MXPA01006753A - Methods for treating certain diseases using naaladase inhibitors - Google Patents

Abstract

A method for regulating the release of TGF-&bgr;from living cells in vitro or in vivo, comprising bringing the cells into contact with an effective amount of a NAALADase inhibitor. Such methods are believed to be useful for affecting neuroregeneration, cell proliferation, cell differentiation, extracellular matrix formation, myelination, inflammation, immune function, liver function, pancreatic function, angiogenesis, or wound healing;and/or preventing or treating diabetes.

Description

METHODS TO TREAT CERTAIN DISEASES USING NAALADAS INHIBITORS BACKGROUND OF THE INVENTION The present invention relates to a method for regulating the release of transforming growth factor β (beta), commonly known as "TGF-β". Most particularly, the invention relates to the use of TGF-β regulators to prevent and / or treat neural tissue damage resulting from ischemia and reperfusion injury, neurological disorders and other neurodegenerative diseases; to prevent or treat vascular attack; or to treat or prevent other disorders such as arthritis, diabetes, inflammatory disorders, immune system disorders and cancer. Transforming growth factor β ("TGF-β") is recognized as a prototype of multifunctional growth factors. TGF-β regulates a variety of important cellular and tissue functions, such as cell growth and differentiation, angiogenesis, immune function, extracellular matrix production, cellular chemotaxis, apoptosis, and hematopoiesis. Members of the TGF-β superfamily are widely distributed in most adult and embryonic tissues expressing at least one member of the family. Active TGF-β is a disulfide-linked homodimer consisting of two chains of 112 amino acids. Following the entangled disulfide bond between two pro-TGF-β peptides, proieolytic processing at a tetrabasic point divides the mature TGF-β domain from the amino terminal portion of pro-TGF-β, which is called the protein associated with latency ( LAP). However, mature TGF-β remains non-covalently associated with LAP, and this is the latent form of TGF-β that is secreted by most cells in vitro. The latent complex does not bind to the TGF-β receptor and does not elicit a biological response. In vitro treatment of the latent complex with acid, alkali, chaotropic agents or heat releases TGF-β, biologically active, but the in vivo mechanism of activation is not completely clear. Since many cell types express both TGF-β and its receptor, however, the activation of latent TGF-β is thought to be a critical control point in the regulation of various biological actions of TGF-β. The biological actions of TGF-β are mediated by its binding to a heteromeric transmembrane receptor complex of two designated subunits type I (Rl) and type II (Rll), which are approximately 55 and 80 kDa, respectively. The current model of ligand-receptor interaction of TGF-β proposes that Rll, but not Rl, can bind TGF-β. The binding of TGF-β to Rll induces the assembly of an RI-RI heterodimer, Rl transphosphorylation by Rll, and then the activation of signal transduction pathways to elicit a biological response. Approximately six mammalian type II and four type I receptors have been cloned, and demonstrate different specificities and affinities to bind to different members of the TGF-β superfamily. It has recently been shown that disruption of the TGF-β signaling pathway may be involved in the pathogenesis of human cancer. It is known that TGF-β suppresses the growth of epithelial cells, and a disruption of this pathway can lead to uncontrolled proliferation. Disruption at any point of the TGF-β signaling pathway may contribute to the loss of tumor suppressor activity. In the nervous system, it is thought that a loss of neuroprotective actions of TGF-β can result in mutations of components of the signaling system of TGF-β in neurons and may contribute to chronic neurodegenerative diseases. One of the best characterized in vivo actions of TGF-β is its ability to mediate a wound healing cascade, which results in accelerated tissue preparation. At the point of a peripheral wound, the degranulation of platelets releases a bolus of TGF-β, which initiates a number of biological responses. Monocytes, lymphocytes, neutrophils and fibroblasts are brought to the wound site as a result of chemotactic activity of TGF-β. The autoinduction of TGF-β in a number of cell types maintains high levels of growth factor in the fold of the wound, where it induces angiogenesis and production of extracellular matrix to aid in tissue repair.

TGF-β may have similar functions with respect to tissue repair in the central nervous system as it does in peripheral organs. Neuronal injury can result from a variety of injuries, including physical trauma, hypoxia, excitotoxins, cytotoxins, reactive oxygen species, neurotrophic factor deprivation, or infection. The expression of TGF-β frequently increases in areas of neuronal dysfunction. Additionally, TGF-β maintains neuronal survival and reduces the size of the vascular accident in a number of animal or mammalian vascular accident models. A local inflammatory response occurs as part of the wound healing procedure of the central nervous system, and then resolves when the damaged area is repaired. The TGF-β produced by glial cells disappears when the inflammatory response subsides. Under these circumstances, it appears that TGF-β may be effective in reducing neuronal damage or providing neuroprotection against damage, for example, by amyloid plaques of Alzheimer's disease or excitatory lesions. Activation of metabotropic glutamate receptors (mGluR), which are selectively activated by N-acetylaspartylglutamate, in glial cultures has been reported to regulate the release of TGF-β. Bruno et al., "Neutralizing Antibodies for TGF-ß Prevent Neuroprotection Mediated by Group-ll Metabotropic Glutamate Receptors (mGluRs) in Cortical Cultures", Neurosci. Abs., 2299 (1997); and Wroblewska et al., "N-Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells", J. of Neurochemistry, 69: 1, 174-81 (1997). Thus, only some compounds of natural occurrence have been used to increase the activity of TGF-β. However, the topics of synthetic and pure arise whenever materials, proteins or other large molecules derived naturally, in vivo are used. Accordingly, there is a need for relatively small molecules to regulate the release of endogenous TGF-β, both to produce more reliable effects and to exemplify the synthesis of pharmaceutically useful compounds.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for treating a disease or condition in a mammal by administering an effective amount of NAALADase inhibitor to the mammal in need of such treatment. The disease or condition can be selected from the group consisting of neurodegenerative disorders, diseases related to cell growth, infectious diseases, wound healing, diseases related to the immune system, scarring of epithelial tissue, vascular diseases of the collagen, fibroproliferative disorders, connective tissue, inflammatory diseases, respiratory insufficiency syndrome and infertility.

In another embodiment, the disease or condition to be treated includes the impairment of immune function, extracellular matrix formation disorders, diabetes, autoimmune disorders, inflammatory diseases, disorders related to cell growth wherein the cells are selected from the group consisting of cells renal, hematopoietic cells, lymphocytes, epithelial cells, neuronal cells and endothelial cells. In another modality the method includes the treatment of a disease or condition that is evidenced by the level! abnormal of TGF-ß.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings, compound 3 is 2- (phosphonomethyl) pentanedioic acid. Figure 1 is a bar graph showing the effect on the concentration of TGF-β1 of compound 3 in cell cultures during a 20-minute ischemia. Figure 2 is a bar graph showing the effect on TGF-ß2 concentration of compound 3 in cell cultures during 20 minute ischemia. Figure 3 is a bar graph showing the reversal of the neuroprotective effect of compound 3 by neutralizing antibodies to TGF-β.

Figure 4 is a bar graph showing the non-reversal of the neuroprotective effect of compound 3 by neutralizing antibodies to FGF. Figure 5 is a bar graph showing the neuroprotective effect of compound 3 by the pretreatment of TGF-β neutralizing antibodies in rats subjected to medial cerebral arterial occlusion (MCAO). Figure 6 is a bar graph showing the levels of TGF-β1 during ischemia and reperfusion after treatment of rats with MCAO with compound 3, as compared to the vehicle-only treatment.

DETAILED DESCRIPTION OF THE INVENTION Definitions "Compound 1" refers to the pure and impure forms of 2- (2-sulfanylethyl) pentanedione, or the compound prepared by Example 23. "Compound 2" refers to the acid 2- [. { (2,3,4,5,6-pentafluorobenzyl) hydroxyphosphinyl} methyl] pentanedioic acid. "Compound 3" refers to 2- (phosphonomethyl) pentanedioic acid (PMPA).

"Effective amount" refers to the amount required to produce the desired effect. "Therapeutically effective amount" refers to the amount required to treat diseases, disorders, or conditions cited herein or that one skilled in the art knows as being conducive to such treatment and in an amount capable of taking effect, modifying or altering in a manner detectable treatment of the disease, disorder or condition. "Isteros" refers to elements, molecules or ions that have similar or identical physical properties, due to similar or identical arrangements of outer shell electrons. Two isostere molecules may have similar or identical volumes and shapes. Ideally, isostero compounds should be isomorphic and capable of co-crystallization. Among the other physical properties that isostero compounds commonly share, are the boiling point, density, viscosity and thermal conductivity. However, generally certain properties are different: dipole moments, polarity, polarization, size and shape, since the external orbitals can be hybridized differently. The Practice of Medicinal Chemistry, Academic Press, 1996. "Esters of carboxylic acid" include, without limitation, direct derivatives such as hydroxamic acids, acylcyanamides and aciisulfonamides; flat acid heterocycles such as tetrazoles, mercaptoazoles, sulfinylazoles, sulfonylazoles, isoxazoles, isothiazoles, hydroxythiadiazoles and hydroxychromes; and non-planar acid functions derived from sulfur or phosphorus such as phosphinates, phosphonates, phosphonamides, sulfonates, sulfonamides and aciisulfonamides. The Practice of Medicinal Chemistry, Academic Press, 1996. "Metabolite" refers to a substance produced by metabolism or by a metabolic process. "Pharmaceutically acceptable equivalent" includes, without limitation, pharmaceutically acceptable salts, hydrates, metabolites, prodrugs, and isostere carboxylic acids. It is expected that many pharmaceutically acceptable equivalents have an activity in vitro or in vivo equal or similar to that of the compounds of the formulas I-VI. "Pharmaceutically acceptable salt" refers to a salt of the compounds of the invention which possess the desired pharmacological activity and which are not disadvantageously neither biologically nor otherwise. The salt can be formed with inorganic acids, such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate. , hexanoate, hydrochloride, bromohydrate, iodohydrate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate and undecanoate. Examples of a base salt include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine, N-methyl salts -D-glucamine, and salts with amino acids such as arginine and lysine. Also groups containing basic nitrogen can be quaternized with agents including: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halogenides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as benzyl and phenethyl bromides. "Pharmaceutically acceptable prodrug" refers to a derivative of the compounds of the invention that undergo biotransformation before exhibiting its pharmacological effect or effects. The prodrug is formulated with the aim of improving its chemical stability, improving patient acceptance and compliance, improving bioavailability, prolonging the duration of action, improving organ selectivity, improving the formulation (for example, increasing water solubility), and / or reduce side effects (eg, toxicity). The prodrug can be easily prepared from the compounds of the invention using methods known in the art, such as those described in "Burger's Medicinal Chemistry and Drug Chemistry", fifth edition, vol. 1, pgs. 172-178, 949-982 (1995). For example, the compounds of the invention can be transformed into prodrugs by converting one or more of the hydroxy or carboxy groups into esters. "Alkyl" refers to a branched or unbranched saturated hydrocarbon chain comprising the designated number of carbon atoms. For example, a straight or branched C? -C6 alkyl hydrocarbon chain contains from 1 to 6 carbon atoms, and includes, without limitation, substituents such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertbutyl, -pentyl, n-hexyl and the like, unless otherwise indicated. "Alkenyl" refers to an unsaturated, branched or unbranched hydrocarbon chain, comprising the designated number of carbon atoms. For example, a straight or branched C2-C2 alkenyl hydrocarbon chain contains from 2 to 6 carbon atoms and has at least one double bond, and includes, without limitation, substituents such as ethenyl, propenyl, isopropenyl, butenyl , isobutenyl, terbutenyl, n-pentenyl, n-hexenyl and the like, unless otherwise indicated. "Alkoxy" refers to the group -OR, where R is alkyl as defined herein. Preferably, R is a branched or unbranched saturated hydrocarbon chain containing from 1 to 6 carbon atoms. "Aryl" refers to a carbocyclic or heterocyclic aromatic moiety, which may be unsubstituted or substituted. The term includes mono-, bi- and tricyclic rings of 5 to 8 members and fused rings, wherein the ring is unsubstituted or substituted in 1 to 5 positions with halo, haloalkyl, hydroxyl, nitro, haloalkyl, branched-chain alkyl or straight Ci to Ce, branched chain alkenyl or straight from C2 to Ce, Ci to CQ alkoxy, C2 to C6 alkenyloxy, phenoxy, benzyloxy, amino, thiocarbonyl, ester, thioester, cyano, imino, alkylamino, aminoalkyl, sulfhydryl, thioalkyl, and sulfonyl. When the aryl ring is heterocyclic, it may contain from 1 to 4 heteroatoms selected from the group consisting of O, N and S. The term "aryl" includes the case wherein aromatic or tertiary alkylamines are oxidized to a corresponding N-oxide. "Phenyl" includes all isomeric phenyl radicals, optionally mono-substituted or multi-substituted with non-interference substituents selected from the group consisting of amino, imino, alkylamino, aminoalkyl, -NR2 wherein R2 is selected from the group consisting of hydrogen, straight chain alkyl or branched chain from Ci to C6, straight or branched chain alkenyl of C3 to Ce or alkenyl; halo; haloalkyl such as trifluoromethyl and iodoisopropyl; hydroxy; straight or branched chain alkyl of C1 to C6; straight or branched chain alkenyl of C2 to C6; carbonyl or thiocarbonyl; ester or thioester; alkoxy or alkenoxyl; cyano; nitro; sulfhydryl, thioalkyl, or sulfonyl: and C1 to C4 bridging alkyl, as well as when a bridging alkyl substituent forms a heterocyclic ring fused to the aryl group. "Halo or halogen" refers to fluorine, chlorine, bromine and iodine, unless otherwise indicated. "Isomers" refers to compounds that have the same number and type of atoms, and therefore the same molecular weight, but are different with respect to the arrangement or configuration of their atoms.

"Stereoisomers" refers to compounds that have identical chemical constitution, but differ with respect to the arrangement in space of their atoms or groups. "Optical isomers" refers to any of two types of stereoisomers. A type is represented by mirror image structures called enantiomers, which originate from the presence of one or more asymmetric carbon atoms in the compound (glyceraldehyde, lactic acid, sugars, tartaric acid, amino acids). The other type is exemplified by diastereoisomers, which are not mirror images. These occur in compounds that have two or more asymmetric carbon atoms; thus, these compounds have 2n optical isomers, where n is the number of asymmetric carbon atoms. "Enantiomers" refers to stereoisomers that are mirror images that can not be superimposed on one another. "Enriched enantiomer" refers to a mixture in which an enantiomer predominates. "Racemic" refers to a mixture that contains equal parts of individual enantiomers. "Non-racemic" refers to a mixture containing unequal portions of individual enantiomers. "Animal or mammal" refers to a living organism that has sensitivity and power of voluntary movement and requires for its existence of oxygen and organic food. Examples include, without limitation, a mammal such as a member of the human, equine, porcine, bovine, murine, canine or feline species. In the case of a human, the term "animal" can also be referred to as "patient". "Disease" refers to any deviation or interruption of the normal structure or function of any part, organ or system (or combination thereof) of the body, which is manifested by a characteristic series of signs and symptoms whose etiology, pathology and prognosis They can be known or unknown. "Dorland's Illustrated Medical Dictionary" (Dorland's glossary medical dictionary) (W.B. Saunders Co., 27th edition, 1988). "Disorder" refers to any disorder or abnormality of a function, a morbid physical or mental state. "Dorland's Illustrated Medical Dictionary" (W.B. Saunders Co., 27th edition, 1988). The term "release", as used herein, covers the release of ATGF-β in a substance. The term "TGF-β" as used herein refers to transforming growth factor beta. The term "regulation", in the context of endogenous release, refers to the production of a statistically significant increase in the exogenous concentration of TGF-β compared to the concentration occurring in the absence of the compound of the invention. Preferably, this is a physiologically important amount that results in the observation of a desired biological effect either in vitro or in vivo.

The term "Treatment" as used herein covers any treatment of a disease and / or condition in an animal or mammal, particularly a human, and includes: (i) preventing the occurrence of a disease, disorder or condition in an animal who may be predisposed to the disease, disorder and / or condition, but has not yet been diagnosed as having it; (ii) inhibit the disease, disorder or condition, that is, stop its development; and / or (iii) alleviating the disease, disorder or condition, i.e., causing the regression of the disease, disorder and / or condition. "Acid-containing metallic chelating agent" refers to any compound that has (i) a functional group capable of interacting with the metals at the active site of the NAALADase enzyme, and (ii) an acidic portion that interacts at the recognition site of the NAALADase enzyme. "Disease or disorder related to NAALADase" refers to any disease or disorder specifically known in the art as capable of treatment by administration of the NAALADase inhibitor. "Neurodegenerative disorder" refers to conditions and disorders in which neurons have been surgically damaged, chemically, by acute or chronic disease procedures such as diabetes, Alzhaimer, or Parkinson's, Guillain Barre, or similar events and where the neurons are actively stimulated or incited to regrowth or regeneration in a manner similar to the neurotrophic actions of neural growth factors but other than passive treatments known in the art such as the prevention of other neurodegenerative and neuroprotective effects. "Abnormal level of TGF-β" refers to a variation capable of being measured from normal levels of TGF-β as determined by those skilled in the art and which is the causative agent of, related to, mediator of, or evidencing the conditions related to TGF-β, diseases, disorders, or pathologies.

NAALADase Inhibitors Although not limited to any particular theory, it is believed that the NAALADase inhibitors used in the methods of the invention and pharmaceutical compositions modulate TGF-β levels and in particular by increasing the levels of TGF-β , and / or the NAALADase inhibitors are believed to inhibit the activity of myeloperoxidase. A preferred NAALADase inhibitor is a compound of formula I: or a pharmaceutically acceptable equivalent, wherein: Y is CR3R4, NR5 or O; Ri is selected from the group consisting of hydrogen, C1-C9 alkyl, C2-C9 alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, Ar, COOR, NR6R7 and OR, wherein said alkyl, alkenyl, cycloalkyl and cycloalkenyl are unsubstituted or substituted with one or more substituents independently selected from the group consisting of carboxy, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, halogen, hydroxy, nitro, trifluoromethyl, Ci-Cß alkyl, alkenyl of C2-C6, C1-C9 alkoxy, C2-C9 alkenyloxy, phenoxy, benzyloxy, COOR, NRβRz and Ar; R2 is selected from the group consisting of hydrogen, Ci-Cß alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, Ar, halogen and carboxy, wherein said alkyl, alkenyl, cycloalkyl and cycloalkenyl are not substituted or are substituted with one or more substituents independently selected from the group consisting of carboxy, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, halogen, hydroxy, nitro, trifluoromethyl, Ci-Cß alkyl, C2 alkenyl -C6, C1-C9 alkoxy, C2-C alkenyloxy, phenoxy, benzyloxy, NR6R7 and Ar; R3 and R4 are independently hydrogen or C1-C3 alkyl; R5 is hydrogen or C1-C3 alkyl; R, Re and R7 are independently selected from the group consisting of hydrogen, C1-C9 alkyl, C2-C9 alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl and Ar, wherein said alkyl, alkenyl, cycloalkyl and cycloalkenyl are unsubstituted or substituted by one or more substituents independently selected from the group consisting of carboxy, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, halogen, hydroxy, nitro, trifluoromethyl, Ct-Cß alkyl, C2 alkenyl -C6, C1-C9 alkoxy, C2-C9 alkenyloxy, phenoxy, benzyloxy and Ar; and Ar is selected from the group consisting of 1 -naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl, 4-indolyl, 2-furyl, 3-furyl, tetrahydrofuranyl, tetrahydropyranyl, 2-thienyl, 3-thienyl, -pyridyl, 3-pyridyl, 4-pyridyl and phenyl, wherein said Ar is unsubstituted or is substituted with one or more substituents selected from the group consisting of halogen, hydroxy, nitro, trifluoromethyl, CrC6 alkyl, C2- alkenyl C6, C -? - C6 alkoxy, C2-C6 anynyloxy, phenoxy, benzyloxy, carboxy and NR1R2. Preferably, Y is CH2. Preferably, when Y is CH2, then R2 is - (CH2) 2COOH. Most preferably, when Y is CH2 and R2 is - (CH2) 2COOH, then R1 is hydrogen, C? -C alkyl, C2-C alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, benzyl, phenyl or OR, wherein said alkyl, alkenyl, cycloalkyl, cycloalkenyl, benzyl and phenyl are unsubstituted or substituted with one or more substituents independently selected from the group consisting of carboxy, C3-C8 cycloalkyl, C5-C cycloalkenyl, halogen , hydroxy, nitro, trifluoromethyl, C 1 Ce alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyloxy, phenoxy, benzyloxy, NR6R7, benzyl and phenyl. Preferred compounds of formula I are selected from the group consisting of: 2- (phosphonomethyl) pentanedioic acid; 2 - [[(2-carboxyethyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [(Benzylhydroxyphosphinyl) methyl] pentanedioic acid; 2 - [(phenylhydroxyphosphinyl) methyl] pentanedioic acid; 2 - [[((hydroxy) phenylmethyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [(Butylhydroxyphosphinyl) methyl] pentanedioic acid; 2 - [[(3-methylbenzyl) hydroxyphosphinyl] methyl-pentanedioic acid; 2 - [(3-phenylpropylhydroxyphosphinyl) methy1] pentanedioic acid; 2 - [[(4-fluorophenyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [(methyIhydroxyphosphinyl) methyl] pentanedioic acid; 2 - [(phenylethylhydroxyphosphinyl) methyl] pentanedioic acid; 2 - [[(4-methylbenzyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(4-fluorobenzyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(4-methoxybenzyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(3-trifluoromethylbenzyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[4-trifluoromethylbenzyl) hydroxyphosphnol] methyl] pentanedioic acid; 2 - [[(2-fluorobenzyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(2,3,4,5,6-pentafluorobenzyl) hydroxyphosphinyl] methyl] -pentanedioic acid; and pharmaceutically acceptable equivalents. Preferably, the compound of formula I is 2 - [[(2,3,4,5,6-pentafluorobenzyl) hydroxyphosphinyl] methyl] pentanedioic acid or a pharmaceutically acceptable equivalent. Preferably, the compound of formula I is an enantiomer or a mixture enriched with enantiomers. Representative compounds of formula I in which Ri is substituted with COOR include, without limitation: 2 - [[(2-carboxypropyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(2-carboxybutyl) hydroxyphosphinyl] methyl] pentanedioic acid; 2 - [[(2-carboxypentyl) hydroxyphos) nyl] methyl] pentanedioic acid; 2 - [[(2-carboxy-3-phenylpropyl) hydroxyphosphoryl] -pentanedioic acid; 2 - [[(2-carboxy-3-naphthylpropyl) hydroxyphosphinyl] methyl] -pentanedioic acid; 2 - [[(2-carboxy-3-pyridylpropyl) hydroxyphosphinyl] methyl] -pentanedioic acid; 2 - [[(2-benzyloxycarbonyl) -3- (phenylpropyl) hydroxyphosphinyl] methyl] -pentanedioic acid; 2 - [[(2-methoxycarbonyl) -3- (phenylpropyl) hydroxyphosphinyl] methyl] -pentanedioic acid; 2 - [[((3-carboxy-2-methoxycarbonyl) propyl) hydroxyphosphinyl] methyl] -pentanedioic acid; 2 - [[((4-carboxy-2-methoxycarbonyl) butyl) hydroxyphosphinyl] methyl] -pentanedioic acid; and pharmaceutically acceptable equivalents.

Another preferred NAALADase inhibitor is a compound of formula II: or a pharmaceutically acceptable equivalent, wherein: X is a portion of formula (III), (IV) or (V): (III) (IV) (V) m and n are independently 0, 1, 2, 3 or 4; Z is SR13, S03R13, S02R? 3, SOR13, SO (NR13) Ri4 or S (NR13R14) 2R15; B is N or CR? 6; A is O, S, CR? 7R18 or (CR? 7R? 8) mS; R g, R g, Rio, R 11, i 2, R 13, R 14, R 15, R a, R 17 and e are independently hydrogen, straight or branched chain C 1 -C 9 alkyl, straight or branched chain C 2 -C 9 alkenyl, Cs cycloalkyl -Cß, C5-C7 cycloalkenyl, Ari, hydroxy, carboxy, carbonyl, amino, amido, cyano, socian, nitro, sulfonyl, sulfoxy, thio, thiocarbonyl, thiocyano, formaniiido, thioformamido, sulfhydryl, halogen, haloalkyl, trifluoromethyl or oxy, wherein said alkyl, alkenyl, cycloalkyl or cycloalkenyl are independently unsubstituted or substituted with one or more substituents; An is a carbocyclic or heterocyclic moiety that is unsubstituted or substituted with one or more substituents; with the proviso that when X is a portion of formula III and A is O, then n is 2, 3 or 4; when X is a portion of formula III and A is S, then n is 2, 3 or 4; and when X is a portion of formula III and A is (CR? 7Ri8) S, then n is 0, 2, 3 or 4. Possible substituents of said alkenyl, cycloalkyl, cycloalkenyl and An include, without limitation, C1 alkyl. -C9 straight or branched chain, C2-C9 straight or branched chain alkenyl, C1-C9 alkoxy, C2-C9 alkenyloxy, phenoxy, benzyloxy, Cs-Cs cycloalkyl, C5-C7 cycloalkenyl, hydroxy, carboxy carbonyl, amino, amido, cyano, socian, nitro, nitroso, nitrile, isonitrile, imino, azo, diazo, sulfonyl, sulfoxy, thio, thiocarbonyl, thiocyano, formanilide, thioformamido, sulfhydryl, halogen, haloalkyl, trifluoromethyl, and carbocyclic and heterocyclic. The carbocyclic portions include alicyclic and aromatic structures. Examples of useful carbocyclic and heterocyclic moieties include, without limitation, phenyl, benzyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, nanoyl, isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl, oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trityanil, indolizinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, thienium, tetrahydroisoquinolinyl, cinolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazoyl, acridinyl, phenazinyl, phenothiazinyl and phenoxazinyl. Representative compounds of formula 11 wherein X is a portion of formula IV, R8 is - (CH2) 2COOH, Rg is hydrogen, and B is CR16, include without limitation: 2- (dithiocarboxymethyl) pentanedioic acid; 2- (1-dithiocarboxyethyl) pentanedioic acid; and pharmaceutically acceptable equivalents. Representative compounds of formula II wherein X is a portion of formula IV, R8 is - (CH2) 2COOH, Rg is hydrogen, and B is N, include without limitation: 2-dithiocarboxylaminopentanedioic acid; 2 - [(N-methyldithiocarboxy) amino] pentanedioic acid; and pharmaceutically acceptable equivalents. Representative compounds of formula II wherein X is a portion of formula V, include without limitation: 2-benzyl-4-sulfanylbutanoic acid; 2-benzyl-4-sulfanylpentanoic acid; 2- (3-pyridylmethyl) -4-sulfanylpentanoic acid; 2- (3-pyridylmethyl) -4-sulfanylhexanoic acid; 2-benzyl-3-sulfanylpropanoic acid; 2-benzyl-3-sulfanylpentanoic acid; 2- (4-pyridylmethyl) -3-sulfanylpentanoic acid; and pharmaceutically acceptable equivalents. In a preferred embodiment of formula II, the NAALADase inhibitor is a compound of formula VI: Or a pharmaceutically acceptable equivalent, wherein: N is 0, 1, 2 0 3; Z is SH, SO3R13, S02R13, SOR13, or S (N2R13R? 4) R? 5; and A is CR17R? Preferably, Z is SH Most preferably, when Z is SH, then R-? 8 is - (CH2) 2COOH. Preferred compounds of formula VI are selected from the group consisting of: 2- (2-sulfanylethyl) pentanedioic acid; 3- (2-sulfanylethyl) -1,5,5-pentanetricarboxylic acid; 2- (2-Sulfanylpropyl) pentanedioic acid; 2- (2-sulfanylbutyl) pentanedioic acid; 2- (2-Sulfanyl-2-phenylethyl) pentanedioic acid; 2- (2-sulfanylhexyl) pentanedioic acid; 2- (2-Sulfanyl-1-methylethyl) pentanedioic acid; 2- [1- (Sulfanylmethyl) propyl] pentanedioic acid; 2- (3-sulfanylpentyl) pentanedioic acid; 2- (3-Sulfanylpropyl) pentanedioic acid; 2- (3-Sulfanyl-2-methylpropy) pentanedioic acid; 2- (3-Sulfanyl-2-phenylpropyl) pentanediol acid; 2- (3-sulfanylbutyl) pentanedioic acid; 2- [3-Sulfanyl-2- (phenylmethyl) propyl] pentanedioic acid; 2- [2- (Sulfanylmethyl) butyl] pentanedioic acid; 2- [2- (Sulfanylmethyl) pentyl] pentanedioic acid; 2- (3-Sulfanyl-4-methylpentyl) pentanedioic acid; and pharmaceutically acceptable equivalents.

Most preferably, the compound of the formula VI is selected from the group consisting of 2- (2-sulfanylethyl) pentanedioic acid; 2- (2-Sulfanylpropyl) pentanedioic acid; 2- (3-Sulfanylpropyl) pentanedioic acid and pharmaceutically acceptable equivalents. Most preferably, the compound of formula VI is an enantiomer or a mixture enriched with enantiomers. Other inhibitors of NAALADase can be found in the patents of E.U.A. Nos. 5,672,592, 5,795,877, 5,863,536, 5,880,112, the granted patent applications of E.U.A. Nos. 08 / 825,997, 08 / 833,628, 08 / 835,572 and 08 / 842,360 for which the shipping rights have been paid; the full contents of these patents, applications and publications are incorporated herein by reference. The compounds used in the methods and pharmaceutical compositions of the present invention possess one or more centers of asymmetric carbon and therefore may exist in the form of optical isomers and also in the form of racemic or non-racemic mixtures of optical isomers. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional procedures well known in the art, for example, by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluyltartaric acid and camphor sulfonic acid, and then separation of the diastereoisomer mixture by crystallization, followed by release of the optically active bases of these salts. A different procedure for the separation of optical isomers includes the use of a chiral chromatography column, chosen to optimally increase the separation of the enantiomers. Another available method includes the synthesis of covalent diastereoisomeric molecules, for example esters, amides, acetals, ketals and the like, by reacting the compounds used in the methods and pharmaceutical compositions of the invention, with an optically active acid in an activated form, a diol optically active or an optically active isocyanate. The synthesized diastereomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to release the enantiomerically pure compound. In some cases, hydrolysis to the original optically active drug is not necessary before its administration to the patient, since the compound can behave as a prodrug. The optically active compounds of the present invention can also be obtained using optically active starting materials. It is understood that the compounds of the invention encompass optical isomers and also racemic and non-racemic mixtures.

Synthesis of NAALADase inhibitors The NAALADase inhibitors of formula I can be easily prepared by standard techniques of organic chemistry. The precursor compounds can be prepared by the methods known in the art, such as those described by Jackson et al, J. Med. Chem., Vol. 39, No. 2, pp. 430-636. 619-622 (1996) and Froestl et al, J. Med. Chem. Vol. 38, pp. 3313-3331 (1995). Various means for synthesizing NAALADase inhibitors can be found in J. Med. Chem., Vol. 31, pp. 204-212 (1988); J. Med. Chem., Vol. 39, pp. 619-622 (1996); WO 98 / 45,256, published October 15, 1998; WO 98 / 45,257, published October 15, 1998, and WO 98/13044, published April 2, 1998, which are hereby incorporated by reference in their entirety. Some of the NAALADase inhibitors used in the methods of the inventand pharmaceutical composit can be easily prepared by standard techniques of organic chemistry, using the general synthetic standards described in the U.S. Patents. Nos. 5,672,592, 5,795,877, 5,863,536 and 5,880,112, and patent applicat of E.U.A. Nos. 08 / 825,997, 08 / 833,628, 08 / 835,572 and 08 / 842,360 for which the shipping rights have been paid, the total content of which is included hereby by way of reference.

Methods of the Present Invention Cell Growth The present inventprovides methods for stimulating the growth of tissue, glands, or organs in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. In a preferred embodiment, the growth of tissue, glands or organs promotes milk productor weight gain in an animal or mammal. In another embodiment, the present inventprovides methods for treating cell growth related to disorders in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. In a preferred aspect of this embodiment, the treated cells are selected from the group consisting of kidney cells, hematopoietic cells, lymphocytes, epithelial cells and endothelial cells. In another embodiment, the present inventprovides methods for treating neurodegenerative disorders in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. In a preferred aspect of this embodiment, the neurodegenerative disorder is selected from the group consisting of damage to the neural tissue resulting from reperfusinjury of ischemia, myelinat and neuroregenerat In another embodiment, the present inventprovides methods for treating a disease state in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal.

In a preferred aspect of this embodiment, the disease state is selected from the group consisting of stimulatof cell proliferat inhibitof cell growth, regulatof extracellular matrix proteins, arteriosclerosis, autocrine tumors, fibroplasia and keloid format In another preferred aspect, the stimulatof cell proliferatis selected from the group consisting of induced proliferatof fibroblasts in a semi-solid medium, growth of mesenchymal cells and stimulatof chondrogenesis, osteogenesis, and epithelial cell differentiat In another preferred aspect, the inhibitof cell growth is selected from the group consisting of inhibitof the proliferatof epithelial cells, endothelial cells, T and B lymphocytes, and thymocytes, inhibitof adipose express skeletal muscle and hematopoietic phenotypes, neoplasms, non-cytocidal viral infect or other pathogenic infect and autoimmune disorders. An especially preferred aspect is when the noncytocidal viral infector other pathogenic infectis selected from the group consisting of AIDS, herpes, CMV (cytomegalovirus), EBV (Epstein Barr virus), and SSPE (subacute sclerosis panencephalitis). Another preferred aspect is when the disease state is selected from the group consisting of pathogenesis, glomerulonephritis, liver cirrhosis, and pulmonary fibrosis.

Infectious Disease In another embodiment, the present invention provides methods for treating a mammal suffering from an infectious disease caused by a macrophage pathogen, the method comprising administering an effective amount of a NAALADase inhibitor to the mammal. A preferred aspect of this mode is when a macrophage pathogen is selected from the group consisting of bacteria, yeast, fungi, virus, protozoa, Trypanosoma cruzi, Histoplasma capsulatum, Candida albicans, Candida parapsilosis, Cryptococcus neoformans, Salmonella, Pneumocystosis, Toxoplasma, Listeria. , Mycobacteria, Rickettsia, Leishmania, and their combinations. Especially preferred mycobacteria include Mycobacterium tuberculosis and Mycobacterium leprae. Especially preferred toxoplasmas include Toxoplasma gondii. Especially preferred Rickettsia includes R. prowazekii, R. coronii, and R. tsutsugamushi. Preferred infectious diseases treated with this method include simple or multiple skin lesions, mucosal disease, Chagas disease, acquired immunodeficiency syndrome (AIDS), toxoplasmosis, leishmaniasis, trypanosomiasis, schistosomiasis, cryptosporidiosis, Mycobacterium avium infections, Pneumocystis carinii, pneumonia. and leprosy.

Mediation of Disease Resistance and Susceptibility In another embodiment, the present invention provides methods for mediating resistance to disease and susceptibility in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to said animal or mammal. .

Immunosuppression In another embodiment, the present invention provides methods for suppressing the cellular immune response in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. In another embodiment, the present invention provides methods for providing a therapeutic treatment in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. In another embodiment, the present invention provides methods for treating immunosuppression related to an infectious disease in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferably, immunosuppression is related to infection by trypanosome, viral infection, human immunosuppression virus, human T-cell lymphotropic virus (HTLV-1), lymphocytic corimingitis virus, and hepatitis.

Several disorders related to TGF-β Preferred therapeutic treatments include the inhibition of germ cell division, the inhibition of arotamase in developing ovaries, prevention or alleviation of respiratory deficiency syndrome in newborns, treatment of infertility, blocking of autophosphorylation of tyrosine from EGF receptors, and repair of delayed bone growth or traumatic bone injury. In another embodiment, the present invention provides methods for mitigating tissue damage induced by radiation in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferred tissue damage to treat include fibrosis, extracellular matrix remodeling, vascular damage, aberrant angiogenesis, pneumonitis, atherogenesis, osteonecrosis, mucositis, immunosuppression, and functional impairment. Preferred tissues to be treated by this method include liver, lungs, gastrointestinal tract, kidneys, breast, testes, salivary glands, mucosa, skin, and brain. The tissue of the breast is especially preferred.

Wound treatment In another embodiment, the present invention provides methods for inhibiting the formation of scar tissue during wound treatment, the method comprising administering an effective amount of a NAALADase inhibitor to a host suffering from a tissue wound. Preferred tissues to be treated by this method include skin or other epithelial tissues. Preferably, the tissue has been damaged by injuries resulting from an accidental injury, surgical operations, lacerations induced by trauma, wounds that involve the peritoneum by which excessive formation of connective tissue results in abdominal adhesions or other traumas. In a particularly preferred embodiment, the NAALADase inhibitor is administered at an early stage of healing.

Vascular diseases of collagen In another embodiment, the present invention provides methods for treating collagen vascular disease in an animal or mammal, the method comprising administering an effective amount of an inhibitor of collagen.

NAALADase to the animal or mammal. The vascular diseases of the collagen to be treated include progressive systemic sclerosis (PSS), polymyositis, scleroderma, dermatomyositis, eosinophilic fasciitis, morphea, Raynaud's syndrome, interstitial pulmonary fibrosis, scleroderma, or systematic lupus erythematosus.

Fibroproliferative Disorders In another embodiment, the present invention provides methods for treating a fibroproliferative disorder in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferred fibroproliferative disorders to be treated include diabetic nephropathy, kidney disease, proliferative vitreoretinopathy, liver cirrhosis, biliary fibrosis and myelofibrosis. Especially preferred renal diseases include proliferative mesenteric glomerulonephritis, growing glomerulonephritis, diabetic neopathy, renal interstitial fibrosis, renal fibrosis in transplant patients receiving cyclosporine and HIV-related nephropathy.

Connective Tissue Disorders In another embodiment, the present invention provides methods for treating a connective tissue disorder in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferred connective tissue disorders to be treated include scleroderma, myelofibrosis and hepatic, intraocular and pulmonary fibrosis.

Immune mediation In another embodiment, the present invention provides methods for increasing the effectiveness of a vaccine, the method comprising administering an effective amount of a NAALADase inhibitor to an individual about to receive a vaccine or to receive a vaccine. In another embodiment, the present invention provides methods for treating an allergy in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferably, the allergy is selected from the group consisting of allergy to dust and hay fever.

Polyp Formation In another embodiment, the present invention provides a method for inhibiting the formation of polyps in an animal or mammal, the method comprising administering an effective amount of a NAALADase inhibitor to the animal or mammal. Preferably, the polyp can be formed in the nose or intestine.

Inflammatory diseases In another embodiment, the present invention provides methods for treating inflammatory diseases. Without being limited to a particular mechanism, it is noted that the compounds of the present invention operate by two potential modes of action. The first, mediation and regulation of TGF-β, provides an effective treatment of inflammatory diseases. The second, the inhibition of myeloperoxidase, is also believed to provide an effective way to improve inflammatory diseases. Preferably, inflammatory diseases are related to: progressive systemic sclerosis (PSS), polymyositis, scleroderma, dermatomyositis, eosinophilic fasciitis, morphea, Raynaud's syndrome, interstitial pulmonary fibrosis, systemic lupus erythematosus, diabetic nephropathy, renal disease, proliferative vitreoretinopathy, liver cirrhosis , biliary fibrosis and myelofibrosis, mesangial proliferative glomerulonephritis, growing glomerulonephritis, diabetic neuropathy, renal interstitial fibrosis, renal fibrosis in transplant patients receiving ciclosporin and HIV-related nephropathy.

Route of Administration In the methods of the present invention, the compounds can be administered by any effective technique known in the art, including: orally, parenterally, by inhalation aerosol, topically, rectally, nasally, buccally, vaginally, or by a reservoir implanted in dosage formulations containing conventional non-toxic, pharmaceutically acceptable vehicles, adjuvants and excipients. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrastate, intracranial or intraosseous injection and infusion techniques. Invasive techniques are preferred, particularly direct administration to damaged neuronal tissue. In particular, to be therapeutically effective targeting the central nervous system, NAALADase inhibitors preferably must readily penetrate the blood-brain barrier when administered peripherally. Compounds that do not readily penetrate the blood-brain barrier can be effectively administered by an intraventricular route. The NAALADase inhibitors can also be administered in the form of sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic diluents or solvents, parenterally acceptable, for example as solutions in 1,3-butanediol. Among the vehicles and acceptable solvents that may be employed, there is water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any soft fixed oil such as a synthetic mono or diglyceride can be employed. In the preparation of injectables, fatty acids such as oleic acid and its glyceride derivatives, including olive oil and castor oil, are especially useful in their polyoxyethylated forms. These oil solutions or suspensions may also contain long chain alcohol diluents or dispersants. Additionally, the NAALADase inhibitors can be administered orally in the form of capsules, tablets, suspensions or aqueous solutions. The tablets may contain carriers such as lactose and corn starch, and / or lubricating agents such as magnesium stearate. The capsules may contain diluents including lactose and dried corn starch. The aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient. The oral dosage forms may additionally contain sweetening and / or flavoring and / or coloring agents. The NAALADase inhibitors can be administered rectally in the form of suppositories. These compositions can be prepared by mixing the drug with suitable non-irritating excipients that are solid at room temperature, but liquid at the rectal temperature, such that they melt in the rectum to release the drug. Such excipients include cocoa butter, beeswax and polyethylene glycols. In addition, NAALADase inhibitors can also be administered topically, especially when the conditions to be treated include areas or organs easily accessible by topical application, including neurological disorders of the eye, skin or lower intestinal tract. For topical application to the eye or ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic sterile saline with adjusted pH or, preferably, as a sterile isotonic saline with adjusted pH, with or without a preservative such as benzalkonium chloride. Alternatively, the compounds can be formulated into ointments such as those of petrolatum. For topical application to the skin, the compounds can be formulated in suitable ointments containing the suspended or dissolved compound, for example, in mixtures of one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene and polyoxypropylene compounds, emulsifying wax and water. Alternatively, the compounds can be formulated in suitable lotions or creams containing the active compound suspended or dissolved, for example, in a mixture of one or more of the following: mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, Cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical application to the lower intestinal tract can be done in rectal suppository formulations (see above) or in suitable enema formulations. The NAALADase inhibitors used in the methods of the present invention can be administered by a single dose, discrete multiple doses or continuous infusion. Since the compounds are small, easily diffusible and relatively stable, they are well suited for continuous infusion. For continuous infusion, pumping means are preferred, particularly subcutaneous pumping means.

Dosage Dosage levels of the order of about 0.1 mg to about 10,000 mg of the active compound are useful in the treatment of the aforementioned conditions, with preferred levels being from about 0.1 mg to about 1,000 mg. The specific dose level for any particular patient varies depending on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; the combination of drugs; the severity of the particular disease to be treated; and the form of administration. Typically, the in vitro results of the dose-response effect provide a useful guide on the appropriate doses for administration to the patient. Studies in animal models are also helpful. Considerations for determining appropriate dose levels are well known in the art. In a preferred embodiment, the NAALADase inhibitors are administered in lyophilized form. In this case, 1 to 100 mg of a NAALADase inhibitor of the present invention can be lyophilized in individual bottles, together with a vehicle and a pH regulator such as mannitol and sodium phosphate. The compound can be reconstituted in bottles with bacteriostatic water before administration. The NAALADase inhibitors that are used in the methods of the present invention can be administered in combination with one or more therapeutic agents, including chemotherapeutic agents. Table I provides known mean dosages for selected chemotherapeutic agents. The specific dose levels for these agents and other therapeutic agents will depend on considerations such as those mentioned above for the compounds of the present invention.

Administration Regimen For the methods of the present invention, any administration regimen that regulates the time and sequence of drug delivery can be used, and can be repeated as necessary to perform the treatment. Said regimen may include pretreatment and / or co-administration with additional therapeutic agents.

Combination with other treatments In the methods of the invention, the NAALADase inhibitors can be coadministered with one or more additional therapeutic agents, preferably other angiolytic agents, memory enhancing agents or agents capable of treating the main cause of memory impairment.

Examples of angiolytic agents that can be combined with NAALADase inhibitors include without limitation benzodiazepines (ciordiazepoxide, diazepam, clorazepate, flurazepam, halazepam, prazepam, clonazepam, cuazepam, alprazolam, lorazepam, oxazepam, temazepam, triazolam); barbiturates; ß blockers; and buspirona. The NAALADase inhibitors can be co-administered with one or more therapeutic agents either (i) together in a single formulation, or (ii) separately in individual formulations designed for rates of optimal release of their respective active agent. Each formulation may contain from about 0.01% to about 99.99% by weight, preferably from about 3.5% to about 60% by weight, of a NAALADase inhibitor, as well as one or more pharmaceutical excipients, such as wetting agents, emulsifiers and regulators of pH. The neurotropic NAALADase inhibitors of the present invention can be administered with other therapeutic agents. The following examples are illustrative of the present invention and are not intended to be limitations thereof. Unless indicated otherwise, all percentages are based on 100% by weight of the final composition.

EXAMPLES EXAMPLE 1 Cell Culture Data The compounds of the invention were used to produce a neuroprotective effect in both in vitro cell culture models and in vivo vascular accident models. SpecificallyWhen 2-phosphonomethylpentanedioic acid was added to ischemic cultures, the level of TGF-β1 and TGF-β2 increased significantly. These data show that the compounds of the present invention promote the release of considerably increased amounts of endogenous TGF-ßs from glial cells, and this, in turn, provides neuroprotection to surrounding neurons. TGF-β neutralizing antibodies were added to determine if the neuroprotective effect would be blocked. The neuroprotective effect of 2-phosphonomethylpentanodioic acid was blocked by the TGF-β neutralization bodies in this cell culture model. However, when other growth factor antibodies, such as FGF antibody were added, the neuroprotective effect of 2-phosphonomethylpentanedioic acid was not reversed in the culture. This indicates that the compounds are directly related to the levels of TGF-β during the vascular accident.

EXAMPLE 2 In Vivo Vascular Accident Model Data The effect of the neutralizing antibodies of TGF-β on the neuroprotection offered by 2-phosphonomethylpentanodioic acid following middle cerebral arterial occlusion (MCAO) in rats was also studied to give a more relevant model of vascular accident in vivo. Treatment of MCAO rats with 2-phosphonomethylpentanedioic acid caused a significant elevation in TGF-β during occlusion and reperfusion as reported by microdialysis, as shown in Figure 6. These data demonstrated that the inventive compounds exemplified by 2-phosphonomethylpentanedioic acid, provided neuroprotection, at least in part by regulating endogenous transformation growth factors. Additionally, the antibodies that neutralize TGF-β, significantly attenuated the neuroprotective effect of 2-phosphonemylpentanedioic acid, in vivo as shown in figure 5. In this way, it was observed that the regulation of TGF-ßs may have implications, not only in its utility in the vascular accident, but also in other neurological and psychiatric diseases. In addition, this mechanism may have implications in myelination, prostate cancer, inflammation, diabetes and angiogenesis.

EXAMPLE 3 In Vivo Toxicity of Naaladasa Inhibitors The toxicological effect in vivo of the inhibition of NAALADase has been examined in mice. The results show that the NAALADase inhibitors are non-toxic to mice, suggesting that they would be similarly non-toxic to humans when administered in therapeutically effective amounts. A representative description can be found in the patents of E.U.A. Nos. 5,672,592, 5,795,877, 5,863,536 and 5,880,112; and the granted patent applications of E.U.A. Nos. 08 / 825,997, 08 / 833,628, 08 / 835,572 and 08 / 842,360, for which the shipping rights have been paid; the full contents of these patents and applications are incorporated herein by reference. To examine the toxicological effect of the compounds of the invention in vivo, a group of mice were injected with 2-phosphonomethylpentanedioic acid in doses of 1, 5, 10, 30, 100, 300 and 500 mg / kg of body weight. Mice were observed twice a day for 5 consecutive days. The survival rate at each dose level is given below in Table 1. The results showed that the compound of the invention was not toxic to the mice, suggesting that it would likewise be non-toxic to humans when administered in amounts therapeutically effective.

TABLE I EXAMPLE 4 In Vitro Inhibition of Naaladasa Activity Several compounds used in the methods of the invention and pharmaceutical compositions have been subjected to in vitro inhibition test of NAALADase activity. Some results are described in the patents of E.U.A. Nos. 5,672,592, 5,795,877, 5,863,536 and 5,880,112, and the patent applications of E.U.A. Nos. 08 / 825,997, 08 / 833,628, 08 / 835,572 and 08 / 842,360 for which the shipping rights have been paid; the full contents of these patents and applications are incorporated herein by reference.

EXAMPLE 5 Myeloperoxidase (human) / inflammation Myeloperoxidase (MPO, EC 1.11.1.7) is a major constituent of azurophil granulations in neutrophils. Their function (s) is (are) still unknown, but when combined with a halide and H202 they form a highly toxic system, which can kill microorganisms, injure host cells and inactivate humoral factors (reviewed by Klebanoff &Clark, 1978; Clark; , 1983). The MPO was first isolated by Agner (1941), and subsequently the enzyme was purified in crystalline form (Agner, 1958; Harrison et al., 1977). Felberg & Schuitz (1972) demonstrated, by polyacrylamide gel electrophoresis, the heterogeneity of MPO isolated from leukocytes of blood collecting several donors. Later, Strauven et al, (1978) obtained four isoenzymes from individual donors, and the relative amounts seemed to vary with the age of the donors. More recently, three forms of HL-60 cells (Yamada et al, 1981a) and healthy donors (Pember et al, 1982) were isolated by cation exchange chromatography. One of the forms required octyltrimethylammonium bromide (Cetab) for extraction, and a variable distribution was observed in high density, low density azurophil granulations as well as differences in degranulation (Kinkade et al, 1983; Pember & Kinkade, 1983; Olsen & Little, 1984). Differences in enzymatic activity and inhibition-sensitivity towards 3-amino-1,2-triazole were also observed (Pember al., 1983). However, since absorption ratios (A A) and specific enzymatic activities indicate enzymatic purity, the interpretation of these differences was in doubt.

Myeloperoxidase (MPO, EC.1.11.1.7) is an enzyme of the oxidereductase class that catalyzes the reaction H202 + Cl - H20 + OCI. The enzyme is a hemoprotein observed in the azurophil granulations of neutrophils and mononuclear phagocytes. The reaction produces hypochlorites with strong antimicrobial activity. It has been reported that the hypochlorite product before the hydroxyl radicals is involved in the lesion measured by reactive oxygen metabolites (ROM). And ROMs have been implicated in many inflammatory disorders including inflammatory bowel disease (IBD). Some drugs such as aminosalicylic acid and sulfasalazine have been shown to purify OH superoxide, OCI, as well as inhibit MPO and alleviate inflammatory diseases.

MPO Test Procedure Mieloperoxydose (MPO) isolated from human polymorphonuclear leukocytes (Calbiochem, Cat. No. 475911) is used. The test compound and / or vehicle was pre-incubated with 0.02 μg of enzyme and 0.0085% hexadecyltrimethylammonium bromide in 100 mM potassium phosphate buffer pH 7.4 for 30 minutes at 25 ° C. The reaction was initiated by the addition of 1 mM guaiacol as substrate in addition to 0.15 H202 and continued for another 5 minutes. The formation of tetraguayacol was measured by the increase in absorbance at 450 nm. The compounds were screened at 10 μM.

Reference data MPO test Compound IC50 (μM) * NDGA (nordihydroguarético acid) 1.0.

Indicates the standard reference agent used. References: Svensson, B.E., Domeij, K., Lindvall, S. and Rydekk, G. Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidase. Biochem. J. 242: 673-680, 1987.

EXAMPLE 6 Wound healing A patient suffers from a wound that needs to heal. The patient may be administered before, during or after the wound procedure, an effective amount of a compound of the present invention. It is expected that after treatment, the patient's wound will heal more quickly and more effectively, with a diminished change in incomplete wound healing.

EXAMPLE 7 Diabetic Neuropathy A patient suffers from diabetes. The patient can be administered an effective amount of a compound of the present invention. It is expected that after treatment, the patient will be neuroprotected to a statistically significant degree and will be less likely to experience diabetic retinopathy than if the patient were not treated in this way.

EXAMPLE 8 Inflammation A patient suffers from inflammation due to an injury or illness. An effective amount of the compound of the present invention can be administered to the patient before, during or after the inflammation.

It is expected that after the treatment, the patient's inflammation will improve more quickly and more effectively. Having thus described the invention, it will be evident that the same can vary in many ways, these variations should not be considered as far from the spirit and scope of the invention and all modifications are intended to be included within the scope of the following claims.