WO2004091522A2 - Method for treating neurodegenerative disorders - Google Patents

Abstract

The invention provides a pharmaceutical composition and a method for treating and/or preventing neurodegenerative disorders such as Alzheimer's disease, dementia, and mild cognitive impairment. In particular, the method involves administering metabolites of an NSAID to a person desiring prophylaxis against or treatment of a neurodegenerative disorder.

Description

COMPOSITION AND METHOD FOR TREATING NEURODEGENERATIVE DISORDERS

Related U.S. Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 60/462,242 filed on April 11, 2003; and U.S. Provisional Application Serial No. 60/480,073 filed on June 19, 2003; which are incorporated herein by reference in their entirety.

Field of the Invention The invention relates to compositions and methods for the prevention and/or treatment of diseases, particularly to a pharmaceutical composition and method for treating neurodegenerative diseases and/or slowing the onset of signs or symptoms of neurodegenerative disease by administering metabolites of a racemic, R-, or S- (NSAID or modified NSAID) to a patient.

Background of the Invention Alzheimer's disease is a neurodegenerative disease that seriously affects a person's ability to carry out normal daily activities. Alzheimer's disease is characterized by a progressive decline of cognitive functions, including loss of declarative and procedural memory, decreased learning ability, reduced attention span, and severe impairment in thinking ability, judgment, and decision making. Mood disorders and depression are also often observed in Alzheimer's disease patients. It is estimated that Alzheimer's disease affects about 4 million people in the USA and 20 million people world wide. Although Alzheimer's disease is an age- related disorder (with an average onset at 65 years), Alzheimer's disease is not a normal part of aging. Furthermore, the causes of Alzheimer's disease are unknown and the incidence of the disease in industrialized countries is expected to rise dramatically as the population of these countries is aging.

Over the past decade many therapeutics have been designed to treat Alzheimer's disease. Estrogen therapy, antioxidant therapy, non-steroidal anti- inflammatory drugs (NSAIDs) treatment, and treatment with inhibitors of acetylcholine esterase (AChE) have been indicated to have beneficial effects in Alzheimer's disease. However, the only FDA approved drugs for the treatment of Alzheimer's disease are inhibitors of AChE (tacrine (Cognex^®), donepezil (Aricept^®), rivastigmine (Exelon^®), and galantamine (Reminyl^®)); and the N-methyl D-aspartate (NMD A) antagonist memantine (Namenda®). AChE inhibitors are only marginally efficacious in treating Alzheimer's disease and have severe side effects. Furthermore, there has not been convincing evidence that any of the known medications for Alzheimer's disease are effective in preventing Alzheimer's disease. Consequently, there is a large unmet need for better and safer drugs in the treatment of Alzheimer's disease.

Summary of the Invention The invention encompasses pharmaceutical compositions and dosage forms of NSAID metabolites and therapeutic and prophylactic uses of NSAID metabolites. In particular, the invention provides pharmaceutical compositions and methods for treating, delaying the onset, or slowing the progression of A/3 ₂ related disorders and neurodegenerative disorders such as Alzheimer's disease, dementia, and mild cognitive impairment (MCI).

The pharmaceutical compositions and dosage forms within the scope of the invention include a therapeutically or prophylactically effective amount of a metabolite of a racemic, R-, or S-NSAID. The pharmaceutical compositions within the scope of the invention also include NSAID metabolites that are structurally modified and formulations of NSAID metabolites useful as therapeutics. The methods of the invention comprise treating or preventing neurodegenerative or A/3 ₂ related disorders by administering to an individual in need of treatment a therapeutically effective amount of a metabolite of a racemic, R-, or S- (NSAID or modified NSAID). The invention further provides methods useful in treating neurodegenerative disorders including, but not limited to, Alzheimer's disease, dementia, Parkinson's disease, Huntington's disease, brain trauma, infarction, hemorrhage, amytrophic lateral sclerosis/Lou Gehrig's disease (ALS), inherited ataxias such as olivopontocerebellar atrophy (spinocerebellar ataxia type 1), and Machado- Joseph disease (spinocerebellar ataxia type 3). The methods are used to treat (or delay the progression or onset of) the diseases or disorders. The methods also slow or prevent the onset (or rate of increase) of signs or symptoms of the diseases or disorders.

In a specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of racemic, R-, or S-flurbiprofen. In another specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of racemic, R-, or S-ibuprofen. yet another embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a modified form of a metabolite of racemic R-, or S- (NSAIDs or modified NSAIDs). The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples, which illustrate preferred and exemplary embodiments.

Brief Description of the Drawings

Figure 1 depicts Aj3 ₂ and A/3 ₀ levels in cell cultures when flurbiprofen, 4'hydroxyflurbiprofen, 3',4'-dihydroxyflurbiprofen, and 3'-hydroxy-4'- methoxyflurbiprofen are administered to the cells. Figure 2 depicts examples of the metabolism of R- and S- flurbiprofen;

Figure 3 illustrates examples of the metabolism of R- and S- ibuprofen;

Figure 4 shows examples of the metabolism of sulindac;

Figure 5 depicts examples of the metabolism of indomethacin;

Figure 6 illustrates examples of the metabolism of meclofenamic acid (meclofenamate);

Figure 7 depicts examples of the metabolism of R- and S- fenoprofen;

Figure 8 depicts examples of the metabolism of diclofenac;

Figure 9 depicts examples of the metabolism of piroxicam; and Figure 10 depicts examples of the metabolism of diflunisal.

Detailed Description of the Invention 1.0. Introduction Alzheimer's disease patients have amyloid plaque buildup, suggesting that abnormal processing of the amyloid β protein (Aβ) may be the primary cause of Alzheimer's disease. Recent evidence suggests that agents capable of lowering levels of the amyloid β protein (Aβ) are effective in preventing or slowing the onset of Alzheimer's Disease. Furthermore, the longer form of Aβ (Aβ₄₂) is much more amyloidogenic than the shorter forms (Aβ₄₀ or Aβ ₉). Therefore, some Alzheimer's disease therapeutics are designed to inhibit the production of Aβ ₂ and/or lower Aβ ₂ levels. For example, NSAIDS have been shown to lower Aβ ₂ levels and to be a useful therapeutic in combating neurodegenerative diseases. (See Weggen et al, Nature, 414:212-216 (2001); U.S. Pat. App. No. 10/012,606). However, the mechanism by which NSAIDs reduce Aβ ₂ levels is unknown.

NSAIDs also have unwanted side effects such as severe ulcerogenicity and gastrointestinal bleeding, which have poorly understood causes and have typically been associated with the ability of NSAIDs to inhibit the activity of COX enzymes (prostaglandin H synthase) in catalyzing prostaglandin synthesis from arachidonic acid. Some studies suggest that that the gastrointestinal side effects of NSAIDs are due to the inhibition of cyclooxygenase-1 (COX-1) enzymes. See Buttgereit et al., Am JMed., 110 Suppl 3A:13S-9S (2001). Other studies show that NSAIDs selectively inhibiting COX-2 do not appear to reduce the incidence of symptomatic side-effects such as nausea, vomiting, epigastric pain heartburn, and abdominal discomfort. See Rainsford, J Physiol Paris, 95:11-9 (2001). Consequently, it is difficult to reduce or eliminate the side effects of NSAIDs while still retaining A/3 ₂ lowing activity.

The present invention is based upon the discovery that metabolites of NSAIDs are capable of decreasing the levels or rate of secretion of Aβ ₂ in cells. As shown in Figure 1, A/3 ₂ levels are reduced in cell cultures when metabolites of flurbiprofen are administered to the cells.

It is also believed that some metabolites of NSAIDs retain therapeutic efficacy in treating neurodegenerative diseases while having fewer side effects than NSAIDs. Some NSAID metabolites have fewer side effects due to reduced COX inhibition activity. For example, the sulfone metabolite of sulindac is ineffective at inhibiting COX enzymes and therefore lacks the side effects caused by COX inhibition. See Gibson et al, Clin. Pharmacol. Ther. 42(1)82-88 (1987) and Duggan et al., J Pharmacol. Exp. Ther. (201)(1):8-13 (1977). Several diclofenac metabolites were also tested and found to lack COX inhibitory activity. See Moser et al., J. Med. Chem. 33:2358-2368 (1990). Thus, levels of Aβ₄₂ can be lowered by administering some metabolites of NSAIDs without the occurrence of side effects caused by COX inhibition. Therefore, administering metabolites of NSAIDs to individuals in need not only treats or slows symptoms of neurodegenerative disorders, but may reduce unwanted side effects caused by treatment with NSAIDs.

Metabolites of NSAIDs may also often have desirable compounding, solubility, delivery, and/or formulation advantages over NSAIDs. For example, metabolites of NSAIDs typically have hydrophilic groups attached to the NSAID. It is believed that increased hydrophilic content NSAID metabolites will make the

NSAID metabolites more soluble and easier to formulate than the more hydrophobic NSAIDs. In addition, manufacturing metabolites of NSAIDs may be less expensive than manufacturing NSAIDs. Thus, the discovery that metabolites of NSAIDs retain Aβ ₂ lowering activity presents several therapeutic advantages in treating neurodegenerative disease.

2.0. Embodiments

The invention provides compositions and methods for treating neurodegenerative disorders and/or reducing A/3₄₂ protein levels. The pharmaceutical compositions and dosage forms of the invention include metabolites of NSAIDs. The methods of the invention include administering to an individual in need of treatment a therapeutically effective amount of metabolites of racemic, R-, or S- (NSAIDs or modified NSAIDs), or pharmaceutically acceptable salts, esters, or hydrates thereof. As used herein, "NSAIDs" refers to non-steroidal anti-inflammatory drugs. NSAIDs are distinct from steroidal drugs with anti-inflammatory properties such as corticosteroids. Typically, NSAIDs are organic acids that have analgesic (pain- reducing), anti-inflammatory, and anti-pyretic (fever-reducing) effects. Some examples of NSAIDs include salicylic acid (Aspirin), ibuprofen (Motrin, Advil), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), idflunisal (Dolobid), nabu-metone (Relafen), etodolac (Lodine), oxaprozin (Daypro), Meclofenamic acid (Meclofen) and indomethacin (Indocin). NSAIDs can be grouped into classes, for example, amino aryl carboxylic acid derivatives (e.g., flufenamic acid, meclofenamic acid); aryl acetic acid derivatives (e.g., indomethacin, sulindac); and aryl propionic acid derivatives (fenoprofen, ibuporofen, carprofen, and flurbiprofen).

Metabolites are typically thought of as the products or intermediates in organic processes that are necessary for life, including the process by which cells convert materials for uptake or excretion. Although metabolism is known to vary in different organisms, metabolites can generally be divided into two categories. The first category (Phase 1 metabolites) refers to products or intermediates occurring from the activity of P450 enzymes on the compound, including hydroxylation or oxidation products. The second category (Phase 2 metabolites) refers to conjugated forms of the compound or conjugation of its Phase 1 metabolites, such as glucuronidated forms of the compound. Thus, NSALD metabolites as used herein, include both Phase 1 and Phase 2 metabolites of NSAIDs from animals. Preferably, the NSAID metabolites for this invention are found in mammals, particularly humans.

Metabolites of NSAIDs can be determined by techniques known in the art such as administering an NSAID to an organism (i.e. an animal) and analyzing a sample from the organism by liquid chromatography-mass spectrometry (LC-MS). Other techniques known in the art such as gas chromatography-mass spectrometry (GC-MS), tandem mass-spectromety (MS-MS), nuclear magnetic resonance (NMR), LC-NMR, and LC-MS-NMR may also be employed to identify metabolites of a compound. In addition, metabolites of compounds can be predicted based on chemical structures of the compounds. Computer software for predicting metabolites is readily available, including such programs as: Topkat, (Artificial Intelligence Applications Institute); Case/Multi-Case (Case Western Reserve University); DEREK (University of Leeds); HazardExpert and Pallas (CompuDrug); MetaboLynx™ (Micromass), and ACD/MS (Advanced Chemistry Development). There are several ways that metabolites are derived from a compound. A common metabolic conversion occurs through the process of introducing hydrophilic functional groups onto the compound to be metabolized. For example, P450 enzymes often oxidate a compound during metabolism. Some common metabolic conversions are: aromatic hydroxylation; aliphatic hydroxylation; N-, O-, and S-dealkylation; N- hydroxylation; N-oxidation; sulfoxidation; deamination; and dehalogenation.

Metabolism also occurs through reductive reactions. For example, hydrolysis is observed in a wide variety of drug metabolism reactions. Specific enzymes that are involved in hydrolysis are proteases, amidases, and esterases.

Metabolism can occur by introducing hydrophilic functional groups to a compound. For example, a glucuronic acid functional group may be added to a compound during metabolism by a process called glucuronidation. A specific transferase, uridine diphosphate glucuronosyltransferase (UGT), catalyzes glucuronidation. Sulfate, glycine, and acetyl groups are also commonly added to compounds during metabolism.

As described above, there are several ways that metabolites are derived from a compound. However, the metabolic pathways described above are exemplary and are not intended to limit metabolites encompassed by the present invention. The present invention includes R-, S-, and racemic NSAID metabolites made through the metabolic processes described above as well as R-, S-, and racemic NSAID metabolites produced through metabolic processes known to one of ordinary skill in the art. Preferably, the metabolites of racemic, R-, or S-NSAIDs of the present invention are capable of reducing AjS ₂ in human cells or reducing A ₂ in an individual. "Reducing A/3₄₂ in human cells" as used herein, refers to reducing the production or secretion of A/3 ₂ in the assay described in Example 1 below. "Reducing A3₄₂ in an individual" as used herein refers to reducing A/3₄₂ in cerebrospinal fluid (CSF) or reducing A ₂ in the blood plasma. The ELISA antibodies described in Example 1 below can be used to dectect A 3 ₂ in cerebrospinal fluid (CSF) and blood plasma.

In one embodiment of the invention, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of R-, S-, or racemic flurbiprofen, ibuprofen, sulindac, indomethacin, meclofenamic acid, fenoprofen, diclofenac, piroxicam, or diflunisal. In another embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of racemic, R-, or S-flurbiprofen. (Figure 2 illustrates examples of the metabolism of R- and S- fiurbiprofen.) Specific examples of racemic, R-, or S-flurbiprofen metabolites are: racemic, R-, or S-3'-hydroxy-4'-methoxy-flurbiprofen, racemic, R-, or S-3',4'- dihydroxy-flurbiprofen, and racemic, R-, or S-4'-hydroxy-flurbiprofen. In another example, racemic, R-, or S-flurbiprofen metabolites may be glucuronidated forms of: racemic, R-, or S-flurbiprofen, racemic, R-, or S-3'-hydroxy-4'-methoxy-flurbiprofen, racemic, R-, or S-3',4'-dihydroxy-flurbiprofen, or racemic, R-, or S-4'-hydroxy- flurbiprofen. Preferably the racemic, R-, or S-flurbiprofen metabolites are capable of reducing Aβ₄₂ in human cells or reducing A 3 ₂ in an individual. For example, Figure 1 shows that administering racemic 3'-hydroxy-4'-methoxy-flurbiprofen, 3 ',4'- dihydroxy-flurbiprofen, and 4'-hydroxy-flurbiprofen to cell cultures lowered levels of A/3₄₂ in the cell cultures. In a specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of racemic, R-, or S-ibuprofen. (Figure 3 depicts examples of the metabolism of R- and S- ibuprofen.) Specific examples of racemic, R-, or S-ibuprofen metabolites are: racemic, R-, or S-1-hydroxyibuprofen, racemic, R-, or S-2-hydroxyibuprofen, racemic, R-, or S-3-hydroxyibuprofen, racemic, R-, or S-carboxyibuprofen, racemic, R-, or S-carboxyhydratropic acid, racemic, R-, or S-2,4'-carboxyphenylpropionic acid, and the racemic, R-, or S-taurine conjugate of ibuprofen (ibuprofen-tau). See Pettersen et al, J. Chromatog. 145:413-420 (1978); Shirley et al, J Pharmacol Exp Ther 269:1166-1175 (1994); and Zwiener et al, Anal Bioanal Chem 372:469-575 (2002). In another example, racemic, R-, or S-ibuprofen metabolites are glucuronidated forms of racemic, R-, or S-ibuprofen, racemic, R-, or S-l- hydroxyibuprofen, racemic, R-, or S-2-hydroxyibuprofen, racemic, R-, or S-3- hydroxyibuprofen, racemic, R-, or S-carboxyibuprofen, racemic, R-, or S- carboxyhydratropic acid, 2,4'-carboxyphenylpropionic acid, or racemic, R-, or S- ibuprofen-tau. Preferably the racemic, R-, or S-ibuprofen metabolites are capable of reducing A/3 ₂ in human cells or reducing A/3 ₂ in an individual.

In another specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of sulindac. Some examples of sulindac metabolites are shown in Figure 4. Specific examples of sulindac metabolites are sulindac sulfide and sulindac sulfone, or glucuronidated forms of sulindac, sulindac sulfide, or sulindac sulfone. Preferably the sulindac metabolites of the present invention are capable of reducing Aj8₄₂ in human cells or reducing A/3 ₂ in an individual. In another specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of indomethacin. (Figure 5 depicts examples of the metabolism of indomethacin.) Specific examples of indomethacin metabolites are: O-desmethyl-indomethacin, N- desbenzoyl-indomethacin, N-desbenzoyl-O-desmethyl-indomethacin, desmethyl- deschloro-benzoyl-indomethacin (DMBI) 4-chlorobenzoic acid, 5-methoxy-2-methyl- 3-indole acetic acid (5MIAA), and indomethacin-O-glucuronide, or glucuronidated forms thereof. Preferably the indomethacin metabolites of the present invention are capable of reducing A/5 ₂ in human cells or reducing A/3 ₂ in an individual. In another specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of meclofenamic acid (meclofenamate). (Figure 6 depicts examples of the metabolism of meclofenamic acid (meclofenamate). In specific examples, the meclofenamic acid metabolites are 3'-hydroxymethyl-meclofenamate, 4'-hydroxy-meclofenamate, 5- hydroxy-meclofenamate, 3'-hydroxymethyl-4'-hydroxy-meclofenamate, or glucuronidated forms of thereof, and glucuronidated meclofenamic acid. Preferably the meclofenamic acid metabolites of the present invention are capable of reducing A/3 ₂ in human cells or reducing A|8 ₂ in an individual.

In another embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of racemic, R-, or S-fenoprofen. (Figure 7 depicts examples of the metabolism of racemic, R-, or S- fenoprofen.) In specific examples, the racemic, R-, or S-fenoprofen metabolites are: racemic, R-, or S-4-hydroxyfenoprofen, racemic, R-, or S-acyl-glucuronide fenoprofen, and racemic, R-, or S-4-hydroxyfenoprofen-acyl-glucuronide. Preferably the fenoprofen metabolites of the present invention are capable of reducing A/3 ₂ in human cells or reducing A/3 ₂ in an individual.

In yet another embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of diclofenac. (Figure 8 depicts examples of the metabolism of diclofenac.) In specific examples, the diclofenac metabolites are: diclofenac-O-glucuronide, 4'- hydroxydiclofenac, 5-hydroxydiclofenac, 3'-hydroxydiclofenac, 3'hydroxy- 4'methoxy diclofenac, 4',5-dihiydroxydiclofenac„or glucuronidated forms thereof. Preferably the diclofenac metabolites of the present invention are capable of reducing A/3 ₂ in human cells or reducing A/5 ₂ in an individual. In a specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of piroxicam. (Figure 9 depicts examples of the metabolism of piroxicam.) In a specific example, the piroxicam metabolite is 5'-hydroxypiroxicam or glucuronidated forms of piroxicam or 5'-hydroxypiroxicam. Preferably the piroxicam metabolites of the present invention are capable of reducing A/3 ₂ in human cells or reducing A ₂ in an individual..

In another specific embodiment, the method comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of diflunisal. (Figure 10 depicts examples of the metabolism of diflunisal.) In specific examples, the diflunisal metabolites are: diflunisal phenol glucuronide, diflunisal sulfate, 3-hydroxydiflunisal, 3-hydroxydiflunisal sulfate, and diflunisal acyl glucuronide. Preferably the diflunisal metabolites of the present invention are capable of reducing A/3 ₂ in human cells or reducing A ₂ in an individual. Another method of the invention comprises administering to an individual in need of treatment a therapeutically effective amount of a metabolite of a modified racemic, R-, or S-NSAID. Modifying a compound, such as an NSAID, can be performed in a variety of ways. Typically, the compound to be modified has chemical additions or substitutions performed. For example, a modified compound may be derived from another compound by having an acid group replaced by an ester group in a chemical substitution. Preferably, the modified compound is functionally similar and/or structurally similar to the compound being modified. For example, the metabolite of a modified NS ALD may be derived from an analogue of flurbiprofen such as 2-(2'-fluro-4-biphenylyl) propionic acid and 2-(2,2'-difluroro-4-biphenylyl) propionic acid, both found in U.S. Pat. No. 3,755,427, which is incorporated herein by reference. Modifications and additions to indole compounds are also typical ways of producing NSAID analogues from which metabolites of the present invention may be derived.

Metabolites of modified NSAIDs include derivatives of NSAIDs, which are functionally similar and or structurally similar to the NSAID from which they are derived. Specific examples of modified NSAIDs can be generated by modifying functional groups of known NSAIDs. For example, derivatives of modified NSAIDs of the present invention include derivatives of NSAIDs with substitutions to the aminocarboxylic acid, arylacetic acid, and arylpropionic acid groups of NSAIDs. Alkyl, hydroxyl alkyl, phenyl, benzyl, or thienyl groups may be added to indoles in various combinations in order to prepare NSAID derivatives. In addition, structural derivatives of NSAIDs can be identified by commercially available computer modeling programs. Derivatives of NSAIDs also include conformationally constrained compounds which mimic the secondary structure of the NSAID. A derivative of an NSAID includes any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to an NSAID that results in an NSAID mimetic.

Modified NSAIDs also include linking a nitrous oxide releasing group to an NSAJD. For example, modified NSAIDs include nifrosated or nitrosylated NSAIDs, NSAJD derivatives, or NSAID analogues. Nitrosation refers to linking a nitrogen monoxide group (NO) to a compound. Nitrosylation refers to linking a nitrogen dioxide group (NO₂) to a compound. Nifrosated and/or nitrosylated NSAIDs and nifrosated and/or nitrosylated NSAID derivatives are known to release nitric oxide, which may increase the efficacy of clearing Aβ deposits in an individual. (See Jantzen et al, Journal ofNeuroscience, 22:2246-2254 (2002)). Examples of nifrosated and/or nitrosylatred NSAIDS are found in U.S. Pat. App. Serial No. 09/938,560, which is incorporated herein by reference. In a specific example of this embodiment, one of the administered compounds may be a metabolite of a nifrosated and/or nitrosylated racemic, R-, or S-flurbiprofen.

Another way a modified NSAID is prepared is by covalently attaching a sulfur-containing functional group containing a hydrocarbyl moiety. Examples of modified NSAIDs attached to sulfur-containing functional groups are found in U.S. Pat. No. 6,355,666, which is incorporated herein by reference. In a specific example, the modified NSAID may have the structure:

X— L— Y— S(O)_n— Y'— Q wherein:

X is an NSAID; L is an optional linker/spacer;

Y and Y' are optionally present, and when present are independently — O — or — NR' — , wherein R' is H or an optionally substituted hydrocarbyl moiety; n is 1 or 2; and

Q is H or an optionally substituted hydrocarbyl moiety. In a specific example, flurbiprofen, fenoprofen, and carprofen are modified by: (1) altering the position of the propionic acid substituent on the phenyl ring, (2) altering the position or type of substituents on the phenyl ring opposite the propionic acid substituent, (3) altering the bond connecting the two phenyl rings, and/or (4) replacing the acetic acid substituent with a carboxylic acid substituent or other derivative.

Specific modifications to meclofenamic acid and flufenamic acid compounds include, but are not limited to: (1) altering the position of the carboxylic acid substituent on the phenyl ring, (2) altering the position or type of substituents on the phenyl ring opposite the carboxylic acid substituent, (3) altering the bond connecting the two phenyl rings, (4) replacing the carboxylic acid substituent with a propionic acid substituent or other derivative.

Sulindac sulfide metabolites and derivatives can be prepared by performing modifications including, but not limited to: (1) replacing the fluoride group with another substituent, (2) replacing the propionic acid derivative with another substituent, (3) replacing the methylthio derivative with another substituent. For example, Moser et al, J. Med. Chem. 33:2358-2368 (1990) discloses methods for the synthesis of diclofenac analogues. Indomethacin metabolites and derivatives can be prepared by performing modifications including, but not limited to: (1) substituting the carboxylic acid or indole nitrogen with another substituent.

The methods of the invention also include administering to an individual in need of treatment a therapeutically effective amount of a modified metabolite of a racemic, R-, or S- (NSAID or modified NSAID). A metabolite may be modified according to the methods of modifying NSAIDs described above, including linking compounds such as an enzyme or coenzyme to the metabolite. Thus, according to the method of the present invention, a modified metabolite may also be administered to an individual in need of treatment. Individuals who desire or are in need of treatment can have a neurodegenerative or A/3 ₂ related disorder, a predisposition to a neurodegenerative or A/3 ₂ related disorder, and/or desire prophylaxis against neurodegenerative or A 3 ₂ related disorders. The therapeutically effective amounts of the compounds administered for treatment are capable of slowing the progression, eliminating, or reducing at least one symptom or sign of the A/3 ₂ related disorder or neurodegenerative disorder. Symptoms or signs include deterioration of cognition, memory, and language. The symptoms may be either mild (prodromal) or moderate to severe (clinically diagnosable). For individuals desiring prophylaxis against an A/3₄₂ related disorder or neurodegenerative disorder, the therapeutically effective amount of the administered compounds is capable of preventing, delaying the onset, or slowing the increase (or rate of increase) of at least one symptom or sign of the disorders. The symptoms may be either mild (prodromal) or moderate to severe (clinically diagnosable). The methods may thus reduce or prevent the progression of mild cognitive dysfunction in old age (a substantial proportion of which represents prodromal or early clinical symptoms of Alzheimer's disease). The methods may also inhibit age-related cognitive decline in an individual at risk of developing clinically diagnosable Alzheimer's disease. In addition, the method for reducing A/3 ₂ protein levels may lower levels of

A/3₄₂ or prevent an increase in A/3 ₂ levels, or slow the rate of increase A/3₄₂ levels, in an individual.

Pharmaceutical composition embodiments include the metabolites of NSAIDS (or modified NSAIDS) administered in the embodiments of the methods of the present invention. The pharmaceutical compositions contain an NSAID metabolite in combination with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may contain NSAID metabolites at an amount of from about 0.01 microgram to about 5000 mg per day, preferably from about 1 microgram to about 2500 mg per day. Preferably, the pharmaceutical compositions of the present invention contain from about 400 mg to about 800 mg, or 1600 mg of NSAID metabolites.

3.0. Preparation of NSALD Metabolites

Many of the compounds of the invention can be obtained commercially. For example, 4'-hydroxydiclofenac is available from BD Biosciences (Franklin Lakes, New Jersey), Ultrafine (UFC Ltd.) (Downington, Pennsylvania), and Sigma-Aldrich (St. Louis, Missouri). N-desbenzoyl-indomethacin (5-methoxy-2-methyl-3- indoleacetic acid) is available from Cole-Parmer (Vernon Hills, Illinois).

Compounds administered in the present invention can also be synthesized. For example, a method for synthesizing substituted propionic acids and derivatives thereof, including racemic 4'-hydroxyfiurbiprofen, is found in U.S. Pat. No. 3,969,402, which is incorporated herein by reference. 4'-hydroxyflurbiprofen can also be synthesized as directed in PCT App. No. PCT/JP98/01770 (Pub. No. WO 98/47880). 3',4'-dihydroxyflurbiprofen and 5'-hydroxy-4'-methoxyflurbiprofen can be synthesized according to the following method:

Compound 1 can be synthesized as reported by Bayly et. al., Bioorg Med

Chem Lett 9, 307-312, 1999, and compound 2 is commercially available. Coupling of 1 and 2 can be performed as described by Littke, et. al., J. Am. Chem. Soc. 122, 4020- 4028 (2000), using potassium fluoride, Pd(dba)₂ and P(t-Bu)₃H-BF . The methyl ester can be saponified using sodium hydroxide in methanolic water, and the methyl ethers deprotected with TMS-iodide (1.5 equivalent) to give a mixture of the mono- methoxide and fully deprotected phenol products. The desired metabolites can be separated by chromatography and resolved by common chiral techniques.

2-hydroxyibuprofen can be synthesized from ibuprofen using methods such as a 1,6-eliminative epoxide cleavage (Kurtz and Houser, J. Org. Chem. 46:202-203J (1981)) or acid additions (Segrestaa et al, Chem. Pharm. Bull. 50:744-748 (2002)). The stereoselective synthesis of carboxyibuprofen may be carried out according to the synthesis performed in Tan et al., Chirality 9(l):75-87 (1997). Ibuprofen metabolites can also be synthesized according to the reaction schemes in U.S. Pat. No. 3,228,831, which is incorporated herein by reference.

Sulindac, sulindac sulfide, and sulindac sulfone can be synthesized according to the methods described in U.S. Pat. Nos. 3,654,349, 6,232,312, 6,028,116, and 4,748,271. Sulindac sulfide and sulindac can also be synthesized according to the reaction schemes in Shuman et al, J. Org. Chem. 42:1914-1919 (1977) illustrated below:

6 + PhCH₂ (CH,)₃0H- + OHCCO.H

(major product from (trace from dehydration and thermal dehydration) isomerization)

10

12

(major product from 10 and thermal isomerization )

Methods for synthesizing the indomethacin metabolite 4-chlorobenzoic acid are found in Travis et al, Organic Letters, 5:1031-1034 (2003) and Bjorsvik et al, J. Org. Chem., 67:7493-7500 (2002). N-desbenzoyl-O-desmethyl indomethacin can be synthesized from the commercially available N-desbenzoyl-indomethacine by deprotecting chemistry techniques standard to one of ordinary skill in the art. Other indomethacin metabolites may be synthesized by performing the reactions outlined U.S. Pat. No. 3,161,654 with the appropriate starting materials.

Meclofenamic acid metabolites and diclofenac metabolites can be synthesized by reacting the appropriate starting materials according to the synthesis schemes in AntiUa and Buchwald, Organic Lett. 3(13):2077-2079 (2001) and J. Am. Chem. Soc. 125:6653-6655 (2003).

Fenoprofen metabolites, including 4-hydroxyfenoprofen, can be synthesized by reacting the appropriate starting materials according to the synthesis schemes in Cristau et al, Org. Lett. 6(6):913-916 (2004); Buck et al., Org. Lett. 4(9): 1623-1626 (2002); and Evans et al., Tetrahedron Lett. 39:2937-2940 (1998).

Diclofenac and the diclofenac metabolites, including 5-hydroxy diclofenac, 3 'hydroxy-4 'methoxy diclofenac, 4',5-dihydroxydiclofenac, and 4'-hydroxy diclofenac; can be synthesized according to the reaction schemes in Moser et al, J. Med. Chem. 33:2358-2368 (1990). Peroxicam metabolites, including 5'hydroxypiroxicam, can be synthesized using according to the method in U.S. Pat. No. 3,591,584 by using the appropriate starting materials.

Diflunisal metabolites can be synthesized according to the method in U.S. Pat. No. 3,714,226 by using the appropriate starting materials. Metabolites of NSAIDs can also be prepared by synthesizing or obtaining a commercially available NSAID and performing chemical transformations on the NS AID until the desired metabolite is prepared. NSAID starting materials are commercially available either in the form of racemic mixtures or as optically pure enantiomers. In all cases, racemic mixtures contain equal amounts of the R- and S- isomers of the NSAID. For example, the following racemates can be obtained through Sigma Chemical Co.: ketoprofen, flurbiprofen, etodolac, suprofen, carprofen, indoprofen and benoxaprofen. Naproxen, marketed as the S-isomer only, is also available from this source. Additionally, many commercial sources exist for the stereospecific R-isomers of many NSAIDs. R-ketoprofen, R-flurbiprofen and R- ketorolac, for example, are available through Sepracor, Inc.; R-naproxen can be obtained as the sodium salt through Sigma Chemical Co.; R-etodolac is available from Wyeth-Ayerst; R-tiaprofenic acid is available through Roussel (France, Canada, Switzerland, Spain, Denmark, Italy); R-suprofen is manufactured by McNiel Pharmaceuticals; R-carprofen is available from Roche; R-pirprofen is available through Ciba (France), described in U.S. Pat. No. 5,177,080.

Methods for synthesizing NSAIDs are also known in the art. For example, a method for synthesizing flurbiprofen is described in U.S. Pat. No. 3,755,427, a method for synthesizing ibuprofen is found in U.S. Pat. Nos. 3,228,831 and 3,385,886, a method for synthesizing sulindac is found in U.S. Pat. Nos. 3,654,349 and 3647,858, a method for synthesizing indomethacin is found in U.S. Pat. No. 3,161,654, a method for synthesizing meclofenamic acid is found in U.S. Pat. No. 3,313,848, a method for synthesizing fenoprofen is found in U.S. Pat. No. 3,600,437, a method for synthesizing diclofenac is found in U.S. Pat. No. 3,558,690, a method for synthesizing piroxicam is found in U.S. Pat. No. 3,591,584, and a method for synthesizing diflunisal is found in U.S. Pat. No. 3,714,226; which are all incorporated herein by reference. Many of the reaction schemes for synthesizing NSAIDs may also be used to synthesize NSAID metabolites by simply varying the starting materials in the reaction. A variety of chemical transformations useful for synthesizing metabolites of

NSAIDs from NSAIDs are known in the art. For example, protecting group methodologies (protection and deprotection) useful in synthesizing NSAID metabolites include those described in L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995). A specific example well known in the art is that sulfur and oxygen protecting groups may be used for protecting thiol and alcohol groups against unwanted reactions during a synthetic step. Additional processes useful for preparing NSAID metabolites and derivatives can be found in published reports such as Dewitt Molecular Pharmacology 55:625-631, (1999); Kalgutkar et al. (2000) PNAS 97:925-930; and Bayly et al. Biorg and Med Chem Letters 9:307-312 (1999.

4.0. Formulation, Dosages, and Routes of Delivery The active compounds according to this invention can be administered to patients to be treated through any suitable routes of administration and can be provided with any pharmaceutically acceptable carrier. Advantageously, the active compounds are delivered to the patient parenterally, i.e., by intravenous, intramuscular, intraperiotoneal, intracisternal, subcutaneous, or intraarticular injection or infusion.

For parenteral administration, the active compounds can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use. Lyophilized compositions may include therapeutically acceptable carriers such as gelatin, DL-lactic and glycolic acids copolymer, D-mannitol, etc. To convert the lyophilized forms into solutions or suspensions, diluent containing, e.g., carboxymethylcellulose sodium, D-mannitol, polysorbate 80, and water may be employed. Lyophilized forms may be stored in, e.g., a dual chamber syringe with one chamber containing the lyophilized composition and the other chamber containing the diluent. In addition, the active ingredient(s) can also be incorporated into sterile lyophilized microspheres for sustained release. Methods for making such microspheres are generally known in the art. See U.S. Patent Nos. 4,652,441; 4,728,721; 4,849,228; 4,917,893; 4,954,298; 5,330,767; 5,476,663; 5,480,656; 5,575,987; 5,631,020; 5,631,021; 5,643,607; and 5,716,640. In a solution or suspension form suitable for parenteral administration, the pharmaceutical composition can include, in addition to a therapeutically, therapeutically, or prophylactically effective amount of a compound of the present invention, a buffering agent, an isotonicity adjusting agent, a preservative, and/or an anti-absorbent. Examples of suitable buffering agent include, but are not limited to, citrate, phosphate, tartrate, succinate, adipate, maleate, lactate and acetate buffers, sodium bicarbonate, and sodium carbonate, or a mixture thereof. Preferably, the buffering agent adjusts the pH of the solution to within the range of 5-8. Examples of suitable isotonicity adjusting agents include sodium chloride, glycerol, mannitol, and sorbitol, or a mixture thereof. A preservative (e.g., anti-microbial agent) may be desirable as it can inhibit microbial contamination or growth in the liquid forms of the pharmaceutical composition. Useful preservatives may include benzyl alcohol, a paraben and phenol or a mixture thereof. Materials such as human serum albumin, gelatin or a mixture thereof may be used as anti-absorbents. In addition, conventional solvents, surfactants, stabilizers, pH balancing buffers, and antioxidants can all be used in the parenteral formulations, including but not limited to dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, and the like. The parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes. The active compounds can also be delivered orally in enclosed gelatin capsules or compressed tablets. Capsules and tablets can be prepared in any conventional techniques. For example, the active compounds can be incorporated into a formulation which includes therapeutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum fragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets. In addition, liquid carriers such as fatty oil can also be included in capsules.

Other forms of oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing the active compounds used in this invention. Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included. In addition, for convenient administration by enteral feeding tube in patients unable to swallow, the active compounds can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil.

Another way the active compounds can be delivered to the patient orally is through the use of biodegradable polymers. See e.g., U.S. Patent Nos. 5,324,518, 5,599,552, and 5,681,873. Biodegradable polymers encapsulate a compound and release it over time. The patient's saliva erodes the biopolymer and thus releases the compound over time. As the biopolymer capsule erodes the compound enters the patient's blood system through the oral mucosa. Importantly, the time of delivery can be adjusted according to erosion time of the selected biodegradable polymer coating. The active compounds can also be administered topically through rectal, vaginal, nasal, bucal, or mucosal applications. Topical formulations are generally lαiown in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, drops and aerosols. Typically, topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like. A special form of topical administration is delivery by a transdermal patch. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al, Annual Review of Medicine, 39:221-229 (1988), which is incorporated herein by reference.

Another method of topically delivering the compounds of the present invention to the patient is achieved by enhancing the permeability of the patient's skin. Enhancing skin permeability can be achieved through either the application of ultrasonic vibrations or chemicals. Low frequency ultrasonic vibrations can be used to make the skin more permeable to the passage of proteins and other compounds. See e.g., U.S. Patent Nos. 6,234,990 and 6,190,315. The ultrasonic vibrations produce pressure waves that create a cavitational effect in the skin. This cavitational effect disorganizes the lipids in the tissue and makes the skin more permeable. Thus, compounds of the present invention can be delivered topically by applying the compounds to an appropriate area of the skin immediately after ultrasonic vibrations have been app Bentley Pharmaceuticals' lipid CPE-215 to a patient's skin or membranes will temporarily saturate the tissue and cause phase separation of the oil and water components of the skin. While the water and oil domains of the tissue are temporarily separated by CPE- 215, both lipophilic and hydrophic drugs are delivered effectively through that area of the skin. Thus, compounds of the present invention can be delivered by topically applying the compounds in combination with skin permeability enhancing chemicals. The active compounds can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant the active compounds in any suitable formulation into the subcutaneous space of the anterior abdominal wall. See, e.g., Wilson et al., J. Clin. Psych. 45:242-247 (1984). Sustained release can be achieved by incorporating the active ingredients into a special carrier such as a hydrogel. Typically, a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material. Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co-L-lactic acid) are suitable for this invention. See, e.g., Phillips et al., J. Pharmaceut. Sci., 73:1718-1720 (1984). The active compounds can also be conjugated, i.e., covalently linked, to a water soluble non-immunogenic high molecular weight polymer to form a polymer conjugate. Preferably, such polymers do not undesirably interfere with the cellular uptake of the active compounds. Advantageously, such polymers, e.g., polyethylene glycol, can impart solubility, stability, and reduced immunogenicity to the active compounds. As a result, the active compound in the conjugate when administered to a patient, can have a longer half-life in the body, and exhibit better efficacy. In one embodiment, the polymer is a peptide such as albumin or antibody fragment Fc. PEGylated proteins are currently being used in protein replacement therapies and for other therapeutic uses. For example, PEGylated adenosine deaminase (ADAGEN^®) is being used to treat severe combined immunodeficiency disease (SCTDS). PEGylated L-asparaginase (ONCAPSPAR^®) is being used to treat acute lymphoblastic leukemia (ALL). A general review of PEG-protein conjugates with clinical efficacy can be found in, e.g., Burnham, Am. J. Hosp. Pharm., 15:210-218 (1994). Preferably, the covalent linkage between the polymer and the active compound is hydro lytically degradable and is susceptible to hydrolysis under physiological conditions. Such conjugates are known as "prodrugs" and the polymer in the conjugate can be readily cleaved off inside the body, releasing the free active compounds.

Alternatively, other forms controlled release or protection including microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the active compounds.

Another preferable delivery form is using Hposomes as carrier. Liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Active compounds can be enclosed within such micelles.

Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, and Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., both of which are incorporated herein by reference. Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomes can reduce the toxicity of the active compounds, and increase their stability. The active compounds of the present invention are administered in a therapeutically, therapeutically, or prophylactically effective amount. A therapeutically, therapeutically, or prophylactically effective amount refers to an amount necessary to achieve the desired therapeutic effect without causing any serious adverse effects in the patient treated. Generally, the toxicity profile and therapeutic efficacy of therapeutic agents can be determined by standard pharmaceutical procedures in suitable cell models or animal models or human clinical trials. As is known in the art, the LD₅₀ represents the dose lethal to about 50% of a tested population. The ED₅₀ is a parameter indicating the dose therapeutically effective in about 50% of a tested population. Both LD₅₀ and ED₅₀ can be determined in cell models and animal models. In addition, the IC₅Q may also be obtained in cell models and animal models, which stands for the circulating plasma concentration that is effective in achieving about 50% of the maximal inhibition of the symptoms of a disease or disorder. Such data may be used in designing a dosage range for clinical trials in humans. Typically, as will be apparent to skilled artisans, the dosage range for human use should be designed such that the range centers around the ED₅₀ and/or IC₅₀, but significantly below the LD₅₀ obtained from cell or animal models.

Typically, the compounds of the present invention can be effective at an amount of from about 0.01 microgram to about 5000 mg per day, preferably from about 1 microgram to about 2500 mg per day. However, the amount can vary with the body weight of the patient treated and the state of disease conditions. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time. The suitable dosage unit for each administration of the compounds of the present invention can be, e.g., from about 0.01 microgram to about 2000 mg, preferably from about 1 microgram to about 1000 mg.

In the case of combination therapy, a therapeutically effective amount of another compound can be administered in a separate pharmaceutical composition, or alternatively included in the pharmaceutical composition that contains a compound according to the present invention. The pharmacology and toxicology of many of such other compounds are known in the art. See e.g., Physicians Desk Reference, Medical Economics, Montvale, NJ; and The Merck Index, Merck & Co., Rahway, NJ. The therapeutically effective amounts and suitable unit dosage ranges of such compounds used in art can be equally applicable in the present invention. It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. The therapeutically effective amount for each active compound can vary with factors including but not limited to the activity of the compound used, stability of the active compound in the patient's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of absorption, distribution, and excretion of the active compound by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan. The amount of administration can also be adjusted as the various factors change over time.

5.0. Examples

5.1. Example 1 : Aβ Secretion Assay

To test compounds and compositions capable of modulating Aβ levels, H4 neuroglioma cells expressing APP695NL and CHO cells stably expressing wild-type human APP751 and human mutant presenilin 1 (PSI) M146L are used. Generation and culture of these cells have been described. See Murphy et al, J. Biol. Chem., 274(17):11914-11923 (1999); Murphy et al, J. Biol. Chem., 275(34):26277-26284 (2000). To minimize toxic effects of the compositions and compounds, the H4 cells are incubated for 6 hours in the presence of the various compositions and compounds. To evaluate the potential for toxic effects of the compositions and compounds, additional aliquots of cells are incubated in parallel with each composition or compound. The supernatants are analyzed for the presence of lactate dehydrogenase (LDH) as a measure of cellular toxicity.

After incubating the cells with the compositions and compounds for a pre- determined time period, sandwich enzyme-linked immunosorbent assay (ELISA) is employed to measure secreted Aβ levels as described in Murphy et al, J. Biol. Chem., 275(34):26277-26284 (2000). For cell culture studies, serum free media samples are collected following 6-12 hours of conditioning, Complete Protease Inhibitor Cocktail added (PIC; Roche), and total Aβ concentration measured by sandwich ELISA antibody 3160, affinity purified poiyclonal antibody raised against Aβl-42.

3160/BA27 sandwich ELISA is used for Aβ₄₀ and 3160/BC05 sandwich ELISA is used for Aβ ₂. HRP conjugated monoclonal antibodies BA27 for detection of Aβ40 and BC05 for detection of Aβ ₂ have been previously described. Suzuki et al, Science, 264(5163):1336-1340 (1994). All measurements are performed in triplicate.

5.2. Example 2: Determination of COX inhibition activity In vitro cellular COX inhibition can be determined using specific assays for inhibition of COX- 1 and COX-2 (Kalgutkar et al. J. Med Chem., 43:2860-2870 (2000)). Another art-known cellular assay for determining COX inhibition is based on the production of prostaglandin-E₂ from exogenous arachidonic acid in cells expressing COX-1, COX-2, or a combination thereof. COX enzymes (prostaglandin H synthase) catalyze the rate-limiting step in prostaglandin synthesis from arachidonic acid. Cell lines are known and available that express at least one form of the enzyme. For example, a human skin fibroblast line can be induced with IL-1 to synthesize COX-2, and a kidney epithelial cell line 293 has been stably transfected to constitutively express COX-1. In these assays, arachidonic acid can be added exogenously to increase signal to readably detectable levels. Thus, the amount of prostaglandin-E₂ in the extracellular medium can be assayed by radioimmunoassay, for measuring COX activity. IC₅₀ values for compounds for COX-1 and COX-2 can be determined by an ordinary skilled artisan. Anti-inflammatory activities of compounds can be determined using the art-known rat/mouse paw edema assay as described in Penning et al. J. Med Chem., 40:1347-1365 (1997).

For a further description of assays, cell line, and techniques capable of assessing COX inhibitory activity and A/3₄₂ lowering activity see, e.g., WO 01/78721, and references cited therein, all of which are incorporated herein in their entirety.

5.3. Example 3: Additional Alzheimer's Assays

The levels of the Aβ peptide can be measured in conditioned medium and in lysates from cultured neuroblastoma cells transfected with an APP expression vector (Proc. Nat Acad. Sci. USA 93:13170 (1996)). Neuronal survival and protection can be assessed with cultured neuronal cells challenged with neurotoxic factors such as the Aβ42 peptide. At various time points, cell death or viability is measured by apoptotic assay or cell counting (J Neurobiol 25:585, (1994); Brain Res. 706:328 (1996)). Neurite extension can be assessed with neuronal cells that are seeded in culture and the number and length of neurites that form after 16 to 20 hrs are recorded (J Neurobiol. 25:585 (1994); J. Neurosci. 14:5461, (1994)).

5.4 Example 4: Oral Pharmaceutical Compositions Oral pharmaceutical compositions of NSAID metabolites can be prepared in tablet and gelatin capsule form. One formulation of oral tablets is performed by mixing 200 mg of 3'4'-dihydroxyflurbiprofen with 400 mg lactose. A suitable amount of water for drying is added and the mixture is dried. The mixture is then blended with 76 mg starch, 8 mg hydrogenated vegetable oil, and 8 mg polyvinylpyrrolidinon. The resulting granules are compressed into tablets. Tablets of varying strengths are prepared by altering the ratio of NSAID metabolites in the mixture or changing the total weight of the tablet.

Another formulation of oral tablets is performed by mixing 200 mg of 2- hydroxyibuprofen with 400 mg lactose. A suitable amount of water for drying is added and the mixture is dried. The mixture is then blended with 80 mg starch, 10 mg hydrogenated vegetable oil, and 10 mg polyvinylpyrrolidinon. The resulting granules are compressed into tablets. Tablets of varying strengths are prepared by altering the ratio of NSAID metabolites in the mixture or changing the total weight of the tablet. A formulation of gelatin capsules can be prepared by mixing 200 mg of 3'- hydroxy-4'-methoxy-flurbiprofen with 500 mg of microcrystalline cellulose and 100 mg of corn starch. 800 mg of magnesium stearate is then blended into the mixture and the resulting blend is encapsulated into a gelatin capsule. Doses of varying strengths can be prepared by altering the ratio of NSAID metabolites to pharmaceutically acceptable carriers or changing the size of the capsule. Another formulation of gelatin capsules can be prepared by mixing 200 mg of

3'-hydroxymethyl-meclofenamate with 400 mg of microcrystalline cellulose and 75 mg of corn starch. 650 mg of magnesium stearate is then blended into the mixture and the resulting blend is encapsulated into a gelatin capsule. Doses of varying strengths can be prepared by altering the ratio of NSAID metabolites to pharmaceutically acceptable carriers or changing the size of the capsule.

5.5. Example 5: Treating Neurodegenerative Disorders Neurodegenerative disorders such as Alzheimer's disease can be treated by administering to an individual in need a therapeutically effective amount of NSAID metabolites. One method of treating neurodegenerative disorders involves administering 400 mg of NSALD metabolites daily to an individual in need. Through the administration of NSAID metabolites, an individual in need will have slowed or stopped the progressive decline of cognitive functions. The slowing or stopping of the decline of cognitive functions can be determined through measuring the loss of declarative and procedural memory, the decrease in learning ability, the reduction in attention span, and the impairment in thinking ability, judgment, and decision making.

5.6. Example 6: Preparation of 4'-hydroxyflurbiprofen 4'-hydroxyflurbiprofen can be made according to following method. 175g of

4-iodoanisol, 160g of 4'-bromo-3'-nitroacetophenone, and 140 g copper powder are mixed and gradually heated for 5 hours at 80°C and for 4 hours with a gradual raise in temperature from 80°C to 110°C. Methylene dichloride is removed from the mixture upon cooling by evaporation. The remaining compound is 4-acetyl-4'methoxy-2- nitrobiphenyl, which is then slowly added for 45 minutes to a solution of 300g stannous chloride, 400mL hydrochloric acid, and 600mL ethanol. The resulting solution is refluxed for 3 hours and the ethanol is removed by evaporation. The remaining mixture is added to a solution of 560g sodium hydroxide in water and ice to form a solid product. The solid product is extracted into methylene dichloride, dried over ahydrous sodium sulphate, evaporated, and recrystallized with ethanol to form 4-acetyl-2-amino-4'-methoxybiphenyl. lOg of 4-acetyl-2-amino-4'-methoxybiphenyl is added to a mixture of 28 mL tetrahydrofuran, 10 mL water, and 40 mL hydrofluoroboric acid (42% acid by volume). The remaining solution is added to 3g of sodium nitrite in water at a reaction temperature of 5°C. After stirring for 20 minutes, diazonium fluroborate is removed by filtration and washed with hydrofluoroboric acid and methanol/ether. Diazonium fluroborate is suspended in xylene and heated until decomposition takes place at 70°C. The mixture is then refluxed for 45 minutes and hot benzene is used to extract the residue after removing xylene by distillation. Aqueous sodium carbonate and water are used sequentially to wash the extract, and recrystallization with ethanol gives 4-acetyl-2-fluoro-4 'methoxybiphenyl.

4g of 4-acetyl-2-fluoro-4'methoxybiphenyl is added to a solution of .75 g sodium in 45 mL isopropanol. The solution is stirred, cooled to 5°C, and ethyl chloroacetate is added dropwise. After 5 hours of stirring, the solution is kept overnight at room temperature. Isopropanol is removed form the solution by evaporation and the resulting mixture is refluxed for 45 minutes with a mixture of 1.8mL 18N aqueous sodium hydroxide and 25 mL 10% (by volume) aqueous ethanol. Ethanol is removed by distillation and the resulting mixture is diluted to 200mL. 8.75 g of sodium metabisulphite is added to the solution and the resultant mixture is heated for 6 hours. After cooling, 20 mL of ether and 3mL 18N sodium hydroxide are added to the solution. The layers of the solution are separated and the ethereal layer is washed with dilute acetic acid and water. The residue is dried over anhydrous sodium carbonate, evaporated, and distilled to give 2-(2-fluro-4'-methoxy-4- biphenylyl)propionaldehyde.

2.5 g of 2-(2-fluro-4'-methoxy-4-biphenylyl)propionaldehyde is added to 10 mL of ethanol. The mixture is added to a solution of 2 g sodium acetate and lg hydroxylamine sulphate in 10 mL of water. After stirring for 2 hours, refluxing for 5 minutes and collilng with ice, the solid oxime is collected from the solution by filtration and washed with ethanol. 2.5g of the solid oxime is mixed with 55mg nickel sulphate and 15 mL water. After heating the mixture to a boil, 2 mL 18N sodium hydroxide and 2 mL water are added and the resulting mixture is refluxed for 24 hours. Upon cooling to room temperature, dilute hydrochloric acid is added to precipitated an acid that is extracted into ether. The resulting ethereal extract is removed with aqueous potassium carbonate and the precipitated extract is then re- extracted into ether. The ethereal extract is washed with water, dried with anhydrous sodium sulphate, and evaporated to form 2-(2-fluro-4'-methoxy-4- biphenylyl)propionic acid after recrystallization from 1 : 1 benzene/light petroleum. 0.4 g of 2-(2-fluro-4'-methoxy-4-biphenylyl)propionic acid is mixed with 9mL 50% acid (by volume) hydrobromic acid and 3 mL glacial acetic acid. After refluxing for 3 hours, the mixture is cooled and the remaining solid product is separated, washed with water and dried at 100°C to give 4'-hydroxyflurbiprofen (also referred to as 2-(2- fluro-4 ' -hydroxy-4-biphenylyl)propionic acid.

5.7. Example 7: Preparation of 2'-hydroxyibuprofen Synthesis of racemic, R-, or S- 2'-hydroxyibuprofen can be performed according to the following reaction scheme from Kurtz and Houser, J. Org. Chem. 46:202-203J (1981):

Scheme I

Ibuprofen (compound II) undergoes a benzylic bromination with N- bromosuccinimide (NBS) to form compound III. Compound III then undergoes a dehydrobromination in DMF with LiBr to give compound IV. Compound IV is treated with m-chloroperbenzoic acid (m-CPBA) to form compound V, which forms compound VI by treatment with potassium tert-butoxide. Compound VI is converted to racemic 2-hydroxyibuprofen (compound VII) by catalytic hydrogenation with Pd/C or selectively converted to R- or S- 2-hydroxyibuprofen by treatment with an asymmetric rhodium catalyst.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of delaying the onset of neurodegenerative disorders in an individual in need of treatment comprising administering to the individual a therapeutically effective amount of a metabolite of a racemic, R-, or S-NS AID, or pharmaceutically acceptable salt, ester, or hydrate thereof.

2. A method of delaying the onset of or treating Alzheimer's Disease in an individual comprising administering to the individual a therapeutically effective amount of compounds selected from the group consisting of metabolites of: racemic, R-, or S- flurbiprofen, ibuprofen, sulindac, meclofenamic acid, fenoprofen, diclofenac, piroxicam, diflunisal, and indomethacin, and pharmaceutically acceptable salts, esters, and hydrates thereof.

3. The method of Claim 2, wherein the metabolite is a human metabolite.

4. The method of Claim 2, wherein the metabolite is selected from the group consisting of: metabolites of racemic flurbiprofen, R-flurbiprofen, and S- flurbiprofen.

5. The method of Claim 2, wherein the metabolite is selected from the group consisting of: racemic, R-, or S- 3'-hydroxy-4'-methoxy-flurbiprofen; racemic, R-, or S-3',4'-dihydroxy-flurbiprofen; and racemic, R-, or S-4'-hydroxy-flurbiprofen

6. The method of Claim 2, wherein the metabolite is R-flurbiprofen.

7. The method of Claim 2, wherein the metabolite is S-flurbiprofen.

8. The method of Claim 2, wherein the metabolite is racemic flurbiprofen.

9. The method of Claim 2, wherein the metabolite is selected from the group consisting of: metabolites of racemic ibuprofen, R-ibuprofen, and S-ibuprofen.

10. The method of Claim 2, wherein the metabolite is capable of reducing the A/3 ₂ in human cells or reducing A/3 ₂ in an individual.

11. The method of Claim 2, wherein the metabolite is capable of reducing

A8 ₂ in human cells or reducing A in an individual by a greater percentage than

A/3₄₀.

12. A method of reducing A/3₄₂ in an individual comprising administering to the individual a therapeutically effective amount of a metabolite of a racemic, R-, or S-NSAID, or pharmaceutically acceptable salt, ester, or hydrate thereof.

13. The method of Claim 12, wherein the metabolite is a human metabolite.

14. The method of Claim 12, wherein the metabolite is selected from the group consisting of: metabolites of racemic flurbiprofen, R-flurbiprofen, and S- flurbiprofen.

15. The method of Claim 12, wherein the metabolite is selected from the group consisting of: racemic, R-, or S- 3'-hydroxy-4'-methoxy-flurbiprofen; racemic, R-, or S-3 ',4 '-dihydroxy- flurbiprofen; and racemic, R-, or S-4'-hydroxy-flurbiprofen

16. The method of Claim 12, wherein the metabolite is R-flurbiprofen.

17. The method of Claim 12, wherein the metabolite is S-flurbiprofen.

18. The method of Claim 12, wherein the metabolite is racemic flurbiprofen.

19. The method of Claim 12, wherein the metabolite is selected from the group consisting of: metabolites of racemic ibuprofen, R-ibuprofen, and S-ibuprofen.

20. The method of Claim 12, wherein the metabolite is capable of reducing the Aj8₄₂ in human cells or reducing A/3₄₂ in an individual.

21. The method of Claim 12, wherein the metabolite is capable of reducing Aj8 ₂ in human cells or reducing A/3 ₂ in an individual by a greater percentage than

A/3₄₀

22. A pharmaceutical composition comprising a therapeutically effective amount of a metabolite of a racemic, R-, or S-NS AID, or pharmaceutically acceptable salt, ester, or hydrate thereof; in combination with a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of Claim 22, wherein the metabolite is selected from the group consisting of metabolites of: racemic, R-, or S- flurbiprofen, ibuprofen, sulindac, meclofenamic acid, fenoprofen, diclofenac, piroxicam, diflunisal, and indomethacin, and pharmaceutically acceptable salts, esters, and hydrates thereof.

24. The pharmaceutical composition of Claim 22 wherein the metabolite is selected from the group consisting of: metabolites of racemic flurbiprofen, R- flurbiprofen, and S-flurbirofen.

25. The pharmaceutical composition of Claim 22 wherein the metabolite is selected from the group consisting of: metabolites of racemic ibuprofen, R-ibuprofen, and S-ibuprofen.

26. The pharmaceutical composition of any of Claims 22-25, wherein the metabolite, or pharmaceutically acceptable salt, ester, or hydrate thereof, is in an amount of from about 400 mg to about 1600 mg.