-propane sulfonate. 4-(2- hydroxyethyl)- I -piperazi6ne-ethanesulfonate. 3-(N-morpholIino)p ro pane sul fon ic acid, sodium tetrahlydrothiophene-l .1 -dioxide-3 ,4-disulfate trihy.drate. sodium 4- hydroxybutane- I -suifonate, sodium I ,3,5-pentanetriol trisulfate, 2-aminloethli hydrogen sulfate, phosphonofor-mic acid, phosphionoacetic acid, indigo carmine. 0 phantnaceuticalLy acceptable salts dicreof. 00
10. 18. The use of claim 14, wherein said anionic compound is 3- IND aminopropanesulfonic acid, or a pharmaceutically acceptable salt thereof..
11. 19. Use of an anionic compound for the manufacture of a medicament for inhibiting an inflammatory process. The use of claim 19, wherein said anionic compound has the structure: wherein Y- is an. anionic group at physiological pH; Q is a carrier molecule; X+ is a cationic group; and n1 is an integer seiected such that the biodistribution of the compound for an intended target site is not prevented while maintaining activity of said compound.
12. 21. The use of claim 19, wherein said anionic compound has the structue: x RI I wherein Z is )V-2 or R 4 RI and R2 are each independently hydrogen, ai substituted or unsubstituted aliphatic group. an aryl group, a heterocyclic group,- or a salt-forming cation; R- 3 is hydrogen, lower alkyl, aryl, or a salt-forming. cation:, R 4 is hydrogen, lower alkyl, aryl or amino; X is, independently for'each' occurrence, 0 or S; Y I and V 2 are each independently hydrogen Maogen-. alkyl, amino. hydroxy, aLkoxy, or aryloxy; and n is an integer from 0 to 12.
13. 22. The use of claim 19, wherein said anionic compound is selected from the IND3 group conSisting of 1 ,4-butndjol disulfate, sodium I 5 -pcntanedisult'onale, taurine (sodium 2 -amino-ethicsulforitc), hornotaunine (3- 0 anLopro panesulfonic acid). 3 -(cycIohexv~arnino)- I -propane sulfonate. 4-(2- 0hvdroxyelthyj -pi peraine-ehanes ul fo natce, 3 -(N-morpholino)p 'rpariesulfornic acid. sodium tetralivdrothiophene.. I -dioxidc-3 .4-disul face trihydrace. sodium 4- hydroxvbu tane- I -suifonate. sodium 1.3 ,S-pentatietrio I trisulfate. 2-arni nety hy.,dro6zeri sulfate. phosphonoforrnic acid, phosphionoacetic acid, indigo c armne, pharmnaceutically acceptable salts thereof. 00is3
14. 23. The use afcai 22, wherein said anionic compoundis3 IND ~~amiiuproparicsulfon-ic acid, or a sl hro N1
15. 24. The use of claim 22, wherein said i fam iatoxy process cauies AIlzheirner. The use of claim 24, wherein said inflammatory processis due to avivation of macrophages by an amyloidogenic protein or peptide, or a fragment thereof.
16. 26. The use of claim 25, wherein the anayloidogenic protein or peptide is AP3. DATED this 13th Day of October Sheiston IP Attorneys for Neurochem (international) Limited