WO2012170839A2

WO2012170839A2 - Treatment of neuroinflammatory disease with selective cox1 inhibitors

Info

Publication number: WO2012170839A2
Application number: PCT/US2012/041586
Authority: WO
Inventors: Karen J. Brunke; John CAUFIELD; Keni NII; Makoto OHMIYA
Original assignee: Cardeus Pharmaceuticals, Inc.; Astellas Pharma, Inc.
Priority date: 2011-06-09
Filing date: 2012-06-08
Publication date: 2012-12-13
Also published as: WO2012170839A3

Abstract

Highly selective COX1 inhibitors can be used to treat a neuroinflammatory disease.

Description

Treatment of Neuro inflammatory Disease with Selective COXl Inhibitors

[0001] This application claims_the benefit of U.S. Patent Application Nos.

61/495,267 filed June 9, 2011, and 61/502,657, filed June 29, 2011, each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] The invention provides new treatments and pharmaceutical formulations for the treatment of neuro inflammatory disease, such as Alzheimer's Disease (AD), Parkinson's Disease Dementia (PDD), and other types of dementia, as well as other microglial neuro inflammatory diseases such as Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), Multiple Sclerosis (MS), and other related disorders and so relates to the fields of biology, chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.

Description of Related Disclosures

[0003] It is estimated that dementia affects more than 10 % of individuals over 65 years of age, and nearly 50 % of those over 85 years of age. In two-thirds of those affected, the underlying cause of dementia is Alzheimer's Disease. AD is an age- related, progressive neurodegenerative disease that currently lacks any highly efficacious therapy, and no methods for preventing the disease have been developed. Another leading dementia is Parkinson's Disease Dementia which occurs in the late stages of Parkinson's Disease. This dementia affects 200,000-400,000 individuals in the US and more worldwide.

[0004] Although acetylcholinesterase inhibitors such as donepezil, galantamine, and rivastigmine have been used clinically based on the cholinergic neuron as the target of therapy, their efficacies are limited and manifestation of undesired side effects is frequent, perhaps associated with peripheral cholinergic stimulation. Therefore, a new generation of anti-dementia drugs with novel mechanisms of action is needed.

[0005] Despite studies on the involvement of various neurotransmitters, their receptors, and associated second messengers in AD and other neuroinflammatory diseases, the causes of cognitive impairment and neuronal degeneration in these diseases are not fully understood. Thromboxane A2 (TXA₂), which is synthesized by

cyclooxygenase-1 (COXl) enzymatic conversion of arachidonic acid to prostaglandin H2 which is then enzymatically converted to TXA₂ by thromboxane synthase, is increased in AD and vascular dementia patients in both plasma and brain [Vital Trial Collaborative Group, J. of Int. Med. (2003) 254: 67-75; Iwamoto, et al, J. Neurol (1989) 236: 80-84]. The increased thromboxane A2 level is significantly associated with cognitive (MMSE, ADAScog) dysfunction in AD and vascular dementia patients [Vital Trial Collaborative Group, 2003, supra]. COX1 expression levels and enzyme activity were increased in temporal cortices samples from AD patients compared to nondemented control brains [Kitamura, et al. (1999) BBRC 254 (3): 582-586]. This finding gives a biochemical basis to explain the increased level of thromboxane A2 in AD brains. (Thromboxane B2 (TXB₂), the inactive metabolite of TXA₂, is measured in all studies due to the short half- life of TXA₂.) However, there is very limited information on the use of selective thromboxane A2 or COX1 inhibitors even in animal studies of the disease. One study with a highly selective cyclooxygenase-2 (COX2) inhibitor in AD patients showed no improvement of disease over 6 months [Aisen et al. (2003) JAMA 289: 2819-2826]. Other reviews report that only COX-nonselective NSAIDs rather than COX-selective agents have any beneficial effects [Townsend and Pratico (2005) FASEB J 19: 1592- 1601].

[0006] Independent epidemiological studies [Etiman et al. (2003) BMJ

327: 128-132] indicated that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of developing AD. Most NSAIDs inhibit both COX1 and COX2. A wide variety of NSAIDs has been developed, and while some of these, such as aspirin and indomethacin, are more COX1 selective than COX2 selective, most of the approved NSAIDS are more COX2 selective than COX1 selective.

[0007] Unfortunately, COX1 preferential NSAIDs do not penetrate the central nervous system (CNS) and brain to any significant extent and so would be unlikely to have any beneficial effect on AD or other neuroinflammatory disease.

Aspirin, a classic NSAID, irreversibly inhibits COX1 and results in decreased levels of prostaglandins and thromboxane A2 in the plasma, but it is poorly brain penetrant like the other COX1 preferential NSAIDs and showed no benefit at low dose in prevention or treatment of AD.

[0008] Choi et al. [(2008) FASEB J 22: 1491-1501] reported that COX1 null mice show more resistance to LPS-induced neuronal death. Choi and Bosetti [(2009) Aging 1(2): 234-244] also reported that COX1 null mice show reduced

neuro inflammation in response to beta-amyloid. They suggest that "COX1 may represent a viable therapeutic target to treat neuroinflammation." However, the importance of neuroinflammation to disease remains controversial in the field; for example,

glucocorticoids (potent anti-inflammatory agents) show no effect on AD (Aisen et al. (2000) Neurology 54:588-593).

[0009] U.S. Patent 6,927,230 describes triazole compounds that have COX inhibitory activity and recites that these compounds may have beneficial effect in patients suffering from any of a wide and diverse set of diseases and conditions, including AD, but subsequent development and reports have focused on their use in treating vascular disease (see, e.g., U.S. Patent Application Publication No. 2011/0034504).

[0010] Thus, there remains a need for more efficacious therapies for dementia generally and AD and PDD specifically and other neuroinflammatory diseases as well. The present invention meets these needs.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides pharmaceutical formulations and unit dose forms for use in a method of treating and/or preventing a neuroinflammatory disease, a cardiovascular disease, or pain by administering a therapeutically effective dose of a highly selective COXl inhibitor that crosses the blood brain barrier. A "highly selective COXl inhibitor" is any compound that inhibits COXl at least 25-fold preferentially over its ability to inhibit COX2. In various embodiments, the highly selective COXl inhibitor is a compound that inhibits COXl at least 50-fold, 100- fold, or 500-fold, or 1200-fold over its ability to inhibit COX2. In various embodiments, the compound is a triazole compound described in U.S. Patent No. 6,927,230 or an active metabolite, prodrug, ester or salt form of such compound. In various embodiments, the compound is a compound of Formula 1 :

or an active metabolite, prodrug, ester, or salt thereof,

Formula 1

wherein Y and Z are independently CH or N,

R¹ is lower alkyl which is optionally substituted with halogen,

R is hydrogen, lower alkyl or lower alkoxy, and

R is hydrogen, lower alkyl or lower alkoxy.

[0012] In one embodiment, the compound in the pharmaceutical formulations of the invention is ASP6537 or an active metabolite, prodrug, ester or salt form of such compound. ASP6537 has the structure shown below and can be named, using ChemDraw Ultra, as 3 yphenyl)-lH-l,2,4-triazole.

[0013] In a second aspect, the present invention provides a method for treating and/or preventing a neuroinflammatory disease, a cardiovascular disease, or pain by administering a therapeutically effective dose of a highly selective COX1 inhibitor, both as a monotherapy and in combination with other classes of drugs. In various embodiments, the highly selective COX1 inhibitor is a compound of Formula 1 or an active metabolite, prodrug, ester, or salt thereof. In one embodiment, the therapeutically effective dose reduces blood plasma thromboxane A2 levels by at least forty percent (40%), and in various embodiments, thromboxane A2 levels are decreased by at least 50%, at least 75%, at least 90%, or at least 99%. In one embodiment, the therapeutically effective dose reduces microglial activation by at least twenty percent (20%), and in various embodiments, microglial activation is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or a or at least 95%. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily). In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily). In another embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily). In one embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD.

[0014] In one embodiment, the methods of the invention provide important new treatments for AD, ALS, HD, MS, PDD, PD, and other dementias and

neuro inflammatory diseases. In this embodiment of the methods of the invention, a compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits thromboxane A2 synthesis in the brain to improve cognition and inhibits microglial activation to delay progression of the disease. Current therapies, which include cholinesterase inhibitors and NMDA glutamate receptor antagonists, work on specific types of neurons, but the highly selective COX1 inhibitors used in the methods of the invention are not targeted to a single type of neuron and work across multiple neuron types. In addition, the inhibition of neuroinflammation through reduction in microglia activation has disease modifying potential by reducing or even stopping neuronal death. Therefore, the methods, pharmaceutical formulations, and unit dose forms of the invention are useful alone and in combination with other therapies for treatment of dementias and other neuro inflammatory diseases.

[0015] In one embodiment, the methods of the invention provide important new treatments for cardiovascular disease. In this embodiment of the methods of the invention, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits platelet aggregation. In one embodiment, a compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is administered to prevent or treat cardiac disease in a patient unable to take aspirin for that indication. In one embodiment, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered to prevent or treat cardiac disease in a patient that is also taking an non-steroidal anti-inflammatory drug (NSAID). In one embodiment, the present invention provides a pharmaceutical formulation of a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, and an NSAID for the treatment of patients with cardiovascular disease and pain.

[0016] In one embodiment, the methods of the invention provide important new treatments for the treatment of chronic pain. In this embodiment of the methods of the invention, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits thromboxane A2 synthesis. In one embodiment of these methods, a fast-acting pain relieving medication is administered to the patient prior to the administration of a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, for treatment of acute pain. In one embodiment of the methods of the invention, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits both thromboxane A2 synthesis and microglia activation for either acute or chronic pain.

[0017] Accordingly, the present invention provides new pharmaceutical formulations, unit dose forms and methods for delivering a therapeutically effective dose of a highly selective COX1 inhibitor to a patient suffering from a neuroinflammatory disease or condition, cardiovascular disease, and/or chronic pain. In various embodiments, the pharmaceutical formulation contains a triazole COX inhibitor (such as those described in U.S. Patent No. 6,927,230; U.S. Patent Application Publication Nos. 2008/0213383 and 2011/0034504; and PCT Application No. JP2011/062172, each of which is incorporated herein by reference). In various embodiments, the pharmaceutical formulation contains a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, and the therapeutically effective dose ranges from 3 to 400 mg daily, which daily dose is administered in one (QD administration) or two (BID administration) doses. In various embodiments, the unit dose forms of described herein contain one-third, one- half, or all of the daily dose. Illustrative unit dose forms include 1 mg, 3 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 400 mg dose forms. In one embodiment, the unit dose form contains ASP6537.

[0018] These and other aspects and embodiments of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 shows the results of a study, described in Example 1, of the levels of TXB₂ in the brain and plasma of aged and young rats. [0020] Figure 2 shows the results of a study, described in Example 1, demonstrating in Panel A that ASP6537 decreases TXB₂ levels in the brains aged rats in a dose-dependent manner given a single dose while aspirin given at daily doses over 7 days had no effect on TXB₂ levels in the brains of aged rats. In the figure, ** indicates a p value <0.01 compared to vehicle-treated group. In Panel B, single doses of either ASP6537 or aspirin in the plasma of aged rats caused dose-dependent decreases in TXB₂ levels.

[0021] Figure 3 shows the results of a study, described in Example 2, showing the role of thromboxane A2 in cognition in a mouse spontaneous alternation model of cognition. Part A shows that the TXA₂ agonist U-46619 decreases spontaneous alternation behavior, and Part B shows that the addition of the TXA₂ antagonist SQ-29548 returns this behavior to normal. The study involved intracerebroventricular (icv) delivery of agonist and/or antagonist directly to the ventricles of the brain, bypassing the blood brain barrier, allowing high and rapid drug delivery to the brain. Then, test animals were placed in a Y-shaped maze for a set time, and the number of arms entered and the sequence of entries were recorded, and a score was calculated to determine alternation rate (degree of arm entries without repetitions). A high alternation rate is indicative of sustained cognition as the animals must remember which arm was entered last so as not to reenter it.

[0022] Figure 4 shows the results of a study, described in Example 3, comparing the effects of ASP6537 and donepezil in the scopolamine-induced deficits model using the mouse Y-maze test. In the figure, each value shows the mean ± standard error of mean. C: control group (vehicle + scopolamine), ip: intraperitoneal, N: normal group (vehicle + saline), po: Oral. Vehicle=0.5% methylcellulose. ###: P< 0.001 vs. normal group using Student's test. *: P<0.05, **: P< 0.01 vs. control group using

Dunnett's test.

[0023] Figure 5 shows the results of a study, described in Example 3, comparing the effects of ASP6537 and donepezil in the MK-801 -induced deficits model using the mouse Y-maze test. In the figure, each value shows the mean ± standard error of the mean. C: control group (vehicle + MK-801), ip: intraperitoneal, N: normal group (vehicle + saline), po: oral. Vehicle=0.5% methylcellulose. ###: P<0.001 vs. normal group using Student's test. *: P<0.05 vs. control group using Dunnett's test. [0024] Figure 6 shows the results of a study, described in Example 3, showing the effect of ASP6537 (designated AS 1516537 in this figure) on spatial memory deficits and average velocity in aged rats in the Morris water maze. Panel A shows that the cumulative latency in finding the platform was significantly longer in aged compared to young rats, and that ASP6537 reduces this latency. Panel B shows that treatment with ASP6537 did not alter the average velocity. Panel C compares the daily changes in spatial memory deficits between aged rats, treated rats (3 different doses and dosed for 4 days), and young rats. In this figure: po is orally administered; SE is standard error; SEM is standard error of mean; each column and bar represents the mean ± SEM; the number of rats N is shown in parentheses; ## is P<0.01, statistically significant compared with young rats (Student's t-test); ** is P<0.01, statistically significant compared with vehicle- treated aged rats (Dunnett's multiple comparison test).

[0025] Figure 7 shows the results of a study, described in Example 4, demonstrating that combination therapy with donepezil and ASP6537 has an additive therapeutic effect over either drug along in improving the performance of transgenic mice in the Y-maze test.

[0026] Figure 8 shows the results of a study, described in Example 5, showing that ASP6537 reduces PGE2 levels in aged rats in a dose-dependent manner at higher doses of ASP6537 with dose shown on y-axis being in units of mg/kg.

[0027] Figure 9 shows the results of a study, described in Example 5, showing that ASP6537 inhibits microglial activation in aged rats, reducing activation to levels of young rats. The histological staining procedure used to generate the data uses polyclonal antibodies to Ibal (ionized calcium-binding adaptor molecule- 1) antigen peptide which detects microglia activation. Figure 9 shows the increase of activated microglia in different parts of the brain in aged rats. The experiment was performed by loading ASP6537 at a concentration of 10 mg/mL into the Alzet Osmotic pump model 2mL2 (0.03 mg/uL, 5 uL/hr; 2 week). The number 10 on the x-axis with respect to ASP6537 refers to the concentration of compound in the pump.

[0028] Figure 10 shows the results of a pharmacokinetic study, described in Example 6, of ASP6537 after oral administration to aged rats. Each data point is the mean plus/minus the standard deviation for three animals. Squares indicate brain and triangles plasma concentrations. [0029] Figure 11 shows the results of a brain penetration study, described in Example 6, of ASP6537 after intravenous administration to monkeys. A common measure for PET (positron emission tomography) scan is standardized uptake value (SUV), shown on the y axis in Figure 10. Standardized uptake values (SUVs) are a measure of the concentration of a radiotracer in a defined region divided by the injected dose normalized for body weight at a fixed time after tracer injection.

[0030] Figure 12 shows the results of a human clinical study in healthy volunteers, described in Example 7, showing the effect of a single orally administered suspension dose of ASP6537 on thromboxane B2 concentration in serum. ASP6537 is referred to as FK881 in this figure.

[0031] Figure 13 shows the results of a human clinical study in healthy volunteers, described in Example 7, showing the effect of a single orally administered suspension dose of ASP6537 on reduction of serum thromboxane A2 levels, as determined by measuring thromboxane B2 levels (referred to as "inhibition" in the figure) in comparison with pre-dose baseline. ASP6537 is referred to as FK881 in this figure.

[0032] Figure 14 shows the results of a human clinical study in healthy volunteers, described in Example 7, showing the effect of a single orally administered tablet dose of ASP6537 on thromboxane B2 concentration in serum. ASP6537 is referred to as FK881 in this figure.

[0033] Figure 15 shows the results of a human clinical study in healthy volunteers, described in Example 7, showing the effect of a single orally administered tablet dose of ASP6537 on reduction of serum thromboxane B2 levels (termed inhibition) in comparison with pre-dose baseline. ASP6537 is referred to as FK881 in this figure.

[0034] Figure 16 shows the results of a study, described in Example 11, demonstrating the benefit of ASP6537 over aspirin for concomitant dosing with ibuprofen. In the figure, ** indicates a p value of < 0.01 (Student t-test vs. vehicle).

DETAILED DESCRPTION OF THE INVENTION

[0035] The present invention provides pharmaceutical formulations and unit dose forms for use in a method of treating and/or preventing a neuro inflammatory disease, cardiovascular disease, or pain by administering a therapeutically effective dose of a highly selective COX1 inhibitor that crosses the blood brain barrier. In various embodiments, the highly selective COX1 inhibitor is a compound of Formula

Formula 1

wherein

Y and Z are independently CH or N,

R¹ is lower alkyl which is optionally substituted with halogen,

R is hydrogen, lower alkyl or lower alkoxy, and

R is hydrogen, lower alkyl or lower alkoxy.

[0036] In some embodiments, Y is CH. In some embodiments, Y is N. In some embodiments, Z is CH. In some embodiments, Z is N. In some embodiments, Y is CH and Z is CH. In some embodiments, Y is N and Z is CH. In some embodiments, Y is CH and Z is N.

[0037] In some embodiments, R¹ is lower alkyl. In some embodiments, R¹ is chosen from -CH₃, -CH₂F, -CF₃, -CH₂CH₃, -CH₂CF₃, and -CH(CH₃)₂. In some embodiments, R¹ is chosen from -CH3.

[0038] In some embodiments, R is lower alkoxy. In some embodiments,

2

R is methoxy.

[0039] In some embodiments, R is lower alkoxy. In some embodiments,

R is methoxy.

2

[0040] In some embodiments, R is methoxy, R is methoxy, Y is CH, and

Z is CH.

[0041] In some embodiments, the compound of Formula 1 is chosen from

3-methoxy- 1 ,5-bis(4-methoxyphenyl)- 1H- 1 ,2,4-triazole,

3-ethoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4-triazole,

1 ,5-bis(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)- 1H- 1 ,2,4-triazole,

2-methoxy-5-(l-(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)-lH-l,2,4-triazole-5- yl)pyridine,

2-methoxy-5-(5-(4-methoxyphenyl)-3-(2,2,2-trifluoroethoxy)- 1H- 1 ,2,4-triazol- 1 - yl)pyridine, 1 ,5-bis(4-methoxyphenyl)-3-(2-propoxy)-l H- 1 ,2,4-triazole,

1 ,5-bis(4-methoxyphenyl)-3-(fluoromethoxy)- 1H- 1 ,2,4-triazole,

3-methoxy- 1 ,5-bis(4-methoxyphenyl)- 1H- 1 ,2,4-triazole,

or an active metabolite, prodrug, ester, or salt thereof.

[0042] In one embodiment, the compound is ASP6537, i.e., 3-methoxy- l,5-bis(4-methoxyphenyl)-lH-l,2,4-triazole, or an active metabolite, prodrug, ester, or salt thereof.

Definitions

[0043] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0044] A "salt" may be prepared for any compound having a functionality capable of forming a salt, for example, an acid or base functionality. Salts may be derived from organic or inorganic acids and bases. Compounds that contain one or more basic functional groups, e.g., amino or alkylamino, are capable of forming salts with pharmaceutically acceptable organic and inorganic acids. These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting a purified compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- napthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, and tosylate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their acid addition salts. See, e.g., Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66: 1-19.

[0045] Compounds that contain one or more acidic functional groups are capable of forming salts with pharmaceutically acceptable bases. The term "salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds described herein. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Illustrative examples of some of the bases that can be used include sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, N⁺(Ci_-4 alkyl)₄, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethyl enediamine, ethanolamine, diethanolamine, piperazine and the like. Also provided are salts wherein one or more basic nitrogen-containing groups are quarternized. Water or oil-soluble or dispersible products may be obtained by such quatemization. See, for example, Berge et al., supra.

[0046] The term "prodrug" refers to a substance administered in an inactive or less active form that is then transformed (e.g., by metabolic processing of the prodrug in the body) into an active compound. The rationale behind administering a prodrug is to optimize absorption, distribution, metabolism, and/or excretion of the drug. Prodrugs may be obtained by making a derivative of an active compound that will undergo a transformation under the conditions of use (e.g., within the body) to form the active compound. The transformation of the prodrug to the active compound may proceed spontaneously (e.g., by way of a hydrolysis reaction) or it can be catalyzed or induced by another agent (e.g., an enzyme, light, acid or base, and/or temperature). The agent may be endogenous to the conditions of use (e.g., an enzyme present in the cells to which the prodrug is administered, or the acidic conditions of the stomach) or the agent may be supplied exogenously. Prodrugs can be obtained by converting one or more functional groups in the active compound into another functional group, which is then converted back to the original functional group when administered to the body. For example, a hydroxyl functional group can be converted to a sulfonate, phosphate, ester or carbonate group, which in turn can be hydrolyzed in vivo back to the hydroxyl group. Similarly, an amino functional group can be converted, for example, into an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl functional group, which can be hydrolyzed in vivo back to the amino group. A carboxyl functional group can be converted, for example, into an ester (including silyl esters and thioesters), amide or hydrazide functional group, which can be hydrolyzed in vivo back to the carboxyl group.

[0047] The term "ester" refers to a compound formally derived from a carboxylic acid and an alcohol, phenol, heteroarenol, or enol by linking with formal loss of water from an acidic hydroxy group of the former and a hydroxy group of the latter.

[0048] Terms such as " active metabolite" and the like refer to a derivative of the highly selective COX1 inhibitor that retains a detectable level, e.g., at least about 10%, at least about 20%, at least about 30% or at least about 50%, of at least one desired activity of the parent compound,. Determination of a desired activity may be

accomplished as described herein. Such metabolites can be generated in the

gastrointestinal tract, in blood or in one or more subject tissues. Such metabolites are detected using standard analytical methods, e.g., GC-MS analysis of an optionally radiolabeled parent compound and its metabolites, in blood, urine or other biological samples after it is administered to a patient by one or more routes as disclosed herein.

[0049] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers include excipients and diluents. [0050] The highly selective COX1 inhibitors described herein can be enriched isotopic forms, e.g., enriched in the content of ²H, ³H, ⁿC, ¹³C and/or ¹⁴C. In one embodiment, the compound contains at least one deuterium atom. Such deuterated forms can be made, for example, by the procedure described in U.S. Patent Nos.

5,846,514 and 6,334,997. Such deuterated compounds may improve the efficacy and increase the duration of action of compounds disclosed and/or described herein.

Deuterium substituted compounds can be synthesized using various methods, such as those described in: Dean, Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development, Curr. Pharm. Des., 2000; 6(10); Kabalka et al., The Synthesis of Radiolabeled Compounds via

Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans,

Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.

Treatment and Prevention of Neuro inflammatory Diseases and Conditions

[0051] The over expression of COX1 and increased synthesis of thromboxane A2 (TXA₂) have been observed in the AD brain [Iwamoto et al. (1989) J Neurol 236: 80-84] in the cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV) dementia [Griffin et al. (1994) Ann Neurol 35 (5): 592-597] in gliomas, mengiomas and metastatic brain lesions [Castelli et al. (1989) Cane Res 49: 1505-1508]; and in the CSF from traumatic brain injury [Mustafa et al. (2003) Neurosurg Quart 13(2): 59-63]. Those individuals with AD had both higher plasma and brain thromboxane A2 levels [as measured by its inactive metabolite, thromboxane B2 (TXB₂), due to the short half-life of TXA₂] especially in frontal and temporal cortex regions of the brain [Iwamoto et al. (1989) J Neurol 236: 80-84]. Increased levels of thromboxane A2 occur as a result of increased synthesis through a pathway mediated by COX1 involving enzymatic conversion of arachidonic acid to prostaglandin H2 which in turn is enzymatically converted to thromboxane A2 via thromboxane synthase. The elevated level of TXB₂ observed in the plasma correlated with elevated levels in the brain in the AD patients and also in aged rats (Example 1, Figure 1). Thus, aged rats make a good model for studying effects on plasma and brain levels of TXB₂ in dementias. In addition, as shown in Example 1 and Figure 2 A, ASP6537 can suppress brain thromboxane B2 (TXB₂) levels in aged rats by suppressing brain TXA₂ synthesis with suppression reaching statistical significance at the effective dose needed to improve cognitive function in aged rats. The effective dose for cognition improvement in aged rats in the water maze model was 1 mg/kg of ASP6537 as shown in Example 3, Figure 6. Of interest, the level of

thromboxane A2 (Example 1 , Figure 2A) where cognition improvement was

demonstrated in this model was approximately 40% lower than that seen in untreated aged rats. As seen in Figure 1 and described in Example 1, a 40% reduction in

thromboxane A2 levels in aged rats reduced the thromboxane A2 level in the brain to that observed in the brains of young rats (Example 1, Figure 1). Thus, the thromboxane A2 level after treatment with ASP6537 at a dose giving 40% reduction in brain thromboxane A2 levels in aged rats correlates with the levels observed in the brains of young, healthy, cognitively unimpaired animals.

[0052] ASP6537 dose dependently lowers the levels of both brain and plasma thromboxane A2 as discussed in Example 1 (Figures 2 A and 2B). Aspirin while effective at dose-dependent TXA₂ reduction in the plasma does not reduce the level of TXA₂ in the brain. In Example 1 , Figure 2A, aspirin dosed daily for 7 days did not reduce TXA₂ in the brain while a single dose of ASP6537 dose-dependently reduced TXB₂. As mentioned earlier, TXB₂ is the inactive metabolite of TXA₂. TXB₂ is measured in the experiments in Figures 1 and 2.

[0053] As dosing with ASP6537 both reduces TXA₂ in the plasma and

TXA₂ in the brain, reduction of TXA₂ in the plasma (in the absence of drugs such as aspirin which reduce TXA₂ levels in the plasma) is a surrogate for reduction of TXA₂ in the brain and provides a clinically relevant marker for ASP6537 action in the brain. TXA₂ is measured by measurement of its inactive metabolite, TXB₂.

[0054] The present invention arose in part from the discoveries that thromboxane A2 is an inhibitory neurotransmitter that can decrease cognition and that highly selective COXl inhibitors can improve cognitive function based on inhibition of brain TXA₂ synthesis in AD as well as other dementias and that certain triazole compounds, such as ASP6537 and other compounds of Formula 1, , or an active metabolite, prodrug, ester, or salt thereof, which are potent and selective COXl inhibitors, can penetrate the blood-brain barrier and improve cognition. Example 2 and Figure 3 describe a study demonstrating the effect of decreasing TXA₂ levels, as indicated by measuring thromboxane B2 levels, on cognition. U-46619 is an agonist of TXA₂. U- 44619 works by binding the thromboxane A2 receptor and mimicking thromboxane; increased U-44619 has the same effect as increased thromboxane A2. SQ-29548 is a thromboxane A2 receptor antagonist. S-29548 blocks the binding of thromboxane A2 and/or agonist and may compete off bound thromboxane A2, U-44619 (or other agonist) from the thromboxane A2 receptor. In this study, test animals were placed in a Y-shaped maze and the number of arms of the maze and the rate of entry measured. A high alternation rate is indicative of sustained cognition as the animals must remember which arm was entered last so as not to reenter it. In the presence of the TXA₂ agonist, spontaneous alternation behavior of mice is decreased, demonstrating the negative effects of increased thromboxane A2 (see Figure 3A). However, as shown in Figure 3B, when the TXA₂ antagonist SQ-29548 is added (blocking the effect of thromboxane A2, U- 44619 or other agonist), the spontaneous alternation behavior of mice returns to normal. This finding is direct evidence of the role of thromboxane A2 in the increased cognitive impairment typical of Alzheimer's Disease and other dementias.

[0055] TXA₂ synthesis is regulated through the upstream effect of COX1 in neurons. COX1 catalyzes arachidonic acid conversion to prostaglandin H2 which is then enzymatically converted to TXA₂ by thromboxane synthase. Inhibition of COX1 activity results in decreased TXA₂ synthesis and TXA₂ levels in neurons. While the present invention is not to be limited by any theoretical mechanism of action, the suppression of brain TXA₂ is believed to be one of the mechanisms for the beneficial effect of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, on cognitive function in aged rats.

[0056] As shown in Example 3 and Figures 4, 5, and 6 below,

administration of ASP6537 improves cognitive impairments induced by scopolamine and MK-801 in mice and ameliorates the learning disability seen in aged rats. Learning deficits induced by scopolamine, a muscarinic acetylcholine receptor antagonist, are typically exploited in some animal models of dementia as well as for cognition tests in humans. The test looks for reversal of cognitive deficit induced by scopolamine.

Donepezil has been reported to be effective in this animal model. The measurement of spontaneous alternation behavior in a Y-maze is recognized as one of the models for measurement of short term (working) memory performance. A criticism of such scopolamine reversal models pertains to the lack of versatility in the model, as scopolamine's actions are limited to the blockade of brain function mediated via cholinergic (muscarinic) receptors. Scopolamine, however, is relatively nonselective pharmacologically with respect to receptor subtypes, and the drug does not discriminate very much with respect to brain region. Scopolamine certainly would have little direct effect on non-cholinergic neuronal pathways, although cholinergic neurons have functional interactions with a wide variety of neurotransmitter systems that could be affected indirectly by a drug. ASP6537 would therefore have its main effect on the cholinergic neurons in this model. As seen in Example 3 and Figure 4, ASP6537 reverses the scopolamine effect as well or better than donepezil in this model. Scopolamine (0.5 mg/kg, ip) significantly decreased spontaneous alternation rate, the marker of

scopolamine-induced working memory deficit. ASP6537 (1 and 3 mg/kg po) and donepezil (0.25 and 0.5 mg/kg po) significantly attenuated the scopolamine-induced deficits. Both ASP6537 and donepezil ameliorated scopolamine-induced cognitive deficits, indicating that both ASP6537 -and donepezil were effective in the cholinergic cognitive impairment model in mice.

[0057] The effects of ASP6537 and donepezil were compared in the MK-

801 -induced deficit model using the mouse Y-maze test as shown in Example 3, Figure 5. MK-801 is a non-competitive antagonist of N-methyl-D-aspartate (NMD A), a glutamate receptor. Animals treated with MK-801 show various memory/learning deficits because of the blockage of the NMD A receptors. Unfortunately, the dose range over which MK- 801 induces cognitive impairment without causing sensory, locomotor, or toxicological side effects is small. On the basis of published evidence and the present findings, MK- 801, administered s.c. or i.p. into rodents in doses up to 0.1 mg/kg, appears to fulfill the criteria of a cognition impairer in rodents, without causing sensorimotor impairments and/or signs of intoxication. In addition, MK-801 -treated rodents appear to fulfill the criteria of a valid animal model of cognitive dysfunctions, with robust effects across species, housing conditions, and testing paradigms [van der Staay et al. (2011) Behav Brain Res 220(1): 215-229]. It is well-known that the clinical efficacy of donepezil, a currently available anti-dementia drug, is limited in effect in dementia patients. The result of the study described in Example 3, and Figure 5, demonstrates the existence of memory deficits resistant to donepezil. This preclinical result is consistent with the lack of clinical efficacy seen with donepezil with respect to glutamatergic neurons. These results demonstrate that ASP6537 improved working memory deficits caused by glutamatergic disturbance. Because donepezil is not effective in this model, this study demonstrates ASP6537 has beneficial effect in the treatment of cognitive disorders across multiple neuron types including glutamatergic and cholinergic neurons (the cholinergic data were described in Example 3, Figure 4). [0058] Aged rodents have proven to be a useful tool in studying age- related cognitive decline, particularly with regard to hippocampal function. A number of maze tests have been developed to evaluate hippocampal function in aged rodents, including the Morris water maze test for evaluating learning and memory deficits. In this test, a rat is placed in a pool of water about two meters in diameter. In the middle of the pool, about 1-2 cm below the surface, is a hidden platform. Because the rat is not able to see the platform, it can only discover it by accident. When a rat is placed in the pool for the first time, it will swim in random directions, often toward the pool's perimeter. Over time though, they swim towards the middle and discover the platform. If this test is done repeatedly with the same rat, eventually it will remember the location and find the platform in less time. After enough trials, the rat will swim directly to the platform. With cognitive decline, it takes longer for the aged rats to find the hidden platform than with younger rats. Improvement in finding the platform using therapeutic agents correlates with improved learning and spatial memory.

[0059] The therapeutic effect of ASP6537 on spatial memory deficits in aged rats was demonstrated in the Morris water maze in a study described in Example 3 and Figure 6. The cumulative latency in finding the platform was significantly longer in aged rats (25 months old) compared with young rats (9 weeks old), as shown in Figure 6 A. Treatment with ASP6537 significantly shortened the cumulative latency at the dose of 1 mg/kg as shown in Figure 6A, while the average velocity of aged rats was not affected by ASP6537 (Figure 6B). In Figure 6C, the daily latency on each of four days of the trial was graphed versus time for untreated aged rats, aged rats at three different doses of ASP6537 and untreated young rats. The aged rats showed a sharp contrast with the young rats in speed in finding the platform over time (escape latency) and the ASP6537- treated aged rats showed an intermediate learning behavior. In contrast to these results, the ability of donepezil hydrochloride, an acetylcholinesterase inhibitor, to enhance spatial memory was assessed in aged rats using the Morris water maze. As in the

ASP6537 study, control aged rats (24-25 months) showed longer escape latency when they were trained in the water maze task in comparison with young rats. Daily treatment with donepezil hydrochloride (0.1-3 mg/kg, po) failed to improve the latency in aged rats, essentially giving the same result as visualized with aged rats. These results demonstrate that donepezil hydrochloride exerts no ameliorating effect on the spatial memory deficits seen in aged rats. This result supports ASP6537 working across neuron types in the brain to improve cognition.

[0060] Additional studies of ASP6537 in combination with donepezil provide additional demonstration that ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, work across multiple neuron types. Thus, ASP6537 was demonstrated to provide additional benefit when administered in combination with donepezil as donepezil only works on cholinergic neurons by inhibiting acetylcholinesterases and increasing the available acetylcholine. The study described in Example 4 and Figure 7 shows additive cognitive benefit of such coadministration, consistent with ASP6537 working across neuron types. Figure 7 shows the beneficial effect of combining donepezil and ASP6537 in animal models of behavior using a transgenic mouse model for APP (Tg2576) in the Y-maze test to assess cognition.

Tg2576 is a transgenic animal model of Alzheimer's disease with a mutant human gene for amyloid precursor protein (APP). The result of this mutation is much higher levels of truncated beta amyloid peptides 40 and 42. The elevated peptides correlate with memory and learning deficits at younger ages than in age matched mouse controls.

[0061] Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. COX-1 is a major source of PGE2 in microglia. PGE2 is a marker of inflammation in humans and animals alike. In the aged rat study described in Example 5 and Figure 8, the results are consistent with reduction of neuro inflammation by inhibition of COX1 activity by ASP6537. As shown in Figure 8, PGE2 levels were reduced by ASP6537 in a dose dependent manner at the higher doses in aged rats with dose shown on y-axis being in units of mg/kg. This demonstrates that, in aging rats, PGE2 is reduced by inhibition of its synthesis due to upstream inhibition of COX1 activity in microglia.

[0062] Another property of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is their ability to inhibit COX1 present in microglia and to reduce or block activation of microglia. Neuroinflammatory diseases have as a hallmark chronic over activation of microglia and are termed glial diseases for this reason. Microglia activation occurs in the aging population

[Scheuitemake et al. (2012) Neurobiol Again 33(6): 1067-1072] and in particular in those with neuroinflammatory diseases [Streit et al. (2004) J Neuroinflammation 1 : 14-17]. However, in some neuroinflammatory diseases, such as multiple sclerosis, there is no association with aging. A common observation in all neuroinflammatory disease is that microglia are activated many fold. Activation of microglia leads to production of prostaglandin E2 (PGE2), a proinflammatory mediator. Over activation of microglia using a gram negative bacteriotoxin in animal models directly leads to neuronal death via neuroinfiammation [Choi et al. (2008) FASEB J 22: 1491-1501]. There are no effective agents on the market today to reduce neuroinfiammation. ASP6537 through its ability to inactivate microglia and reduce PGE2 levels lowers the levels of the mediators responsible for neuroinfiammation. The presence of activated microglia and the efficacy of an agent in inhibiting microglia activation can be determined using imaging studies. Imaging can be performed using the PET agent [11C]-(R) PK11195, a specific ligand of PBBS (peripheral benzodiazepine-binding sites) [Cagnin et al. (2006) Acta Neurol Scand 114 (Suppl 185): 107-114]. In addition, more sensitive and even more quantitative ligands for use in PET/CT imaging of microglia activation and neuroinfiammation are in development such as [11C] PBR28, [11C] DAA1106, [18F] FEDAA1106, and [11C] Vinpocetine [Ching et al. (2012) Insights Imaging 3: 111-119].

[0063] Reduction in microglia activation correlates with reduction in the proinflammatory mediators responsible for neuroinfiammation. Reduction in

neuroinfiammation directly correlates with reduction in neuronal degeneration and destruction. Thus, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, have disease modifying potential through their ability to reduce neuronal destruction in neuroinflammatory diseases by inhibiting microglial activation as described in Example 5 and Figure 9. Clinical dosing of ASP6537 for inactivation of microglia is the same as clinical dosing for reduction of TXA₂ (as measured by detection of levels of thromboxane B2 in plasma in individuals not taking other platelet aggregation inhibitors such as aspirin which would also lower plasma TXA₂) as described above for neuroinflammatory diseases. The dosing in Example 5 and Figure 9 provides an inhibition effect that returns the level of activation in old rats to that seen in younger animals; importantly, 100% inhibition of microglia activation does not occur at the therapeutically effective doses employed in this study. As some microglia activation may be required to phagocytize dead neurons and permit microglia to perform the role they perform in the younger animals, dosing which gives less than 100% inhibition is desirable. Thus, in accordance with the methods of the invention, 20-95% inhibition of microglia activation provides the desired beneficial effect. As elevated levels of microglia activation are correlated with increased neuroinflammation, these studies demonstrate that ASP6537 can reduce neuroinflammation by inhibiting microglial activation, and that such reduction in neuroinflammation by ASP6537 occurs in multiple regions (across multiple neuron types) in the brain.

[0064] The beneficial effects of the therapeutic methods described herein can be demonstrated by assessing symptoms in patients such as evaluation of cognition, disease specific neuronal tests, daily living activities, and overall clinical response. In addition, because of the delay, arrest, and improvement of symptoms detected as measurable changes such as cognition improvement (in dementias), motor coordination (in motor neuron diseases), and reduced neuroinflammation in neuroinflammatory diseases as well as the delay, decrease, or arrest of neuronal destruction in all these diseases, the treatment methods of the invention are disease modifying in nature. As an example, because COXl inhibition also lessens or arrests the neuronal damage in AD in response to amyloid beta and tau through overall decreased neuroinflammation, there is a potential for long term disease modification by "sparing" neurons from the damage caused by amyloid beta, tau, and neuroinflammation. A disease modifying effect occurs when the pharmacologic treatment delays the underlying pathological or

pathophysiological disease processes, modifying the progression of the disease, and is accompanied by an improvement of clinical signs and symptoms of the condition. The effectiveness of this treatment paradigm for AD is supported by the study described in Example 5 and shown in Figure 9 for microglia inactivation as well as the studies described in Example 3 and shown in Figures 4, 5, and 6 for cognition improvement caused by thromboxane A2 synthesis inhibition and inhibition of microglia activation (resulting in reduction of neuroinflammation).

[0065] ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, exhibit high penetration of the CNS and the brain. Brain penetration for ASP6537 is significant, in that brain levels are 196% of plasma levels based on quantitative whole body autoradiography (QWBA) in rats and as high as 400% of plasma levels in monkeys based on following radioactive tracers as described in Example 6 and shown in Figures 10 and 11. ASP6537 will reach the COXl target in the neurons and microglia to decrease thromboxane A2 levels and

neuroinflammation based on its ability to penetrate the brain. [0066] In human clinical studies, single doses of ASP6537 have been demonstrated to reduce the levels of plasma thromboxane B2 (TXB₂), as described in Example 7 and shown in Figures 12-15. Multiple doses of ASP6537 predictably and effectively reduced TXB₂ as well based on a separate study in humans. From these human and animal studies, an optimal dosing regimen for using ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, to suppress TXA₂ synthesis (as measured by its inactive metabolite, TXB₂) in the brain is through once daily (QD) or twice daily (BID) dosing. The brain dose response curve has been observed to parallel the plasma dose response curve for reduction in thromboxane A2 levels. Clinical studies of ASP6537 in healthy individuals demonstrate that Ctrough (the lowest plasma dose of the drug prior to the next dose) and drug exposure and half-life at steady state demonstrate that a dose of 200 mg QD administered daily for 13 days resulted in 95-99% reduction in plasma thromboxane A2 levels from baseline at a point 24 hours after the last dose (versus about 75% reduction with a single dose).

[0067] In various embodiments, for the treatment of AD, clinical dosing of

ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day. In one embodiment for the treatment of AD, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily). In another embodiment for the treatment of AD, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily). In another embodiment for the treatment of AD, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily). In one embodiment for the treatment of AD, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD. In another embodiment for the treatment of AD, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD. In another embodiment for the treatment of AD, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD.

[0068] Nonsteroidal anti-inflammatory drugs (NSAIDs) currently fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both cyclooxygenases-1 and 2 (COXl, 2), which act as key enzymes catalyzing the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibits COX2. Inhibition of the inducible COX2 isoform is believed to be responsible for some therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COXl inhibition has as one of its primary effects the inhibition of platelet aggregation. COXl inhibition has also been suggested to be responsible for undesired side-effects of NSAID administration on the gastrointestinal (GI) system. The results reported herein, however, demonstrate that the highly selective COXl inhibitor ASP6537 has a positive therapeutic effect with no GI side effects in the therapeutic dose ranges provided by the present invention (see Example 8).

[0069] There are currently no highly selective COXl inhibitors approved for any disease indication, and there is no COXl selective inhibitor approved for AD and/or other dementias and/or neuroinflammatory diseases. Another property that makes ASP6537 an excellent drug for treatment of neuroinflammatory diseases and dementia is its high selectivity for binding of COXl rather than COX2. The selectivity of COXl binding over COX2 binding is demonstrated in Example 8, Table 1. These results showed that ASP6537 had COXl selective inhibition activity in human whole blood of 650-fold COXl selective in comparison with COX2.

[0070] Another property that makes ASP6537 and other compounds of

Formula 1, or an active metabolite, prodrug, ester, or salt thereof, good therapeutics is their limited potential for drug-drug interactions (DDIs) based on the evidence of multiple pathways for metabolism via CYP3A4, CYP2A9 and glucoronidation. This attribute is especially important when ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is used in combination therapy or otherwise provided to a patient taking other medications. Another property that makes ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, excellent for combination therapy as well as monotherapy is their desirable low side effect profile due to low binding affinity to a wide variety of receptors, ion channels and transporters, as described in Example 9, Table 2.

[0071] Microglia participate in all phases of the multiple sclerosis (MS) disease process (Jack et al. (2005) J Neurosci Res 81(3): 365-373). Phagocytosis by microglia/macrophages is a hallmark of the MS lesion; however, the extent of tissue damage and the type of cell death dictate subsequent innate responses. Microglia/macrophages are armed with a battery of effector molecules, such as reactive nitrogen species, that may contribute to CNS tissue injury, specifically to the injury of oligodendrocytes that is associated with MS. A therapeutic challenge is to modulate the dynamic properties of microglia/macrophages so as to limit potentially damaging innate responses, to protect the CNS from injury, and to promote local recovery. As shown in Example 5 and Figure 9, ASP6537 inhibits microglia activation about 75% at the dose used in that study. ASP6537 can promote both local recovery through normal

phagocytosis and mitigate damage through reduction in effector molecule release in MS when dosed at a level that provides 75% inhibition of microglial activation. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day for the treatment of MS. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily) for the treatment of MS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily) for the treatment of MS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily) for the treatment of MS. In one embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD for the treatment of MS. In another embodiment,

ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD for the treatment of MS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD for the treatment of MS.

[0072] Inflammation, including microglial activation and T cell infiltration, is a neuropathological hallmark of amyotrophic lateral sclerosis (ALS), a rapidly progressing neurodegenerative disease. Transgenic mice display similar inflammatory reactions at sites of motoneuron injury as detected in ALS patients, enabling the observation that this inflammation is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity [Henkel et al. (2009) J Neuroimmune Pharmacol 4 (4): 389-398]. The microglial and T cell activation states influence the rate of disease progression. Initially, microglia and T cells can slow disease progression, while they later contribute to the acceleration of disease. Thus, inflammation plays a central role in ALS, and manipulating microglial effector functions can modify disease progression and outcome of this devastating disease. As shown in Example 5 and Figure 9, ASP6537 inhibits microglia activation about 75% at the dose administered in this animal study.

[0073] In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day for the treatment of ALS. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily) for the treatment of ALS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily) for the treatment of ALS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily) for the treatment of ALS. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD for the treatment of ALS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD for the treatment of ALS. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD for the treatment of ALS.

[0074] Parkinson's Disease (PD) is characterized by loss of dopaminergic neurons from the substantia nigra. In recent years, the involvement of neuroinflammatory processes in nigral degeneration has gained increasing attention. Not only have activated microglia and increased levels of inflammatory mediators been detected in the striatum of PD patients, but a large body of animal studies points to a contributory role of

inflammation in dopaminergic cell loss. Notably, the substantia nigra in PD is reported to have one of the highest concentrations of microglia in the brain. Long term treatment with non-steroidal anti-inflammatory drugs has been shown to reduce the risk of developing PD [Hald et al. (2007) Subcell Biochem 42(5): 249-279]. As described in Example 5 and shown in Figure 9, ASP6537 and other compounds of Formula 1, , or an active metabolite, prodrug, ester, or salt thereof, with their ability to inhibit microglia activation, can benefit those with PD. In addition, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be administered in combination with other agents used for treating PD. Early in PD, administration of ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, can reduce neuroinflammation and prevent dopaminergic neuronal loss and can be administered before there is a need to use levodopa. Later in the disease, microglia activation continues, and inhibition of microglia activation in accordance with the methods of the invention provides reduction in neuroinflammation and prevention of further neuronal loss. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, for the treatment of PD ranges from 3 mg/day to 400 mg/day. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily) for the treatment of PD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily) for the treatment of PD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily) for the treatment of PD. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD for the treatment of PD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD for the treatment of PD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD for the treatment of PD.

[0075] Parkinson's Disease frequently progresses to Parkinson's Disease

Dementia (PDD) with, on average, 24.5% of PD patients progressing to PDD. Significant microglial activation is present in the brains of patients with neurodegenerative dementias such as PDD even at early stages of the disease, with a spatial distribution reflecting different clinical phenotypes [Cagnin et al. (2006) supra]. So, while microglia in PD are activated in the substantia nigra, PDD microglial activation is frequently visualized in other parts of the brain, such as the cortex. There are as many or more cholinergic receptors lost in PDD than in AD, and PDD is known to affect additional neurons as well. The studies described in Example 3 and shown in Figures 4, 5, and 6 demonstrate that ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be used to treat PDD (cognition improvement based both on

thromboxane A2 synthesis inhibition and microglia activation inhibition). In addition, as described in Example 5 and shown in Figure 9, microglia activation inhibition is particularly beneficial in patients with PDD because, in addition to inhibition of neuroinflammation around cholinergic and other neurons involved in dementia, there would also be therapeutic benefit from inhibiting the inflammation from PD

(dopaminergic neurons in the substantia nigra). In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, for treatment of PDD ranges from 3 mg/day to 400 mg/day. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily) for treatment of PDD. In another embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily) for treatment of PDD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily) for treatment of PDD. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD for treatment of PDD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD for treatment of PDD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD for treatment of PDD.

[0076] Huntington's Disease (HD) is characterized by the progressive death of medium spiny dopamine receptor bearing striatal GABAergic neurons. In addition, microglial activation in the areas of neuronal loss has recently been described in postmortem studies. Activated microglia are known to release neurotoxic cytokines, and these may contribute to the pathologic process. [(11)C](R)-PK11195 PET studies show that the level of microglial activation correlates with severity of HD. These studies support that microglia contributes to the ongoing neuronal degeneration in HD and demonstrate that [(11)C](R)-PK11195 PET is a useful marker for evaluating the efficacy of therapeutic agents in this relentlessly progressive genetic disorder [Pavese et al. (2006) Neurol 66 (11): 1638-1643]. Additional studies demonstrate that microglial activation is an early process in HD pathology, occurring before the onset of symptoms. The close spatial and temporal relationship between microglial activation and neuronal dysfunction further supports the pathogenic link between the two processes in HD [Tai et al. (2007) Brain Res Bull 72 (2-3): 148-151]. Huntington's disease is an autosomal dominant condition, which means that only one parent must have this gene for a child to inherit HD, which has a prevalence of 30,000 in the US, and 150,000 individuals are at risk of developing the disease in the US. Early detection and treatment could slow or prevent disease progression. The study described in Example 5 and shown in Figure 9

demonstrates the inhibition of microglia activation with ASP6537, and the study described in Example 3 and shown in Figures 4, 5, and 6 demonstrate that ASP6537 and other compounds of Formula 1 are useful in the treatment of the dementia and cognitive impairment associated with HD. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, for treatment of HD ranges from 3 mg/day to 400 mg/day. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily) for treatment of HD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily) for treatment of HD. In another embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily) for treatment of HD. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD for treatment of HD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD for treatment of HD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD for treatment of HD.

[0077] Thus, the present invention represents a significant advance in the treatment of significant unmet medical needs. Neuro inflammatory-related dementias treatable in accordance with the methods described herein in addition to AD, PDD, HD, MS and ALS include adrenoleukodystrophy, antiphospholipid syndrome, Binswanger's disease, CADASIL, dementia associated with brain infection or inflammation (as may be caused by, for example and without limitation, infectious or agents such as bacteria, fungi, parasites, mycobacteria, atypical mycobacteria, prions and the like), dementia associated with inherited conditions (such as Alexander disease, ataxia syndrome, Canavan disease, cerebrotendinous xanthomatosis, DRPLA, fragile X-associated tremor, glutaric aciduria type 1, Krabbe's disease, Kuf s disease, Maple syrup urine disease, neuroacanthocytosis, Niemann Pick disease, organic acidemias, Pelizaeus-Merzbacher disease, Sanfilippo syndrome, spinocerebellar ataxia, and urea cycle disorders), dementia associated with other diseases (such as Behcet's disease, corticobasal degeneration, cerebral multiple sclerosis, transmissible spongiform encephalopathies, including but not limited to Creutzfeldt- Jakob disease, variant Creutzfeldt- Jakob disease, Gerstmann- Straussler-Scheinker Syndrome, Fatal Familial Insomnia, and Kuru, progressive supranuclear palsy, sarcoidosis, Sjogren's syndrome, and systemic lupus erythematosus), dementia due to alcoholism or other metabolic disturbance, dementia pugilistica, dementia with Lewy bodies, familial AD, frontotemporal lobar degeneration

(frontotemporal dementia), Gaucher's disease, homocystinuria, hypothyroidism, MELAS, metachromatic leukodystrophy, moyamoya, Niemann-Pick disease, normal pressure hydrocephalus, pantothenate kinase-associated neurodegeneration, SCA17, Tay-Sachs disease, Wilson's disease, and vascular dementia.

[0078] In addition, more general neuroinflammatory diseases treatable in accordance with the methods described herein include alpha-mannosidosis, ataxia telangiectasia, autism, beta-mannosidosis, brain infection of bacterial, fungal,

mycobacterial, including atypical mycobacterial, parasitic, or viral origin, chronic inflammatory demyelinating neuropathy, chronic inflammatory demyelinating

polyradiculoneuropathy (Guillain Barre syndrome), Cockayne syndrome, a congenital metabolic disorder, corticobasal degeneration, drug-induced demyelination, fucosidosis, Hunter's syndrome, Hurler's syndrome, infantile neuronal ceroid lipofuscinosis, interstitial cystitis, Maroteaux-Lany syndrome, migraine, multiple sclerosis, neuroborreliosis, polymyositis, Pompe's disease, primary lateral sclerosis, prion-induced neuropathy, including but not limited to mad cow disease, progressive supranuclear palsy, radiation induced demyelination, Sanfilippo syndrome, Scheie's syndrome, Schilder's disease, Schindler's disease, Sly syndrome, spinal cord injury, spinal muscular atrophy, spinocerebellar ataxia type 3, subacute combined degeneration of spinal cord secondary to pernicious anaemia, tabes dorsales, temporal arteritis, transmissible spongiform

encephalopathies (including but not limited to Creutzfeldt- Jakob disease, variant

Creutzfeldt- Jakob disease, Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, and Kuru), traumatic brain injury, vasculitis, and woman's disease.

[0079] Various embodiments of the combination formulations and therapies for the neuroinflammatory diseases described herein combine a highly selective COXl inhibitor with drug therapies that are the standard of care for the disease being treated. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is combined with a glucocorticoid(s), such as Solu-Medrol or Decadron, as examples, for treatment of flares during multiple sclerosis (MS). Glucocorticoids can be administered at their regular dose or at lower doses in combination with a highly selective COXl inhibitor. ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, are also combinable with other drugs commonly used for the treatment of MS such as interferon beta, glatiramer, mitoxantrone, nataluzimab, intravenous immunoglobulin, and fingolimod as well as new drugs as they become available. For example, ASP6537 and other

compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, can be used in such combination therapies (and combination unit dose forms, if desired) at a dose in the range of 5-200 mg, which can be administered either QD or BID. In one embodiment, glucocorticoids are administered at a standard or lower dose in combination with ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, dosed at 30-200 mg BID. In similar fashion, combination therapies of the invention include those in which a highly selective COXl inhibitor is coadministered with any other agent approved for the treatment of the neuroinflammatory disease suffered by the patient to be treated.

[0080] In the combination therapies of the invention, any other class of drug known to be useful for treatment of neuroinflammatory disease may be administered in combination with ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof. For example, such other classes of drugs suitable for use in combination with ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, include classes of drugs used for treatment of dementia (AD, PDD, vascular dementia, Lewy body dementia, HIV dementia, frontotemperol dementia, Huntington's dementia, prion disease dementia, Wernicke-Korsakoff syndrome dementia, leukodsytrophy dementia, traumatic brain injury dementia and other dementias), which include but are not limited to: cholinesterase inhibitors, NMDA glutamate inhibitors, 5-HT₆ receptor antagonists and agonists, drugs changing the formation and deposition of amyloid beta through multiple mechanisms, including drugs that interfere with cleavage of amyloid beta precursor, and antibodies that lower the amyloid beta level in plasma. In addition to these classes of drugs, additional new classes of drugs suitable for use in combination with ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof in the methods of the invention include classes of drugs that interfere with neuroinflammation, amyloid beta formation and deposition, synuclein formation and deposition, and tau protein formation. In the case of a disease such as multiple sclerosis, the COX1 inhibitors of this invention can be used in combination with corticosteroids, beta interferons, and other immune suppressing/altering drugs such as glatiramer, fingolimod, natalizumab, mitoxatrone and similar compounds. In the case of Parkinson's Disease, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be combined (coadministered) with levodopa with or without carbidopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine, and anticholinergic agents and other drugs to treat Parkinson' as well as new drugs for treatment of PD as they become available. In the case of Huntington's disease, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be combined (co-administered) with drugs which help to prevent involuntary movements such as tetrabenazine or certain

antipsychotic drugs such as haloperidol and clozapine or drugs to help with dystonia, chorea and muscle rigidity such as clonazepam and diazepam or other drugs used to treat Huntington's disease as well as new drugs as they become available for treatment of Huntington's disease. In the case of ALS, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be combined (co -administered) with drugs which are used in the treatment of ALS such as riluzole, baclofen and antidepressants or other drugs used to treat ALS as well as new drugs as they become available for treatment of ALS. Similarly, for other neuroinflammatory diseases,

ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be combined with drugs commonly used to treat such diseases. [0081] In one embodiment, a cholinesterase inhibitor is used in

combination with a ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, to treat AD, PDD, Lewy body dementia, vascular dementia, frontotemporal dementia, mild cognitive impairment or another dementia. For example and without limitation, donepezil is a cholinesterase inhibitor suitable for such use and can be administered at the recommended doses of 5 or 10 mg QD. Thus, in one embodiment, the highly selective COX1 inhibitor ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered in combination with donepezil, and donepezil is administered at one of the approved doses (on the donepezil label), and ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is given at a dose in the range of 5-200 mg QD or BID, including, for example at a dose in the range of 5-200 mg QD or 5-200 mg BID. In once daily dosing embodiments, donepezil can be administered, for example and without limitation, at either 5 or 10 mg, and ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, at 5-200 mg. In all of these embodiments, donepezil (or any other cholinesterase inhibitor) and ASP6537 (or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof) can be given as distinct tablets, capsules, dissolving tablets or capsules, sustained release formulations, or via any other formulation or means as distinct and separate formulations or the two drugs can be combined in an admixed or combination formulation provided by the invention.

[0082] For example, as provided herein, the two drugs can be formulated into a single tablet, capsule, dissolving tablets or capsules, sustained release formulations, or via any other formulation or means. In one embodiment, the COX1 inhibitor is ASP6537 and the other drug is donepezil. In a combination unit dose form convenient for once or twice daily dosing, donepezil can be present in an amount of either 5 or 10 mg and ASP6537 can be present in an amount in the range of 5-200 mg. In some

embodiments convenient for use in once daily dosing, donepezil can be present in an amount of either 5 or 10 mg and ASP6537 can be present in an amount in the range of 50- 200 mg.

[0083] In one embodiment, the two drugs are formulated as sustained release formulations. Donepezil, for example, is marketed in a 23 mg extended release formulation, which can be used in the methods of the invention. ASP6537 can be administered, for example and without limitation, at doses between 10-400 mg for an extended released formulation. Donepezil and ASP6537 can also be combined together in a unit dosage form formulated for extended release at the doses described above. In various embodiments, these unit dose forms and combination therapies are administered to treat AD, PDD, Lewy body dementia, vascular dementia, frontotemporal dementia and mild cognitive impairment or another dementia.

Treatment of Cardiovascular Disease

[0084] In one embodiment, the methods of the invention provide important new treatments for cardiovascular disease. In this embodiment of the methods of the invention, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits platelet aggregation. In one embodiment, a compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is administered to prevent or treat cardiac disease in a patient unable to take aspirin for that indication. In one embodiment, a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is administered to prevent or treat cardiac disease in a patient that is also taking a non-steroidal anti-inflammatory drug (NSAID). In one embodiment, the present invention provides a pharmaceutical formulation of a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, and an NSAID for the treatment of patients with cardiovascular disease and pain.

[0085] Acetylsalicylic acid (aspirin) inhibits both the COX- 1 -dependent production of thromboxane A2 (TXA₂) in platelets as measured with its inactivate metabolite thromboxane B2 (TXB₂) and COX2-dependent production of anti-aggregatory prostaglandin 12 (PGI₂) in vessel walls, resulting in the "aspirin dilemma." A disturbance of the balance between TXA₂ and PGI₂, in favor of TXA₂, has been implicated in the vasospasm, hyperaggregability, and thromboembolism associated with many

cardiovascular diseases. Agents that could normalize the TXA₂/PGI₂ balance are likely to be beneficial in the treatment of these disorders. As shown in the examples below, ASP6537 can overcome the aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect. ASP6537 also differs from aspirin in that, unlike aspirin, ASP6537 is a reversible COX1 inhibitor. Reversibility has the advantage that patients may stay on drug nearer surgery than the irreversible COX1 inhibitor aspirin, which is discontinued a week prior to surgery to lower bleeding risk. Other NSAIDs which are reversible COX inhibitors do not generally act to inhibit platelet aggregation due to short half-lives (although naproxen has shown anti-platelet activity in some studies) preventing their reaching high enough, or long lasting enough, levels of platelet aggregation inhibition. The use of aspirin and other nonselective NSAIDs is also frequently associated with GI complications, which had been attributed to inhibition of COX1 in gastric mucosa and the resultant reduction of gastric mucosa protective PGs. Recently, however, it was reported that single administration of not only selective COX2 inhibitors, but also selective COX1 inhibitors, induced no or less ulcerogenic effects than nonselective NSAIDs (Kakuta et al, 2008, J Med Chem. 51 : 2400-11; Tanaka et al, 2002, Aliment Pharmacol Ther. 16: 90-101; and Wallace, 1999, Am J Med. 107: 11S-6S; discussion 6S-7S), suggesting that aspirin and other nonselective NSAIDs induce GI effects through inhibition of both COX1 and COX2.

[0086] Platelet adhesion and aggregation play important roles in the pathogenesis of thrombosis, particularly arterial thrombosis, and the efficacy of numerous antiplatelet agents in preventing development or recurrence of thrombotic events has been evaluated. Acetylsalicylic acid (aspirin) is generally effective in the treatment of acute coronary syndrome (ACS), such as myocardial infarction (MI) and transient ischemic attacks, and is considered standard drug for treating those conditions. However, several drawbacks associated with the use of aspirin have been reported, such as insufficient efficacy, unclear dose-response effects, pharmacodynamic interactions with other NSAIDs, and GI complications. An equally or more effective compound without these adverse effects would therefore be of great benefit to patients with ACS. ASP6537 did not induce ulcer formation at 100 mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of >100 mg/kg. Therefore, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, offer an alternative to aspirin in patients suffering adverse effects or the aspirin dilemma.

[0087] A study comparing the in vitro and in vivo TXA₂/ PGI₂ inhibitory effects of ASP6537 and aspirin demonstrated that the normalization of TXA₂/PGI₂ balance by ASP6537 is superior to that of aspirin using guinea pigs and rats. In addition, the antithrombotic and ulcerogenic effects of ASP6537 and aspirin were compared in guinea pigs. In this study, ASP6537 (3-methoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4- triazole) was synthesized at Astellas Pharma, Inc. Aspirin and ibuprofen sodium salt were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Arachidonic acid (AA) was purchased from Biopool (Bray, Co. Wicklow, Ireland). Type I collagen from equine tendon (Collagen reagent Horm ) was obtained from Moriya (Tokyo, Japan). Adenosine 5 '-diphosphate (ADP) was obtained from MC Medical (Tokyo, Japan). Male Hartley guinea pigs (SLC Japan, Inc., Tokyo, Japan), male Wistar rats (Clea Japan Inc., Tokyo, Japan), and male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Inc.,

Yokohama, Japan) were used in this study. All animals were fasted overnight before experiments.

[0088] TXA₂ synthesis was evaluated in this guinea pig study using platelet-rich plasma. ASP6537 and aspirin were dissolved in DMSO and diluted with Tris-saline buffer. Platelet-rich plasma (PRP) was prepared from citrate-anticoagulated blood obtained from fasted guinea pigs under diethyl ether anesthesia. Platelet aggregation (PA) in the PRP was induced by the addition of 0.5 μg/mL collagen.

Following the completion of the aggregation response, indomethacin (10 μΜ final concentration) was added to the reaction mixture, which was then centrifuged at 10,000 x g for 5 min at 4 °C. The amount of the stable metabolite of TXA₂, TXB₂, in the supernatant was measured according to a standard procedure using a TXB₂ EIA kit (Cayman Chemical) following the manufacturer's guidelines.

[0089] PGI₂ synthesis was evaluated in this guinea pig study using isolated aortic strips. Under anesthesia with diethyl ether, thoracic aortas obtained from fasted guinea pigs were cut into slices (4.0-5.1 mg wet weight). The slices were immediately incubated at 37 °C for 30 min in Tris-saline buffer to avoid endogenous PGI₂ production. After replacement of the buffer, the slices were incubated with vehicle or test drug at 37 °C for 15 min. AA (20 μΜ) was then added to the mixture, which was further incubated at 37 °C for 15 min. After addition of 10 μΜ indomethacin, the reaction mixture was centrifuged at 10,000 x g for 5 min at 4 °C. The amount of 6-keto-PGFla in the supernatant was measured according to a standard procedure using a 6-keto-PGFla EIA kit (Cayman Chemical) following the manufacturer's guidelines.

[0090] The inhibitory effects of ASP6537 and aspirin on the synthesis of

TXA₂ and PGI₂ in vitro in tissue were as follows. ASP6537 inhibited platelet TXB₂ production with a geometric mean IC₅o value of 0.00358 μΜ, which was 5,000-fold higher than the value determined for aspirin (18.3 μΜ). ASP6537 and aspirin inhibited PA with a geometric mean IC₅₀ value of 0.00835 and 42.3 μΜ, respectively. In addition, the effects of these two drugs on TXB₂ production correlated well with those on PA. Although ASP6537 also inhibited vessel 6-keto-PGFla production, the IC₅₀ ratios of TXB₂ to 6-keto-PGFla production for ASP6537 and aspirin were 20.8 and 0.738, respectively. Thus, ASP6537 preferentially inhibited TXA₂ (as measured by measuring TXB₂) synthesis over that of 6-keto-PGFla, and this inhibition was markedly more selective than that of aspirin.

[0091] Another study demonstrated the selective inhibition of TXA₂ over

PGI2 using ASP6537 in comparison with aspirin. In this study, ASP6537 (1 , 10, and 100 mg/kg/day) and aspirin (30, 100, and 300 mg/kg/day) were suspended in a 0.5% methylcellulose (MC) solution just prior to use and then administered orally to seven- week-old male Wistar rats (Clea Japan, Inc.) for 7 days. On days 1, 3, and 7, urine was collected over a 24-h period and was then subjected to selective two-step solid-phase extraction. The amount of the stable metabolite of PGI₂, 2,3-dinor-6-keto PGFla, in the urine samples was measured using an EIA kit (Cayman Chemical).

[0092] The effects of ASP6537 and aspirin on urinary concentrations of

2,3-dinor-6-keto PGFla in rats were as follows. ASP6537 significantly reduced urinary 2,3-dinor-6-keto PGFla at a dose of 100 mg/kg on days 1, 3, and 7 of administration, but had no significant effect at the two lower doses examined. Aspirin significantly reduced urinary 2,3-dinor-6-keto PGFla at a dose of 100 mg/kg or higher on day 1 , and by days 3 and 7, had significantly reduced urinary PGI₂ metabolite concentrations at all examined doses.

[0093] Disruption of TXA₂/PGI₂ balance leads to an increased risk of thrombosis. Although COXl is known to regulate the production of TXA₂ by platelets, it remains unclear which isoform dominates the production of endothelial PGI₂. In the in vitro prostanoid synthesis study in guinea pigs, ASP6537 reduced PGI₂ production from isolated aorta, indicating that COXl is responsible for PGI₂ production, at least in part, under these study conditions. However, the inhibitory effect of ASP6537 on TXA₂/PGI₂ production was 28 fold more selective compared to that of aspirin. In addition, ASP6537 reduced urinary PGI₂ metabolite excretion in normal rats, indicating that COXl is also involved in PGI₂ production under normal conditions in vivo. ASP6537 and aspirin inhibit PA in normal rats at doses of 3 and 100 mg/kg, respectively. Here, a 33 -fold greater dose of ASP6537 was required for an inhibitory effect on urinary PGI₂ metabolite excretion to be observed compared with that necessary for the disruption of PA, whereas aspirin inhibited both urinary PGI₂ metabolite excretion and PA at an identical dose. The selective inhibition of ASP6537 on TXA₂ and PA over PGI₂ is attributable to the observed COX1/COX2 selectivity. Both COX1 and COX2 appear to be involved in vascular PGI₂ production under normal physiological conditions, and ASP6537 has a superior ability to aspirin for maintaining TXA₂/PGI₂ balance.

[0094] Another way to demonstrate the superiority of ASP6537 over aspirin is by examining plasma prostanoid concentrations in aged rats. Twenty-eight- month-old male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Inc.) were used as the aged group, and 11 -week-old male rats were used as the young group in a study to demonstrate this effect. ASP6537 and aspirin were orally administered 1 h before blood collection. Heparinized blood was immediately transferred into a plastic tube containing indomethacin (10 μΜ final concentration). Plasma was then prepared, and the amount of TXB₂ and 6-keto PGFla was measured according to standard procedures using EIA kits (Cayman Chemical).

[0095] The effects of ASP6537 and aspirin on plasma prostanoid concentrations in 28-month-old (aged group) and 11 -month-old (young group) male F344/DuCrlCrlj rats were as follows. In untreated rats, both TXB₂ and 6-keto PGFla increased significantly in the aged group compared with the young group. The effects of ASP6537 and aspirin on plasma TXB₂ concentrations in aged rats were as follows. Both ASP6537 and aspirin decreased the amount of TXB₂ in a dose-dependent manner, although a significant reduction was observed at a dose of 1 mg/kg for ASP6537, whereas a dose of 10 mg/kg was required for aspirin to achieve a significant reduction.

[0096] The effects of ASP6537 and aspirin on plasma 6-keto PGFla concentrations in aged rats were as follows. ASP6537 did not decrease the amount of 6- keto PGFla in plasma, even at a dose of 100 mg/kg, whereas aspirin decreased 6-keto PGFla levels in a dose-dependent manner, with statistical significance reached at doses of 100 mg/kg or higher.

[0097] In the aged rat experiments, the plasma concentrations of TXA₂ and PGI₂ metabolites significantly increased with aging. This phenomenon may be due to the increased expression of COX1 and its products in platelets and endothelial cells. Similar increases in the urinary metabolites of TXA₂ and PGI₂ by elderly people have been reported in the clinical setting. The study described here reveals that, although both ASP6537 and aspirin decrease plasma TXA₂ metabolites selectively, ASP6537 has a greater dissociation between the inhibitory effects on the production of PGI₂ and TXA₂ metabolites. Vascular COX2 is markedly increased with aging, while it is present at almost undetectable levels in young rats. Moreover, induced COX2 dominates over COX1 in catalyzing PGI₂ biosynthesis due to its preferential coupling with PGI₂ synthase. In this study, once again ASP6537 exerted greater selective inhibition of TXA₂ over PGI₂.

[0098] In another study, ASP6537 demonstrated superior anti-thrombotic properties to aspirin. One hour after the oral administration of ASP6537 or aspirin to fasted male Hartley guinea pigs, the animals were anesthetized with intraperitoneal injection of ketamine (40 mg/kg; Katalar^®, Sankyo Co., Tokyo, Japan) and xylazine (5 mg/kg; Seractal^®, Bayer Co., Leverkusen, Germany). The left carotid artery was detached and a Doppler flow probe (DBF-10R, 1.5 mm diameter; Primetech Co., Tokyo, Japan) was placed around the carotid artery. The carotid blood flow was monitored using a Doppler blood flow velocimeter (PDV-20; Crystal Biotech America, Hopkinson, MA, USA). The artery was electrically stimulated (2 mA) for 30 sec and blood flow was continuously monitored for 20 min. The time at which the blood flow velocity decreased to zero was recorded as the time to occlusion (TTO) of the vessel. If blood flow continued for longer than 20 min, 20 min was the value recorded for statistical analysis.

[0099] In the control-group animals in this study, carotid blood flow decreased gradually after electrical stimulation and reached a stable level of zero within 10 min. ASP6537 prolonged the TTO in a dose-dependent manner. Significant prolongation of TTO was observed at doses of 3 mg/kg or greater for ASP6537, whereas aspirin tended to prolong the TTO, but not significantly, even at a dose of 300 mg/kg.

[00100] ASP6537 also was shown to exert a potent antithrombotic effect in a guinea pig model of electrically induced carotid arterial thrombosis. This thrombosis model has been widely applied for the evaluation of antithrombotic agents in several animal species. In the guinea pig model, ASP6537 had a clear, dose-dependent antithrombotic effect, and significant prolongation of the TTO was observed at a dose of 3 mg/kg, which is equal to the PA-inhibiting dose of ASP6537 in guinea pigs. In contrast, aspirin did not show a significant antithrombotic effect, even at 300 mg/kg, although the PA-inhibiting dose of aspirin in guinea pigs is 100 mg/kg. A similar ineffectiveness of aspirin has been consistently reported by others using electrically induced thrombosis models. Although the reason for this discrepancy is unknown, the aspirin dilemma may be one of the reasons for the insufficient antithrombotic effect in such models. In the study described here, ASP6537 had a much higher selectivity for COX1 and a superior ability for maintaining TXA2/PGI2 balance than aspirin. Taken together, these findings demonstrate that ASP6537 can overcome the aspirin dilemma, is effective for the prevention of cardiovascular events.

[00101] Pharmacodynamic interactions (drug drug interactions) of aspirin, particularly with NSAIDs, are well documented. The platelet aggregation inhibition activity of aspirin can largely disappear when coadministered with common NSAIDs, such as ibuprofen. This problem is overcome when ASP6537 is used rather than aspirin in combination with NSAIDs, such as ibuprofen, in accordance with the methods described herein. Provided are methods, formulations, and unit dose forms of such combinations of this COX1 inhibitor ASP6537 (and others of its class) with an NSAID that provides the platelet aggregation characteristics described in Example 11 and shown in Figure 16.

[00102] ASP6537 may be taken in combination with an NSAID as described above at standard doses and intervals. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day. In one embodiment a dose range between 5-200 mg QD is an effective dose of ASP6537 and other compounds of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof. In another embodiment, a dose of 10-200 mg BID is an effective dose of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, for treatment. In another embodiment, the dose range of 5 to 150 mg BID is used for treatment of AD with ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof. In another embodiment, the dose range of 5 to 100 mg BID is used for treatment of AD with ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof.

[00103] Thus, provided are novel formulations of drug combinations, i.e., a drug combination of ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, and an NSAID, including but not limited to ibuprofen and naproxen, and unit dose forms comprising such formulations, including unit dose forms in which the NSAID is present at an approved dose and the ASP6537 or other compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is present at a dose described herein, and methods for treating pain with them, including treating pain in patients taking aspirin, which patients discontinue taking aspirin once treatment in accordance with the methods described herein is initiated. If the NSAID in the combination formulation is approved at a dose for three times per day (TID) administration, then the unit dose can contain a dose of ASP6537 or other compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, that is equal to a QD dose described herein, a BID dose described herein, or one-third of a QD dose described herein. Such formulations and unit dose forms may be used in any patient population requiring NSAID treatment (i.e., in pain) and wanting to take a drug other than aspirin for prevention of cardiovascular disease (i.e., the patient population is not limited to dementia patients or patients with neuroinflammatory disease).

Treatment of Pain

[00104] In one embodiment, the methods of the invention provide important new treatments for the treatment of chronic or acute pain. In this embodiment of the methods of the invention, a compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits thromboxane A2 synthesis. In one embodiment of these methods, a fast-acting pain relieving medication is administered to the patient prior to (or concurrently with) the administration of ASP6537 or a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, for treatment of acute pain. Agents for fast acting pain relief include but are not limited to morphine, fentanyl, hydromorphone, Percocet, Vicodin, Darvocet, Tylenol, Feverall, Ibuprofen, Naproxen and Celebrex as well as generic equivalents and new agents as they become available. Some of these are administered through IV routes and pumps while others are given orally.

[00105] Data from a first safety/tolerability/pharmacokinetic study in healthy volunteers demonstrate that ASP6537 is well tolerated at doses up to 1000 mg, and completely inhibits thromboxane B2 synthesis and arachidonic acid-induced platelet aggregation at single doses of 50 mg and above. Pharmacokinetic data from single dose studies indicate the time of maximum plasma concentration (Tmax) of approximately 1.5 hours, an alpha (distribution) half-life (ti_/2) of approximately 2.3 hours, terminal ti_/2 of approximately 12 to 16 hours and linear pharmacokinetics. With these properties, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be used for the management of acute and/or chronic pain and have significant advantages over both conventional NSAIDs and selective COX2 inhibitors. In a Phase 2 human study for acute pain, the analgesic efficacy of ASP6537 at 50, 100, 200, and 400 mg administered orally was evaluated and compared to placebo and ibuprofen dosed at 400 mg as the active comparator. The dose levels were selected based on pharmacokinetic and pharmacodynamic data from a safety, tolerability, and

pharmacokinetic study. In this study, ASP6537 effectively controlled pain but had a relatively slow rate of onset. Therefore, especially given that it penetrates the CNS and inhibits microglia activation, ASP6537 will work as a treatment for chronic pain, especially chronic pain from spinal cord injury or arthritis, and acute pain, such as postsurgical pain. In various embodiments, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is co-administered with another pain management drug that has immediate onset. For treatment of pain from spinal injury, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester or salt thereof, may be co-administered with methylprednisolone, dexamethasone or other anti-inflammatories; neuroprotectors such as tirilazad; and pain killers such as gabapentin, nortriptyline, amitriptyline, opiates and clonidine. In addition, other analgesics, narcotics and nonsteroidal anti-inflammatories, especially ones with fast rates of onset, may be used in combination with ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester or salt thereof.

[00106] Nonsteroidal anti-inflammatory drugs (NSAIDs) currently fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both

cyclooxygenases-1 and 2 (COXl, 2), which act as key enzymes catalyzing the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibits COX2. Inhibition of the inducible COX2 isoform is believed to be responsible for some therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COXl inhibition has as one of its primary effects the inhibition of platelet aggregation. COXl inhibition has also been suggested to be responsible for undesired side-effects of NSAID administration on the gastrointestinal (GI) system. The results reported herein, however, demonstrate that the highly selective COXl inhibitor ASP6537 has a positive therapeutic effect with no GI side effects in the therapeutic dose range.

[00107] A number of adverse events are associated with traditional

COX1/COX2 inhibitory NSAIDs, the most common of which occur in a dose-dependent fashion in the upper GI tract and include discomfort, ulcers, and bleeding. A US survey estimated that 10% to 20% of patients prescribed traditional NSAIDs may experience dyspepsia during treatment, and that 50,000 to 100,000 hospitalizations each year were related to NSAID use. [00108] In the early 1990s, a second isoform of COX was identified and named COX2, with the previously known COX denoted as COXl . COXl mRNA and protein are expressed constitutively in most tissues and cells, particularly in the normal gastric mucosa and platelets, and help produce PGs. In contrast, COX2 mRNA and proteins are predominantly expressed in inflamed tissues, rapidly producing

proinflammatory PGs after proinflammatory stimulation. Since the discovery of COX2, COXl -derived prostaglandin E2 (PGE2) has been found to be involved in constitutive defense mechanisms operating under physiological conditions, and inhibition of COXl is believed by some to be causative of adverse events associated with traditional NSAIDs, which inhibit both isozymes. Selective COX2 inhibitors, such as rofecoxib and celecoxib, known as coxibs, were developed to minimize side effects and show reduced risk of GI toxicity in both animal models and humans.

[00109] However, others believe that, while PG reduction in the stomach does not induce GI damage when COXl alone is inhibited, simultaneous inhibition of both COX isotypes can result in the formation of gastric ulcers. Although COXl is mainly expressed in the GI tract both in animals and humans and almost all PGE2 synthesis was reduced in the GI tissues of COXl deficient mice, these animals exhibited no GI injury. In addition, the more selective COXl inhibitor SC560 was found to not induce GI damage in rats when administered as a monotherapy. However, severe gastric lesions developed when rats received concurrent treatment with SC560 and a COX2 inhibitor (Gretzer et al., 2001, Br. J. Pharmacol. 2001; 132: 1565-73). However, as described in Example 8, ASP6537 does not cause GI injury in multiple animal models. This finding, discussed below, is based on the high selectivity for COX 1 activity exhibited by ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof,.

[00110] As shown in Example 8 and Table 1, ASP6537 [3-methoxy-l,5- bis(4-methoxyphenyl)-lH-l,2,4-triazole], exhibits a 650-fold selectivity to COXl over COX2 in human whole blood assay (hWBA), while SC-560 shows only slight selectivity in the same assay. In the recombination enzyme assay, ASP6537 exhibits a 1600-fold selectivity to COXl over COX2. ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, are in the same chemical class as biarylheterocycles if grouped in one of the three main classes of NSAIDs (Perrone, 2010, Curr Med Chem. 17:3769-805), similar to other COXl selective inhibitors such as SC- 560, but unlike other NSAIDs in this class, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, have a unique heterocycle and exert a potent analgesic effect without inducing gastrointestinal toxicity.

[00111] The ulcerogenic effect of ASP6537 was measured in fasting rats at two doses (100 mg/kg and 320 mg/kg) and evaluated after 5 hours by examination of the stomachs. No ulcers were observed at either dose. Despite the fact that the pathological relationship between COX isozymes and NSAID-induced GI injury remains unknown, the need for both isoforms to maintain a healthy stomach environment may be explained. Non-selective NSAIDs have been shown to exert GI toxicity through a systemic mechanism via reduction of blood flow around the stomach by inhibiting COXl and attracting neutrophils, which can cause mucosal injury by COX2 inhibition.

[00112] As described in Example 12, adjuvant inoculation in rats induces upregulation of COX2 and microsomal PGE synthase followed by high levels of PGE2 production. However, it has been demonstrated that PGI2 in the spinal cords of adjuvant arthritis rats, one of the main factors for pain of this model is derived from COXl .

Adjuvant arthritis rats show hyperalgesia on mechanical stimulation and are recognized as a model of RA, as the pain induced in this model resembles that experienced by human RA patients. In fact, the analgesic activities of NSAIDs in adjuvant arthritis have been found to be correlated with clinical doses for pain control, suggesting that selective COXl inhibitors may be useful in treating inflammatory pain in a clinical setting. The results from the chronic pain model demonstrate that ASP6537 is effective in treatment of chronic pain.

[00113] As described in Example 12, results from an acute pain model show that ASP6537 in vivo exhibited an ameliorating effect on acute pain as measured in acetic acid-induced writhing reaction test in mice and on chronic pain as measured in the modified Randall-Selitto method in adjuvant arthritis rats. ASP6537 showed dose- dependent analgesic activity in both models, and potencies were comparable to those of diclofenac. Although rofecoxib was potently effective in treating chronic inflammatory pain, the compound did not inhibit writhing, even at 100 mg/kg.

[00114] COX2 inhibitors used in clinical settings, such as rofecoxib

(Vioxx), have been reported to have weaker analgesic effects than conventional NSAIDs. For example, the clinical dose of rofecoxib used to treat chronic pain is only 12.5-25 mg, while that for treating postoperative pain is 50 mg. Given that acute pain in clinical settings, such as postoperative pain, may be induced more often by COX1 than COX2, ASP6537 would be useful in achieving pain relief in patients experiencing postoperative pain (especially when used in combination with pain relief medications with fast onset) and RA, given the equivalent efficacy of ASP6537 to that of conventional NSAIDs in relieving both acute and chronic pain.

Pharmaceutical Formulations and Unit Dose Forms

[00115] The present invention provides pharmaceutical formulations and unit dose forms of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof. Generally, ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, can be provided for use in accordance with the methods of the invention in pharmaceutical formulations suitable for oral administration once or twice daily and in unit dose forms that conveniently provide a daily dose in the range of from 3 to 400 mg. In various embodiments, the daily dose ranges from 5 to 400 mg. Suitable unit dose forms include those containing 1, 3, 5, 10, 20, 25, 50, 75,100, 150, 200, 250, and 400 mg of ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof. In various embodiments, these pharmaceutical formulations of the invention are provided as a sustained release formulation in a unit dose form that is a tablet or capsule.

[00116] In other embodiments, however, the present invention provides pharmaceutical formulations of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, suitable for administration by non-oral routes. For example, the present invention provides, patch, depot and pump, nasal spray, and sustained release formulations that deliver a therapeutically effective amount of a highly selective COX1 inhibitor via transdermal, intramuscular or subcutaneous injection or injection directly into the cerebrospinal fluid, inhalation, or oral routes of administration, respectively are provided. In some embodiments, the highly selective COX1 inhibitor is formulated as an oral disintegrating tablet or oral dispersable tablet.

[00117] Certain of these embodiments are particularly useful in dementia or patients advanced in disease (as examples, such as ALS or PD) who may be unable to manage their own drug administration effectively, such that significant cost of care benefit is provided by dosage forms that require less supervision at early stages of disease, and also may be particularly useful in later stage dementia patients who have difficulty swallowing pills and tablets. For lower doses, delivery directly to the CSF through nasal passages, transdermal patch delivery, and subcutaneous or IM

administration of a depot formulation are particularly useful. For higher doses, direct delivery to the CSF (for example, through direct injection via pump or depot) or oral administration of immediate or pulsatile release dose forms are useful. Direct delivery to the CSF can bypass systemic side effects and allow for lower overall dosing.

[00118] In some embodiments in which higher therapeutically effective doses are required than can be delivered transdermally or by depot or sustained release formulation, a therapeutically effective dose can be achieved by using an immediate release oral formulation in combination with a transdermal, depot or sustained release formulation. This embodiment may have particular benefit in that there appears to be a trough effect with highly selective COX1 inhibitors in treating dementia, such that sustained, continuous delivery of a low dose may aid in keeping cognition at its highest level even though additional doses or other formulations of the drug, i.e., immediate release or pulsatile release formulations, may provide additional benefit.

[00119] In one embodiment, the present invention provides a formulation of a compound of Formula 1, or an active metabolite, prodrug, ester or salt thereof, suitable for intravenous bolus, intraperitoneal, subcutaneous, or oral administration that comprises the active ingredient dissolved in an solution compatible with hydrophobic compounds such as polyethylene glycol 400 (PEG 400) or 0.5% methylcellulose. ASP6537 (also known as FK881) is practically insoluble in water or isotonic saline solutions. It is sparingly soluble in polyethylene glycol 400. To prepare the formulation, the pH is adjusted to approximately, neutral, between pH 6 and pH 8. The product is asceptically filtered and filled into vials. The product can either be injected directly for a bolus administration, or provided as an intravenous administration over time. Intravenous administration can be performed directly and the dosing controlled using a drip or a pump, or via a Y-connector to a bag with an intravenous catheter and the dosing controlled using a drip or a pump.

[00120] In another embodiment, the present invention provides a formulation of a compound of Formula 1 , or an active metabolite, prodrug, ester or salt thereof, suitable for oral administration and prepared in a unit dose form such as a tablet or capsule. One example is a 50 mg tablet that contains: ASP6537 (active ingredient), 50 mg; Hydroxypropyl methylcellulose 2910 (binder), 50 mg; Lactose(filler), 87.2 mg; Microcrystalline cellulose (filler), 24 mg; Croscarmellose sodium (disintegrant), 24 mg; Light anhydrous silicic acid (glidant), 2.4 mg; and Magnesium stearate (lubricant), 2.4 mg. A suitable coating for the 50 mg tablet includes Hydroxypropyl methylcellulose 2910, 4.5 mg; Macrogol 6000, 1.2 mg; Titanium oxide, 1.7 mg; yellow ferric oxide, 0.12 mg; Carnuba Wax , trace. Another example is a 100 mg tablet that contains: ASP6537 (active ingredient), 100 mg; Hydroxypropyl methylcellulose 2910 (binder), lOOmg;

Lactose(filler), 174.4 mg; Microcrystalline cellulose (filler), 48 mg; Croscarmellose sodium (disintegrant), 48 mg; Light anhydrous silicic acid (glidant), 4.8 mg; and

Magnesium stearate (lubricant), 4.8 mg. A suitable coating for the 100 mg tablet includes Hydroxypropyl methylcellulose 2910, 9.0 mg; Macrogol 6000, 2.4 mg; Titanium oxide, 3.4 mg; yellow ferric oxide, 0.24 mg; and Carnuba Wax , trace.

[00121] A process for producing the tablets provided by the invention can involve several sequential steps, such as: 1. Milling: drug substance is milled by a jet mill; 2. Blending- 1 : drug substance is blended with Hydroxypropyl Methylcellulose 2910 and/or lactose by a diffusion mixer; 3. Sizing- 1 : the blended powder is sized by a sizing machine; 4. Blending-2: the sized powder is blended by a diffusion mixer; 5. Dispersing: the blended powder is dispersed with purified water by a convection mixer; 6. Drying: dispersing solution is dried in a vacuum dryer; 7. Sizing-2: the dried mass is sized by a sizing machine; 8. Sizing-3: the powder is sized by a pin mill; 9. Melting: milled powder is melted by a direct heating static solids bed dryer; 10. Sizing-4: the melted powder is sized by a sizing machine; 11. Sizing-5: the powder is sized by the pin mill to obtain granules; 12. Blending-3: the powder is blended by a diffusion mixer; 13. Sizing-6: the granules and external ingredients are sized by a sizing machine; 14. Blending-4: the sized powder and external ingredients are blended by a diffusion mixer; 15. Blending-5:

blending granules are blended with Magnesium stearate by a diffusion mixer; and 16. Tableting: the final blended granules are tableted by a rotary-tableting machine. Another suitable process provided by the invention involves (a) combining steps 2 through 5 into a high shear granulating process; and/or (b) combining steps 6 though 9 as one

drying/melting step; and/or (c) using a Fitzmill to size the meted cake.

[00122] A suitable process for film coating the tablets in accordance with the invention can involve sequential steps, such as: 1. Film-coating: (step a) - hydroxypropyl methylcellulose 2910 is dissolved into purified water as film-coating solution 1; (step b) - hydroxypropyl methylcellulose 2910 and Macrogol 6000 are dissolved into purified water as preliminary film-coating solution; (step c) - titanium oxide and yellow ferric oxide are dispersed into purified water as film-coating suspension; (step d.) - both the preliminary film-coating solution and the suspension obtained from the steps b and c are mixed as film-coating solution 2; (step e) - the tablet cores are spray- coated with the film-coating solution 1 and then are spray-coated with the film-coating solution 2; and 2. Polishing: film-coated tablets are polished with Carnauba Wax in a film-coating machine. The tablets can then be conveniently packed in a plastic bottle with a desiccant.

[00123] Thus, there are a variety of formulations and unit dose forms useful in the methods of described herein. In general, therapeutically effective doses reduce brain levels of TXA₂ (as can be measured by measuring reduced levels of TXB₂) to normal or near normal levels and result in improved cognition in patients in need of treatment. In various embodiments, clinical dosing of ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, ranges from 3 mg/day to 400 mg/day. In one embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg BID (10 to 400 mg daily). In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg BID (10 to 300 mg daily). In another embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg BID (10 to 200 mg daily). In one embodiment, ASP6537 or another compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 200 mg QD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 150 mg QD. In another embodiment, ASP6537 or another compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is dosed in the range of 5 to 100 mg QD.

[00124] In general, the formulation will also comprise one or more pharmaceutically acceptable carriers.

General Synthesis of Compounds

[00125] The compounds of this invention can be prepared from readily available starting materials using, for example, the following general methods and procedures. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley, and Sons, 5th Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts (1999) Protecting Groups in Organic Synthesis, 3rd Edition, Wiley, New York, and references cited therein.

[00126] Furthermore, the methods of this invention may employ compounds that contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, and the like.

[00127] The substituted triazoles described herein can be prepared by the synthetic

1 2 3

protocols illustrated in Scheme 1, where R , R , R , Y and Z are as defined herein and each LG is independently a leaving group, such as halogen.

Scheme 1

[00128] In Scheme 1, compound 1-1 is reacted with potassium cyanate to provide compound 1-2. In one embodiment, the reaction is conducted in water at a low temperature (i.e., an ice bath) followed by stirring at room temperature for an extended time. The reaction is continued until substantially complete which typically occurs within about 1 to 16 hours. Upon reaction completion, compound 1-2 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.

[00129] Compound 1-4 can be provided by reacting compound 1-2 with compound 1-3, where LG is a leaving group, under standard substitution reaction conditions well known in the art. In one embodiment, the reaction is conducted in the presence of a tertiary amine (e.g., pyridine) in a suitable solvent (e.g. toluene), under elevated reaction temperatures. The reaction is continued until substantially complete which typically occurs within about 1 to 16 hours. Upon reaction completion, compound 1-4 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.

[00130] Compound 1-5 can be provided by reacting compound 1-4 with a base in a suitable solvent such as water, alcohols (e.g., methanol, ethanol, isopropyl alcohol, etc.), tetrahydrofuran, dioxane, chloroform, methylene chloride, dimethyl acetamide, N,N- dimethylformamide or any other organic solvent which does not adversely influence the reaction. The base can be an inorganic or an organic base such as an alkali metal hydroxide, an alkali metal hydrogencarbonate, alkali metal carbonate, alkali metal acetate, tri(lower)alkylamine, pyridine (e.g. pyridine, lutidine, picoline, dimethylaminopyridine, etc.), N-(lower)alkylmorpholine, N-,N-di(lower)alkylbenzylamine, N-,N- di(lower)alkylaniline or the like. When the base, the acid and/or the starting compound are in liquid, they can be used also as a solvent. The reaction is continued until substantially complete which typically occurs within about 1 to 72 hours. Upon reaction completion, compound 1-5 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.

[00131] Compound 1-5 can then be reacted with compound 1-6, where LG is a leaving group, under standard substitution reaction conditions well known in the art to form a compound of formula 1. Typically, the substitution reaction conditions comprise a base in a suitable solvent such as tetrahydrofuran, dioxane, chloroform, methylene chloride, dimethyl acetamide, N,N-dimethylformamide or any other organic solvent which does not adversely influence the reaction. The suitable base may include a tertiary amine (e.g. triethylamine, pyridine, Ν,Ν-dimethylaniline, etc.), an alkali metal hydroxide (e.g. sodium hydroxide, potassium hydroxide, etc.), an alkalimetal carbonate (e.g. sodium carbonate, potassium carbonate, etc.), alkali metal bicarbonate (e.g. sodium bicarbonate, etc.), a salt of an organic acid (e.g. sodium acetate, etc.) and the like. In case that the base is liquid, the base can be used as a solvent. The reaction is continued until substantially complete which typically occurs within about 1 to 16 hours. Upon reaction completion, the compound of formula 1 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.

[00132] In Scheme 1, compounds 1-1, 1-3 and 1-6 are commercially available or may be prepared by procedures, or obvious modifications thereof, described in USP 6,927,230, which is incorporated herein in its entirety.

EXAMPLES

Example 1. Higher Plasma and Brain TXB₂ Levels Correlate with Aging and AD, and ASP6537 Reduces TXB₂ Levels Where Aspirin is Ineffective in Reducing Brain TXB2 Levels

[00133] To study the differences in the levels of TXB₂ in the brain and plasma of old vs young rats, 28 month old male rats (aged rats) were compared with 11 week old male rats with respect to TXB₂ levels. The results, shown in Figure 1, correlated with what has been reported in the literature for TXB₂ levels in brains of people with AD. Those individuals with AD had both higher plasma and brain TXB₂ levels, especially in frontal and temporal cortex regions of the brain [Iwamoto et al. (1989) J Neurol 236: 80- 84]. The elevated level of TXB₂ in the plasma correlated with elevated levels in the brain.

[00134] ASP6537 (administered at doses of 1, 3 and 10 mg/kg, po) significantly decreased TXB₂ levels both in plasma and brain tissue from aged rats, as evidenced by Figure 2. Aspirin decreased TXB₂ levels in plasma but had no effect on TXB₂ levels in the brain. Data shown in Figure 2A is expressed in mean ± standard error from 10 animals as a relative percentage changes to vehicle, which was treated as 100%. For aspirin, data is expressed in mean ± standard error from 6-7 animals as a relative percentage change to vehicle, which was treated as 100%. ASP6573 was administered as a single dose where aspirin was dosed daily for 7 days. In Figure 2B, data shown is expressed ± standard error from 10 animals and is a total concentration of TXB₂ levels in plasma as expressed in pg/mL. Statistical analyses performed using the Dunnett's multiple comparison test.

Example 2. Role of Thromoboxane in Cognition

[00135] Thromboxane B2 is the stable metabolite of TXA₂. U-46619 is an agonist of TXA2. U-44619 works by binding the thromboxane receptor and mimicking thromboxane; increased U-44619 has the same effect as increased thromboxane. SQ- 29548 is a thromboxane receptor antagonist. S-29548 blocks the binding of thromboxane and/or agonist and may compete off bound thromboxane/agonist from the thromboxane receptor. The present experiment was done by intracerebroventricular (icv) delivery of agonist and/or antagonist directly to the ventricles of the brain, bypassing the blood brain barrier, allowing high and rapid drug delivery to the brain. In this study, test animals were placed in a Y-shaped maze for a set time, and the number of arms entered, as well as the sequence of entries, was recorded, and a score was calculated to determine alternation rate (degree of arm entries without repetitions). A high alternation rate is indicative of sustained cognition as the animals must remember which arm was entered last so as not to reenter it. In the presence of this agonist, spontaneous alternation behavior of mice is decreased, demonstrating the negative effects of increased thromboxane (see Figure 3A). However, as shown in Figure 3B, when the TXA₂ antagonist SQ-29548 is added

(effectively blocking the effect of thromboxane or agonist), the spontaneous alternation behavior of mice returns to normal. This finding is direct evidence of the role of thromboxane A2 in the increased cognitive impairment typical of Alzheimer's Disease and other dementias.

Example 3. ASP6537 Superior to Donepezil in Two of Three Cognition Models;

Equivalent or Better than Donepezil in Scopolamine-Induced Deficit Model

[00136] The effects of ASP6537 and donepezil were evaluated in three different models of cognition and learning in mice and aged rats. ASP6537 showed statistically significant responses in all three models while donepezil only showed responses in one model.

[00137] In the scopolamine-induced deficits model, the effects of ASP6537 and donepezil were examined using the mouse Y-maze test. Donepezil is an

acetylcholinesterase inhibitor, and scopolamine is a muscarinic acetylcholine receptor antagonist. Scopolamine (0.5 mg/kg, ip) significantly decreased spontaneous alternation rate, the marker of scopolamine-induced working memory deficit. ASP6537 (1 and 3 mg/kg po) and donepezil (0.25 and 0.5 mg/kg po) significantly attenuated the

scopolamine-induced deficits. Both ASP6537 and donepezil ameliorated scopolamine- induced cognitive deficits, indicating that both ASP6537and donepezil were effective in the cholinergic cognitive impairment model in mice. Scopolamine and ASP6537 were administered 20 and 30 min before the Y-maze test, respectively. The results of this study are presented in bar graph form in Figure 4.

[00138] The effects of ASP6537 and donepezil were compared in the MK-

801 -induced deficit model using the mouse Y-maze test. MK-801 is an antagonist of N- methyl-D-aspartate (NMD A), a glutamate receptor. MK-801 (0.15 mg/kg, ip) significantly decreases alternation rate, a marker of MK-801 -induced working memory deficit. MK-801 and ASP6537 were administered 20 and 30 min before the Y-maze test, respectively. MK-801 and donepezil were administered 20 and 50 min before the Y-maze test, respectively. The results are shown in Figure 5, and in this model, ASP6537 significantly attenuated MK-801 -induced memory deficits at a dose of 1 mg/kg po, while donepezil had no effect.

[00139] The therapeutic effect of ASP6537 on spatial memory deficits in aged rats, an animal model of dementia, was demonstrated in the Morris water maze. Nine-week-old male young Fischer-344 rats and aged rats (aged 25 months) were used. All animals were given food and water ad libitum. The apparatus and experimental procedures were similar to those described previously [Mandel et al. (1989); Behav Brain Res 31 :221-229]. In brief, all animals were trained in the Morris water maze using a two- trial per day regimen. The rat water maze apparatus consisted of a circular pool (150 cm in diameter) that was filled with water to a depth of 30 cm at a temperature of 20 (150 cm in diameter) that was filled with water to a depth clear plastic and supported by a base resting on the bottom of the pool, was placed 1.2-1.5 cm below the surface of the water. For descriptive data collection, the pool was subdivided into four equal quadrants formed by imaging lines that intersected in the center of the pool at right angles called north, south, east and west. The platform always resided in the center of the southwest quadrant. At the start of a trial, the rat was placed at one of the two cardinal starting points, which were located farthest from the platform (north or east) in a semi-random order. However, the same location was not used on 2 consecutive days. When the rats found the platform, they were allowed to remain on it for 15 seconds. If the rats did not find the platform within 90 seconds, they were removed from the water and then placed on the platform for 15 seconds. The second daily trial began approximately 4 minutes after conclusion of the first with the starting location of the second trial being a random choice of the two remaining locations (south or west). After the trial, the rat was rubbed thoroughly with a towel, placed in a drying cage until it was completely dry and then returned to its home cage. Animals were trained in the water maze for 4 consecutive days. Data were collected by an automated on-line video device designed to track the object with the highest contrast, in the field of vision, which was always the white rat on the black background. Escape latency (the time to find the platform), escape distance and velocity (distance / latency) were recorded for each trial with the video tracking system. The daily latency and daily velocity were obtained from the average latencies and velocities of two trials each day for finding the hidden platform. The cumulative latencies for 4 days training were also calculated from the sum of daily latencies in each treatment group.

Furthermore, the average velocity was calculated from the average of daily velocity for 4 days.

[00140] ASP6537 was administered orally 60 min before the first trial on each day of a four day trial. The cumulative latency in finding the platform was significantly longer in aged rats (25 months old) compared with young rats (9 weeks old), as shown in Figure 6A (P<0.01; Student's t-test). Treatment with ASP6537 significantly shortened the cumulative latency at the dose of 1 mg/kg as shown in Figure 6 A (P<0.01; Dunnett's multiple comparison test), while the average velocity of aged rats was not affected by ASP6537 (Figure 6B). In panel 6C, the daily latency on each of four days of the trial was graphed versus time for untreated aged rats, aged rats at three different doses of ASP6537 and untreated young rats. The aged rats showed a sharp contrast with the young rats in speed in finding the platform over time (escape latency) and the ASP6537- treated aged rats showed an intermediate learning behavior. In contrast to these results, the ability of donepezil hydrochloride, an acetylcholinesterase inhibitor, to enhance spatial memory was assessed in aged rats using the Morris water maze. As in the

ASP6537 study, control aged rats (24-25 months) showed longer escape latency when they were trained in the water maze task in comparison with young rats. Daily treatment with donepezil hydrochloride (0.1-3 mg/kg, po) failed to improve the latency in aged rats, essentially giving the same result as visualized with aged rats. These results suggest that donepezil hydrochloride exerts no ameliorating effect on the spatial memory deficits seen in aged rats. Taken together, these results demonstrate ASP6537 works to change memory deficits by exerting an effect on other neurons, for example the hippocampal pyramidal neurons, in addition to the cholinergic neurons. In addition, decreased thromboxane A2 and decreased neuro inflammation by treatment with ASP6537 results in decreased synaptic loss, reduces neurochemical changes, and improves information encoding in the neurons.

Example 4. ASP6537 Demonstrates Additive Cognitive Benefit in Combination with Donepezil

[00141] Previous data presented demonstrated that ASP6537 worked in cognition models in which donepezil had no effect. Surprisingly, ASP6537 can also provide additional benefit when administered in combination with donepezil. The study presented in Figure 7 shows additive cognitive benefit of such coadministration, consistent with ASP6537, working across neuron types. Figure 7 shows the beneficial effect of combining donepezil and ASP6537 in animal models of behavior using a transgenic mouse model for APP (Tg2576) in the Y-maze test to assess cognition.

[00142] Either donepezil alone, ASP6537 alone or donepezil plus ASP6537 were administered to the mice as indicated, and then the mice were tested for their ability to negotiate the Y-maze after administration of drug(s). There was a statistically significant improvement of cognition when the two drugs were administered in

combination as shown in Figure 7. This result indicates that ASP6537 has additive benefit in combination with donepezil and reduces neuroinflammation and levels of thromboxane A2.

Example 5. ASP6537 Reduces PGE2 and Inhibits Activation of Microglia and Release of Proinflammatory Mediators which in turn Reduce Neuroinflammation in the Brain

[00143] As shown in Figure 8, PGE2 was inhibited by ASP6537, in a dose proportional manner at the higher doses in aged rats with dose shown on y-axis being in units of mg/kg. This demonstrates that, in aging rats, PGE2 is reduced by inhibition of COX1 activity. PGE2 is a marker of inflammation in the aged rats, and this data is consistent with reduction of neuroinflammation by inhibition of COX1 activity by

ASP6537.

[00144] Figure 9 shows the effect of ASP6537 on activation of microglia in aged rats. The staining procedure used to generate the data uses polyclonal antibodies to Ibal (ionized calcium-binding adaptor molecule- 1) antigen peptide which detects microglia activation. Figure 9 shows the increase of activated microglia in different parts of the brain in aged rats. The experiment was performed by loading ASP6537 at a concentration of 10 mg/mL into the Alzet Osmotic pump model 2mL2 (0.03 mg/uL, 5 uL/hr; 2 week). The number 10 on the x-axis with respect to ASP6537 refers to the concentration of compound in the pump. Six 28 month-old male rats (termed aged rats) were then implanted with this osmotic pump subcutaneously under anesthesia. At day 15, again under anesthesia, samples were taken from the different parts of the brain, fixed, and frozen sections prepared for histopathology and stained with Ibal . The effective dose on a daily basis for these animals was 3.6 mg/day. For an average aged male rat of 500 g, this is a dose of 1.8 mg/kg/day. As this is continuous steady state delivery without absorption (avoiding bioavailability and Cmax and Ctrough effects), the comparable dose in animals dosed once daily would be in the approximate range of 2-6 mg/kg/day. Looking at bioavailability alone (without Cmax and Ctrough effects), the approximate extrapolated once daily oral dose is 3.6 mg/kg/day in animals to see about 75-80% reduction in microglia activation. In humans, this translates to a dose between 25 mg and 250 mg per day to achieve 75-80% inhibition of microglial activation in line with expected body accumulation of the drug observed at multiple high doses, especially when administered BID, based on prolonged half-life at extended high doses which was observed in humans, and observed Chough effects in single doses in humans. This is within the same dose range of 10-300 mg per day based on thromboxane A2 synthesis inhibition.

[00145] As demonstrated in Figure 9, a dose of 3.6 mg/day of ASP6537 given via pump as described above results in inhibition of microglia activation. The level of activation in old rats returns to that seen in younger animals. It is important to note that 100% inhibition of microglia activation does not occur at this dose. As some microglia activation may be required to phagocytize dead neurons and perform the role they perform in the younger animals, dosing which gives less than 100% inhibition is desirable. Thus, 60-90% inhibition of microglia activation is the desired range. The dose given in these animals achieved this goal. As elevated levels of microglia activation are correlated with increased neuroinflammation, this data is consistent with ASP6537 reducing neuroinflammation in multiple regions in the brain.

Example 6. Pharmacokinetic Studies of Plasma and Brain Concentrations of ASP6537

[00146] In a pharmacokinetic study conducted in aged rats, plasma, brain, and cerebrospinal fluid (CSF) concentrations of ASP6537 reached maximums of 93.2 ng/mL, 183 ng/mL, and 5.73 ng/mL at 0.5 h after single oral administration at a dose of 1 mg/kg, respectively. The study involved collection of blood samples one hour after drug administration followed by CSF collection. Immediately after collection of CSF, the brain was excised and all samples stored at -80 degrees C until analysis. The one hour post- administration collection point was selected as it correlated with the time point after administration at which independent cognition studies were conducted. The results are shown in Figure 10. Brain concentrations were approximately 1.5 to 2.2 times higher than those in plasma throughout the time points measured. These results indicate that ASP6537 diffuses through the blood brain barrier. Penetration of ASP6537 into the brain and plasma of monkeys intravenously administered ASP6537 was examined using an isotopic tracer. A common measure for PET (positron emission tomography) scan is standardized uptake value (SUV), shown on the y axis in Figure 11. Standardized uptake values (SUVs) are a measure of the concentration of a radiotracer in a defined region divided by the injected dose normalized for body weight at a fixed time after tracer injection. As shown in Figure 11, brain and plasma concentrations of ASP6537 were measured in 3 conscious male rhesus macaques using [11C]ASP6537 and positron emission tomography

(PET). [11C]ASP6537 was intravenously administered either alone or mixed with unlabeled ASP6537. A dynamic PET emission scan (95 min) was performed following the administration of [11C]ASP6537. Arterial blood samples were collected during the PET emission scans to measure ASP6537 concentration in plasma. These measurements were then corrected for metabolites using thin layer chromatography (TLC). In this manner, brain and plasma concentrations of ASP6537 were successfully measured in macaques. Results indicated good penetration into the brain at a tracer dose and 0.2 mg/kg of ASP6537. Based on the pharmokinetic and the isotopic tracer studies, ASP6537 is a brain penetrant COX1 inhibitor, especially well-suited for treatment of inflammatory neurological diseases in accordance with the present invention.

Example 7. Reduction of Thromboxane B2 (TXB₂) with Single Doses of ASP6537

[00147] In Figures 12 through 15, human clinical data are presented that demonstrate that even single doses of ASP6537 (referred to as FK881 in those figures) reduce plasma/serum thromboxane A2 levels in healthy individuals. This is important in providing a therapeutically effective dose of ASP6537 in patients with Alzheimer's disease and other dementias where TXA₂ in the brain is elevated (measured as TXB₂, the inactive metabolite of TXA₂). Data from animals demonstrate that the plasma levels correlate with brain levels. If one makes the following dosing assumptions based on the Phase 1 single dose data: 1) the brain concentration is 100% of plasma at all time points in the dosing interval; 2) the brain dose response curve is the same as plasma, and 3) no correction has been made for accumulation in the BID arms, then effect on cognition is dependent on the lowest level of COX-1 inhibition maintained throughout the entire dosing interval, that is, on Ctrough. In accordance with the present invention, a therapeutically effective dose can be administered as a dose that achieves at least TXB₂ inhibition to IC25, including but not limited to a dose that achieves reduction of TXB₂ levels to IC50 or to IC75 or higher (in the absence of other drugs which reduce the level of TXB₂ in plasma such as aspirin). In addition, drug exposure and half-life at steady state from multiple dosing studies in humans demonstrate that the dose ranges provided herein are efficacious for reduction of thromboxane B2 levels. For example, a dose of 200 mg QD administered daily for 13 days resulted in 95-99% reduction in plasma thromboxane A2 levels (measured as TXB₂) from baseline at a point 24 hours after the last dose (versus about 75%) reduction with a single dose). Example 8. High Selectivity of COXl over COX2 Activity without GI Side Effects for ASP6537

[00148] There is growing acceptance that, while PG reduction in the stomach does not induce GI damage when COXl alone is inhibited, simultaneous inhibition of both COX isotypes will result in the formation of gastric ulcers. Although COXl is mainly expressed in the GI tract both in animals and humans and almost all PGE2 synthesis was reduced in the GI tissues of COXl deficient mice, these animals exhibited no GI injury. In addition, the more selective COXl inhibitor SC560 was found to not induce GI damage in rats when administered alone. However, severe gastric lesions developed when rats received concurrent treatment with SC560 and a COX2 inhibitor (Gretzer et al, 2001, Br. J. Pharmacol. 2001; 132: 1565-73).

[00149] As shown in Table 1, ASP6537 [3-methoxy-l,5-bis(4- methoxyphenyl)-lH-l,2,4-triazole], exhibits a 650-fold selectivity to COXl over COX2 in human whole blood assay (hWBA), while SC-560 shows only slight selectivity in the same assay. In the recombination enzyme assay, ASP6537 exhibits a 1600-fold selectivity to COXl over COX2. ASP6537 belongs to a chemical class with biarylheterocycles when NSAIDs are grouped into three main classes (Perrone, 2010, Curr Med Chem. 17:3769- 805), like other COXl selective inhibitors such as SC-560, although ASP6537 exerts a potent analgesic effect without inducing gastrointestinal toxicity. ASP6537 and rofecoxib were synthesized at Astellas Pharmaceutical Company (Tokyo, Japan), and diclofenac sodium salt, indomethacin, and SC-560 were purchased from Sigma-Aldrich Japan (Tokyo, Japan). All drugs were dissolved in dimethylsulfoxide for in vitro assay. For murine in vivo assay, drugs were suspended or dissolved in 0.1% methylcellulose (MC) solution, and in 0.5% MC solution for rats.

[00150] Enzyme immunoassay kits for TXB₂ and PGE2 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Acetic acid was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Lambda-carrageenan and A23187 were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Dried brewers' yeast

(Saccharomyces genera) was purchased from Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Mycobacterium tuberculosis H37 RA strain was purchased from Difco Laboratories (Detroit, MI, USA).

[00151] Fresh human blood was collected from male volunteers. The subjects had not taken any drugs for at least seven days prior and had not consumed any alcohol for at least one day prior to blood collection. Male ddY mice aged five weeks and male Sprague-Dawley (SD) rats aged six weeks were obtained from Japan SLC Inc. (Shizuoka, Japan). Female Lewis rats (7-9 weeks old) were obtained from Charles River Inc. (Yokohama Japan). All animals were acclimated for at least three to seven days before experiments and were maintained in the institutional animal facilities approved by the American Association for Accreditation of Laboratory Animal Care.

[00152] Recombinant human (rh) COX1 and COX2 were expressed in

Chinese hamster ovary cells. The COX enzymes, derived from the supernatant of homogenized cells, were suspended in reaction buffer. After addition of arachidonic acid (10 μΜ), enzyme samples were incubated for 5 min (COX1) or 10 min (COX2) at room temperature. Reactions were terminated by adding indomethacin (final concentration: 1 mM), and PGE2 production was measured using an enzyme immunoassay kit. The IC50 value of ASP6537 for inhibiting rhCOXl activity was 0.064 μΜ, while that for inhibiting rhCOX2 activity exceeded 100 μΜ. These results showed that ASP6537 had COX1 selective inhibition activity (1562-fold or more).

[00153] ASP6537 inhibited COX1 activity more potently than diclofenac

(diclofenac IC50 value for rhCOXl = 0.024 μΜ, rhCOX2 = 0.032 μΜ) or SC560 (IC50 value for rhCOXl = 0.22 μΜ, rhCOX2 = >100 μΜ; selectivity was 454-fold or more). In contrast, the selective COX2 inhibitor rofecoxib exhibited highly selective COX2 inhibition activity in this assay (IC50 value for rhCOXl = >100 μΜ, COX2 = 0.31 μΜ). Table 1 COX Selectivity of NSAIDs.

SC-560: tool compound

[00154] For COX1 , fresh blood was incubated with test drugs for 1 h at 37

°C to achieve clotting. Reactions were terminated by adding indomethacin (final concentration: 2.5 mM) and blood was centrifuged to obtain serum. For COX2, fresh blood containing anticoagulants was incubated with test drugs and lipopolysaccharide

(LPS; 100 μg/mL) for 24 h at 37 °C, after which the samples were centrifuged to obtain plasma. Serum or plasma was mixed with methanol to precipitate protein and then centrifuged to obtain the supernatant. The supernatant was assayed for thromboxane (TXB₂) for COX1 and PGE2 for COX2 using an enzyme immunoassay kit. TXB₂ levels following human whole blood coagulation and PGE2 levels in LPS -challenged human whole blood were measured as biochemical indices for COX1 and COX2 activity respectively. The IC50 value of ASP6537 for inhibiting COX1 activity was 0.0049 μΜ, while that for inhibiting COX2 activity was 3.2 μΜ. These results showed that ASP6537 had COX1 selective inhibition activity in human whole blood (650-fold). The COX1- inhibiting potency of ASP6537 was far superior to that of diclofenac (IC50 value for COX1 = 0.20 μΜ, COX2 = 0.024 μΜ), and ASP6537 had far greater selectivity than SC560 (IC50 value for COX1 = 0.011 μΜ, COX2 = 0.15 μΜ; 14-fold selectivity) in hWBA. The IC50 values of rofecoxib were 38 and 0.25 μΜ for COX1 and COX2 respectively.

[00155] Data from the literature suggests that the recombinant enzyme assay overestimated the selectivity of COX1 versus COX2 with certain NSAIDs. This is shown in the Table 2. Once in the cell, SC-560 and Ketoprofen showed significantly less selectivity for COX1. ASP6537 is 650-1600 more selective for COX1 than for COX2 depending on the assay used. On a cellular basis, the next most selective agent, SC-560, was about 50-fold less selective for COX1 than ASP6537. SC-560 is a research tool and is not an approved drug for use in humans. Also, Ketoprofen and SC-560 have not been shown to penetrate into the brain.

[00156] ASP6537 and aspirin were dissolved in dimethylsulfoxide (DMSO) and diluted with reaction buffer (0.1 M Tris-HCl [pH 7.3] containing 2 μΜ hematin and 5 mM L-tryptophan). Recombinant human COX1 (rhCOXl) was expressed in Chinese hamster ovary cells, and purified as described previously. Recombinant human COX2 (rhCOX2) was purchased from Sigma-Aldrich Co. For the enzyme assays, rhCOXl and rhCOX2 were preincubated with test drugs for 10 min at room temperature. PGI₂ synthesis was initiated by the addition of 10 μΜ AA, and after 5 min (rhCOXl) or 10 min (rhCOX2), PGI₂ production was terminated by adding 1 mM indomethacin. The concentrations of the stable metabolite of PGI₂, 6-keto-PGFla, were then measured using a 6-keto-PGFla EIA kit (Cayman Chemicals Co., Ann Arbor, MI, USA) according to the manufacturer's instructions.

[00157] ASP6537 inhibited AA-induced 6-keto-PGFla production by rhCOXl with a geometric mean IC₅₀ of 0.000703 μιηοΙ/L, but had no detectable inhibitory effects on that by rhCOX2, even at concentrations up to 100 μιηοΙ/L. In contrast, aspirin inhibited AA-induced 6-keto-PGF la production by both rhCOXl and rhCOX2, with geometric mean IC₅₀ values of 37.9 and 61.9 μΜ, respectively. The IC₅₀ ratios of rhCOX2 to rhCOXl for ASP6537 and aspirin were >142,000 and 1.63, respectively, demonstrating that ASP6537 selectivity inhibited rhCOXl over rhCOX2 approximately 87,100 fold more potently than aspirin. These results demonstrate that ASP6537 is one of the most highly selective COX1 inhibitors identified to date.

[00158] Taken together, these results demonstrate that ASP6537 selectively inhibited COX1 over COX2 in recombinant human enzyme assays and hWBA. Although differences between the human enzyme assay and hWBA, such as in reaction time and enzyme volume, may cause differing IC50 values and selectivity to be obtained for tested drugs, these assays are widely used to compare conventional NSAIDs and COX2 inhibitors. In particular, the hWBA is generally accepted as the best method of predicting COX 1/2 selectivity under in vivo physiological conditions because hWBA reflects certain factors such as protein binding, cell-cell interaction, stability, and cell-permeability of drugs in human blood. For example, rofecoxib is shown to have COX2 selective inhibition activity both in hWBA and in clinical settings, as reflected in the vivo selectivity for rofecoxib within 30 mg/kg and 6 hour after p.o. administration (discussed below). The hWBA and in vivo findings (discussed below) for ASP6537 indicates that the compound will possess selectivity for COX1 in a clinical setting and is far more COX1 selective than SC560.

[00159] In rats, ASP6537 dose dependently inhibited carrageenan-induced paw edema (ED30: 22 mg/kg; diclofenac ED30: 3.6 mg/kg, rofecoxib ED30: 26 mg/kg). In vivo COX1/COX2 inhibition of test compounds (ASP6537 and rofecoxib) was evaluated by the modified Smith's methods in normal rats (Massimo, 1972, J Pharm Pharmacol. 1972; 24: 89-102). To investigate COX1 inhibition in vivo, male Sprague Dawley (SD) rats were sacrificed one hour after oral administration of the drugs. Then stomachs were washed and homogenized in methanol containing 10 μΜ indomethacin. Supematants were freeze-dried and dissolved in an assay buffer of PGE2 EIA kit, and the concentration of PGE2 was determined. To investigate COX2 inhibition, male SD rats were dosed aspirin orally (320 mg/kg) for irreversible inhibition of COXs expressed constitutively, and air was injected into their back subcutaneous ly to produce a cavity. Test compounds were administered orally 23 h after the aspirin dosing, and lambda- carrageenan saline suspension (2% [w/v]) was injected into their air cavity 24 h after the aspirin. Six hours later, the exudates in the air pouch were collected, and the

concentration of PGE2 determined. Thus, to investigate COX1/COX2 inhibition in vivo, PGE2 contents in the stomach (COX1 activity) and in inflammatory exudates (COX2 activity) were determined. In this assay, rofecoxib decreased the exudates PGE2 potently without notable effect on gastric PGE2 at 10 mg/kg, while ASP6537 decreased gastric PGE2 significantly at 10 mg/kg (83.6 % inhibition) with no suppression of exudates PGE2 up to 10 mg/kg. These results demonstrate that ASP6537 is a highly selective and orally active COX1 inhibitor.

[00160] The ulcerogenic effect of ASP6537 was measured as follows. After fasting Sprague Dawley (SD) rats for 24 h, oral administrations were performed for different doses (100 mg/kg and 320 mg/kg). On the other hand, SD rats in control group were dosed 0.5% MC solution. Five hours later, the stomachs were removed and immersed in 2% formalin to fix the gastric tissue wall and opened from the pyloric region along the great curvature. Presence or absence of visible mucosal ulceration was then noted and scored using the following criteria: 0, no alteration; 1, large ecchymosis or some small ulcers; 2, five or more small ulcers or one ulcer three millimeters or larger in diameter; 3, a large number of ulcers. No ulcers were observed at either dose of ASP6537. Diclofenac (5.6, 10, 18, 32 mg/kg) and rofecoxib (100, 320 mg/kg) were also tested. All doses of diclofenac resulted in ulceration. The higher dose of rofecoxib resulted in ulcerogenic activity in one of 10 rats tested (but not considered statistically significant).

[00161] This study showed that ASP6537 caused no ulceration in gastric mucosa, even when administered at an extremely high dose (320 mg/kg), while the findings for rofecoxib and diclofenac ulcer induction concurred markedly well with previous reports. Given these findings, ASP6537 will have favorable GI tolerability in clinical use. Despite the fact that the pathological relationship between COX isozymes and NSAID-induced GI injury remains unknown, several possible reasons explain the need for both isoforms to maintain a healthy stomach environment. For instance, each isoform helps to maintain mucosal integrity, and vicarious up-regulation of COX2 after inhibiting COX1 may induce PGs and subsequently prevent GI injury. In addition, nonselective NSAIDs have been shown to exert GI toxicity through a systemic mechanism via reduction of blood flow around the stomach by inhibiting COX1 and attracting neutrophils, which can cause mucosal injury by COX2 inhibition. These results demonstrate that ASP6537 has a substantially different GI side-effect profile than that of classical NSAIDs.

[00162] As was seen in rats, a similar lack of GI issues with ASP6537 was demonstrated with guinea pigs. Male Hartley guinea pigs were deprived of food and water for 36 and 2 h, respectively, prior to the oral administration of ASP6537 or aspirin.

Animals were sacrificed 3 h after administration under deep anesthesia using carbon dioxide gas. In each experiment, stomachs were removed, inflated by injecting 15 mL of 4% formalin, immersed in 4% formalin for 1 h to fix the gastric tissue, and then opened along the greater curvature. The total length (mm) of visible mucosal lesions in each was measured and was used to create an ulcer index. The mucosal lesions were evaluated subjectively in a blinded manner.

[00163] The geometric mean IC₅₀ value and 95% confidence intervals (C.I.) were calculated using logistic regression analysis. Analysis of statistical significance was performed using the Student's paired t-test. When multiple comparisons were made from the same data set, Dunnett's multiple comparison test was used. In the electrically induced carotid arterial thrombosis model experiment, the difference between the vehicle- and each drug-treated group was determined using Steel's test. P < 0.05 was considered to be significant.

[00164] The GI effects of ASP6537 and aspirin using an ulcerogenesis model in guinea pigs were as follows. Aspirin-induced gastric lesions and the ulcer index at a dose of 300 mg/kg were significantly higher than those of control animals (90.0 ± 15.5 vs. 0.5 ± 0.5 mm [control], P<0.01). In contrast, ASP6537 did not induce gastric lesions, even at doses as high as 100 mg/kg.

[00165] In the ulcerogenic experiments, ASP6537 did not cause any apparent ulceration of the gastric mucosa, even at a dose of 100 mg/kg, which represents a 33 -fold higher dose than the antithrombotic dose in guinea pigs. In contrast, aspirin had a potent ulcerogenic effect at the antithrombotic dose. As the inhibition of both COX1 and COX2 may be required for typical NSAID-induced GI toxicity to occur, the observed differences in gastric ulcerogenic properties between ASP6537 and aspirin may be due to the difference in COX1 selectivity. Example 9. Benefit of Use of ASP6537 Used Alone or in Combination Based on Low Levels of Interactions with Other Receptors, Ion Channels and Transporters

[00166] When ASP6537 is used alone or in combination with other drugs

(see table below), its low level of interactions with other receptors, ion channels and transporters demonstrated that it will combine well with other therapeutics.

Table 2. Inhibition of ASP6537 on binding to various receptors, ion channels, and transporters

GABA_A (Agonist site) 0.00 6.14 4.25 67.62 (Muscimol: 1x10^"' mol/L)

GABA_A (Benzodiazepine

central) 0.66 0.00 0.00 73.69 (Diazepam: 3xl0^"7 mol/L)

GABA_B 0.00 0.00 0.00 50.42 (Baclofen: 3x10^"' mol/L)

Galanin 4.94 0.00 24.53 74.08 (Galanin: 1x10 ^s mol/L)

Glutamate (AMPA) 0.00 0.00 0.00 72.32 (AMPA: 3x10 ^s mol/L)

Glutamate (Kainate) 7.77 16.69 22.38 51.68 (Kainic acid: 1x10^"' mol/L)

Glutamate (NMDA agonist site) 0.00 0.00 0.75 65.65 (Glutamic acid: 3x10^"' mol/L)

Glutamate (NMDA Glycine site) 3.38 10.82 4.71 71.06 (MDL105519: 3x10 ^s mol/L)

Glycine (Strychnine sensitive) 5.16 8.62 22.12 62.29 (Strychnine: 3x10^"' mol/L)

Histamine_! (Central) 3.78 5.49 3.97 66.15 (Pyrilamine: lxlO^"s mol/L)

Histamine₂ 0.00 2.87 3.91 64.64 (Cimetidine: lxlO^"fa mol/L)

(a-Methyl-histamine: 3x10 ^s

Histamine₃ 3.59 2.06 2.60 79.68 mol/L)

LeukotrieneB₄ 0.00 0.00 3.73 89.25 (Leukotriene B4: 1x10 ^s mol/L)

LeukotrieneD₄ 0.00 0.53 2.15 86.56 (Leukotriene D4: 1x10 ^s mol/L)

Muscarinic (Non-selective) 0.54 1.32 0.22 62.71 (Atropine: 3xl0^"a mol/L)

Muscarinic_! 2.12 1.01 2.42 81.73 (Atropine: 1x10 ^s mol/L)

Muscarinic₂ 2.70 0.00 0.00 65.54 (Atropine: 1x10 ^s mol/L)

Neurokinin_! 0.00 1.15 8.99 73.38 (Substance P: 1x10 ^s mol/L)

Neurokinin 0.00 0.00 0.00 53.09 (Neurokinin A: 3x10 ^s mol/L)

Neurokinin₃ 2.22 0.60 1.84 81.65 (Neurokinin B: 1x10 ^s mol/L)

NE transporter 4.34 5.25 8.80 51.50 (Desipramine: lxlO^"a mol/L)

Nicotinic 1.82 9.00 2.12 78.09 (Nicotine: 3x10 ^s mol/L)

Opiate (Non-selective) 2.01 2.31 18.35 65.95 (Naloxone: 1x10 ^s mol/L)

Oxytocin 5.42 5.83 6.98 67.00 (Oxytocin: 3xl0^"a mol/L)

PAF 0.46 31.46 36.05 70.94 (PAF: 3x10 ^s mol/L)

K Channel (KA) 0.00 6.03 12.81 62.17 (Dendrotoxin: lxlO^"lu mol/L)

K Channel (KATP) 0.92 1.77 0.00 73.73 (Glibenclamide: 1x10 ^s mol/L)

K Channel (KV) 0.00 1.57 5.42 83.08 (Charybdotoxin: 3x10 ¹⁰ mol/L) K Channel (SKCa) 0.00 0.41 2.71 72.88 (Apamim: lxl0^"a mol/L)

Serotonin (Non-selective) 0.97 2.53 7.58 58.15 (Serotonin: 3x10 ^s mol/L)

Serotonin transporter 1.55 10.46 10.44 77.07 (Imipramime: 1x10 ^s mol/L)

Sigma (Non-selective) 10.17 8.45 10.75 61.19 (Haloperidol: 3xl0^"a mol/L)

Sodium Channel₂ 5.20 7.42 21.29 75.47 (Veratridine: 3xl0^"fa mol/L)

Testosterone 2.00 4.45 11.87 64.70 (Testosterone: 1x10 ^s mol/L)

TXA₂ 0.00 0.00 4.99 64.96 (U44069: 3x10 ^s mol/L)

([Arg8]-Vasopressin: 1x10 ^s

Vasopressin Vj 1.17 27.55 56.69 76.94 mol/L)

(Vasoactive intestinal

VIP 0.00 1.15 3.77 71.40 polypeptide: lxlO^"8 mol/L)

Guinea pig heart

Ca Channel (L,

Benzothiazepine) 1.17 0.00 50.70 72.88 (Diltiazem: 3xl0^"7 mol/L)

Ca Channel (N) 0.00 0.00 4.93 63.05 (ω-conotoxin: lxl0^"lu mol/L)

Sodium Channel₂ 21.78 54.19 83.04 76.91 (Veratridine: lxlO^"6 mol/L)

[00167] The binding affinity of ASP6537 was evaluated for 58 receptors, ion channels, and transporters in human, rats, guinea pigs, or rabbits. Binding inhibition by 50% was shown for sodium channel at 1 microg/mL of ASP6537 and for Ca channel (N), vasopressin VI, and Ca channel (L, Benzothiazepine) at 10 microg/mL of ASP6537.

Example 10. ASP6537 Superior to Aspirin in Cardiovascular-Protective Effects

[00168] As described in Example 8, the results in Table 1 show that the

IC50 ratios of rhCOX2 to rhCOXl for ASP6537 and aspirin were >142,000 and 1.63, respectively, and that ASP6537 inhibited TXA₂ production more selectively than did aspirin in in vitro and in vivo TXA₂/PGI₂ production studies.

[00169] This next study compares the in vitro and in vivo TXA₂/ PGI₂

inhibitory effects of ASP6537 and aspirin and demonstrates that the normalization of

TXA₂/PGI₂ balance by ASP6537 is superior to that of aspirin using guinea pigs and rats.

In addition, the antithrombotic and ulcerogenic effects of ASP6537 and aspirin were compared in guinea pigs. [00170] ASP6537 (3-methoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4-triazole) was synthesized at Astellas Pharma, Inc. Aspirin and ibuprofen sodium salt were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Arachidonic acid (AA) was purchased from Biopool (Bray, Co. Wicklow, Ireland). Type I collagen from equine tendon (Collagen reagent Horm^®) was obtained from Moriya (Tokyo, Japan). Adenosine 5 '-diphosphate (ADP) was obtained from MC Medical (Tokyo, Japan). Male Hartley guinea pigs (SLC Japan, Inc., Tokyo, Japan), male Wistar rats (Clea Japan Inc., Tokyo, Japan), and male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Inc.,

[00171] TXA₂ synthesis in guinea pig platelet-rich plasma. ASP6537 and aspirin were dissolved in DMSO and diluted with Tris-saline buffer. Platelet-rich plasma (PPvP) was prepared from citrate-anticoagulated blood obtained from fasted guinea pigs under diethyl ether anesthesia. Platelet aggregation (PA) in the PRP was induced by the addition of 0.5 μg/mL collagen. Following the completion of the aggregation response, indomethacin (10 μΜ final concentration) was added to the reaction mixture, which was then centrifuged at 10,000 x g for 5 min at 4 °C. The amount of the stable metabolite of TXA₂, TXB₂, in the supernatant was measured according to a standard procedure using a TXB₂ EIA kit (Cayman Chemical) following the manufacturer's guidelines.

[00172] PGI₂ synthesis in isolated aortic strips of guinea pigs. Under anesthesia with diethyl ether, thoracic aortas obtained from fasted guinea pigs were cut into slices (4.0-5.1 mg wet weight). The slices were immediately incubated at 37 °C for 30 min in Tris-saline buffer to avoid endogenous PGI₂ production. After replacement of the buffer, the slices were incubated with vehicle or test drug at 37 °C for 15 min. AA (20 μΜ) was then added to the mixture, which was further incubated at 37 °C for 15 min. After addition of 10 μΜ indomethacin, the reaction mixture was centrifuged at 10,000 x g for 5 min at 4 °C. The amount of 6-keto-PGFla in the supernatant was measured according to a standard procedure using a 6-keto-PGFla EIA kit (Cayman Chemical) following the manufacturer's guidelines.

[00173] The inhibitory effects of ASP6537 and aspirin on the synthesis of

TXA₂ and PGI₂ in vitro in tissue were as follows. ASP6537 inhibited platelet TXB₂ production with a geometric mean IC₅₀ value of 0.00358 μΜ, which was 5,000-fold higher than the value determined for aspirin (18.3 μΜ). ASP6537 and aspirin inhibited PA with a geometric mean IC₅₀ value of 0.00835 and 42.3 μΜ, respectively. In addition, the effects of these two drugs on TXB₂ production correlated well with those on PA. Although ASP6537 also inhibited vessel 6-keto-PGF la production, the IC₅₀ ratios of TXB₂ to 6-keto-PGFla production for ASP6537 and aspirin were 20.8 and 0.738, respectively. Thus, it is clear that ASP6537 preferentially inhibited TXB₂ synthesis over that of 6-keto-PGF la, and this inhibition was markedly more selective than that of aspirin.

[00174] Another study demonstrated the selective inhibition of TXA₂ over

PGI₂ using ASP6537 in comparison with aspirin as next described. ASP6537 (1, 10, and 100 mg/kg/day) and aspirin (30, 100, and 300 mg/kg/day) were suspended in a 0.5% methylcellulose (MC) solution just prior to use and then administered orally to seven- week-old male Wistar rats (Clea Japan, Inc.) for 7 days. On days 1, 3, and 7, urine was collected over a 24-h period and was then subjected to selective two-step solid-phase extraction. The amount of the stable metabolite of PGI₂, 2,3-dinor-6-keto PGFla, in the urine samples was measured using an EIA kit (Cayman Chemical).

[00175] The effects of ASP6537 and aspirin on urinary concentrations of

[00176] Another way to look at the superiority of ASP6537 over aspirin was by examining plasma prostanoid concentrations in aged rats. Twenty-eight-month-old male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Inc.) were used as the aged group, and 11 -week-old male rats were used as the young group in this experiment.

ASP6537 and aspirin were orally administered 1 h before blood collection. Heparinized blood was immediately transferred into a plastic tube containing indomethacin (10 μΜ final concentration). Plasma was then prepared, and the amount of TXB₂ and 6-keto PGFla was measured according to standard procedures using EIA kits (Cayman

Chemical).

[00177] The effects of ASP6537 and aspirin on plasma prostanoid concentrations in 28-month-old (aged group) and 11 -month-old (young group) male F344/DuCrlCrlj rats were as follows. In untreated rats, both TXB₂ and 6-keto PGFla increased significantly in the aged group compared with the young group. The effects of ASP6537 and aspirin on plasma TXB₂ concentrations in aged rats were as follows. Both ASP6537 and aspirin decreased the amount of TXB₂ in a dose-dependent manner, although a significant reduction was observed at a dose of 1 mg/kg for ASP6537, whereas 10 mg/kg was required for aspirin.

[00178] The effects of ASP6537 and aspirin on plasma 6-keto PGFla concentrations in aged rats were as follows. ASP6537 did not decrease the amount of 6- keto PGFla in plasma, even at a dose of 100 mg/kg, whereas aspirin decreased 6-keto PGFla levels in a dose-dependent manner, with statistical significance reached at doses of 100 mg/kg or higher.

[00179] In the next study, ASP6537 demonstrated superior anti-thrombotic properties to aspirin. One hour after the oral administration of ASP6537 or aspirin to fasted male Hartley guinea pigs, the animals were anesthetized with intraperitoneal injection of ketamine (40 mg/kg; Katalar^®, Sankyo Co., Tokyo, Japan) and xylazine (5 mg/kg; Seractal^®, Bayer Co., Leverkusen, Germany). The left carotid artery was detached and a Doppler flow probe (DBF-10R, 1.5 mm diameter; Primetech Co., Tokyo, Japan) was placed around the carotid artery. The carotid blood flow was monitored using a Doppler blood flow velocimeter (PDV-20; Crystal Biotech America, Hopkinson, MA, USA). The artery was electrically stimulated (2 mA) for 30 sec and blood flow was continuously monitored for 20 min. The time at which the blood flow velocity decreased to zero was recorded as the time to occlusion (TTO) of the vessel. If blood flow continued for longer than 20 min, 20 min was the value recorded for statistical analysis.

[00180] In the control-group animals, carotid blood flow decreased gradually after electrical stimulation and reached a stable level of zero within 10 min. ASP6537 prolonged the TTO in a dose-dependent manner. Significant prolongation of TTO was observed at doses of 3 mg/kg or greater for ASP6537, whereas aspirin tended to prolong the TTO, but not significantly, even at a dose of 300 mg/kg. This example demonstrates that ASP6537 functions as a highly selective COX1 inhibitor with a superior ability to aspirin for normalizing TXA₂/PGI₂ balance and can be used to avoid the aspirin dilemma. Example 11. Benefit of ASP6537 over Aspirin for Concomitant Dosing with Ibuprofen

[00181] Pharmacodynamic interactions (drug drug interactions) of aspirin particularly with NSAIDs are well documented. The platelet aggregation inhibition activity of aspirin can largely disappear when combined with a common NSAID, such as ibuprofen. This problem is overcome when ASP6537 is used rather than aspirin in combination with NSAIDs, such as ibuprofen, in accordance with the methods described herein. Provided are methods, formulations, and unit dose forms of such combinations of this COX1 inhibitor ASP6537 (and others of its class) with an NSAID that provides the platelet aggregation characteristics shown in Figure 16, below. The results of a study of the effect on platelet aggregation inhibition using aspirin or ASP6537 in combination with ibuprofen are presented in Figure 16. Ibuprofen (30 mg/kg) or vehicle was orally administered 1 h before administration of vehicle, ASP6537 (30 mg/kg) or aspirin (100 mg/kg). Then, citrated blood was collected 8 h after the last test drug administration, and the percent of platelet aggregation measured. Data represent mean ± SEM of 5 animals. This study demonstrates that ASP6537 combined with ibuprofen shows excellent platelet aggregation inhibition activity in sharp contrast to aspirin combined with ibuprofen where much of aspirin's platelet aggregation inhibition activity disappears.

Example 12. ASP6537 Use for Acute and Chronic Pain

[00182] The acetic acid-induced writhing reaction model in mice is a model of chemical-induced acute pain useful in evaluating analgesic activity. Test drugs were administered to the ddY mice orally 1 h before acetic acid injection. Writhing was induced by an intraperitoneal injection of 0.6% acetic acid (20 mL/kg). Three minutes after injection, the number of writhing reactions was counted for subsequent period of 10 min. The ED50 value of ASP6537 for inhibiting the writhing reaction was 19 mg/kg, comparable to the effects of diclofenac (ED50: 14 mg/kg) and SC560 (67% inhibition at 32 mg/kg). In contrast, rofecoxib did not inhibit this reaction, even at a dosage of 100 mg/kg; however, COX2 inhibitors are widely understood not to inhibit this reaction. Thus, ASP6537 shows activity in a model for acute pain equivalent to diclofenac.

[00183] In addition to acute pain, studies were done in a model of chronic pain. The rat model of adjuvant arthritis has long been used as an animal model for rheumatoid arthritis (RA). Hyperalgesia in adjuvant arthritis in these animals, a source of chronic inflammatory pain, is believed to reflect that in human RA patients. Arthritis was induced in Lewis rats by injecting a suspension of 0.5 mg of M. tuberculosis in liquid paraffin into the right hind foot pads. Eighteen days after injection, analgesic activity in the left hind paws was measured 2 h after administration of test drugs (via the method used by Sakuma, 2001, Inflammation Res. 50: 509-14, modified from that used by Randall- Selitto, 1999, Med Lett Drugs Ther. 41 : 11-2). The analgesic coefficients were calculated as the pain threshold ratio against control groups, and the ED50 value was defined as the dose that raised the analgesic coefficient to 1.5. The ED50 value of ASP6537 for inhibiting hyperalgesia in an adjuvant arthritis rat model was 1.8 mg/kg, while values for diclofenac and rofecoxib were 1.0 mg/kg and 0.8 mg/kg, respectively. SC560 did not show 50% inhibition at 3.2 mg/kg (pain threshold ratio = 1.45 at 3.2 mg/kg). The analgesic effect of ASP6537 was comparable to that of diclofenac and rofecoxib in a rat model of chronic inflammatory pain.

Example 13. Compound Synthesis

[00184] The compounds described herein, and active metabolite, prodrug, ester, or salt thereof, can be prepared by methods known in the art, including those described in U.S. Patent No. 6,927,230, which is incorporated herein by reference. A representative synthesis is provided below.

[00185] (1) To a solution of dimethylcyanamide (10.0 g, 142.7 mmol) in methanol (50 mL) was added dropwise sulfuric acid (14.0 g, 142.7 mmol) over 2 hours at 20-30° C. The mixture was stirred at 20-30° C for 4 hours and then concentrated in vacuo. To the residue was added acetone (50 mL) and stirred at 20-30° C. After crystallized, the mixture was stirred at 20-30° C for 30 minutes, then at 0-10° C for 1 hour and filtered. The crystals were washed with acetone (20 mL) and dried in vacuo to give Ν,Ν,Ο- trimethylisourea sulfate (22.86 g, 80.0% yield) as white granulated solids. 1H NMR (DMSO d₆, ppm) .delta. 2.98 (3H, br), 3.01 (3H, br), 4.01 (3H, s), 8.66 (2H, br).

[00186] (2) To a cooled (0-15° C) solution of Ν,Ν,Ο-trimethylisourea sulfate (20.0 g, 99.9 mmol) in a mixture of methanol (100 mL) and water (1.8 mL) was added dropwise 28% sodium methoxide in methanol (38.55 g, 199.8 mmol) over 2 hours at 20- 30° C and stirred at the ambient temperature for 1 hour. The resulting precipitate was filtered off and washed with methanol (40 mL). The filtrate was concentrated in vacuo and ethyl acetate (180 mL) and triethylamine (10.11 g, 99.9 mmol) were added to the residue. To the mixture was added dropwise a solution of 4-methoxybenzoylchloride (16.15 g, 94.9 mmol) in ethyl acetate (20 mL) over 2 hour, at 20-30° C and then stirred at the same temperature for 2 hours. To the reaction mixture was added water (40 mL) and the aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were concentrated in vacuo and 4-methoxyphenylhydradine hydrochloride (17.44 g, 99.9 mmol), methanol (120 mL) and acetic acid (10 mL) were added to the residue. To the mixture was added dropwise triethylamine (10.11 g, 99.9 mmol) and stirred at 20-30° C for 3 hours then at 40-50° C for additional 3 hours. The reaction mixture was cooled to 20-30° C and stirred for 30 minutes. To the mixture was added dropwise water (120 mL) and stirred for 1 hour. The crystals were filtered, washed with 50% aqueous methanol (40 mL) and dried in vacuo to give crude 3-methoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4- triazole (20.36 g, 65.5% yield) as pale brownish yellow needles.

[00187] (3) To the stirred purified water (100 mL) was added dropwise a solution of 3-methoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4-triazole (20.0 g, 64.2 mmol) obtained above in acetone (300 mL) and stirred at 20-30. degree. C. for 30 minutes. The mixture was concentrated to .about.200 mL in vacuo, stirred at 35-45° C for 1 hour then at 20-30° C for 1 hour and filtered. The crystals were washed with 50% aqueous acetone (40 mL) and dried in vacuo to give pure 3-methoxy-l,5-bis(4-methoxyphenyl)-lH-l,2,4-triazole (18.36 g, 91.8%) yield) as colorless needles. Representative X-ray powder diffraction Peaks (2Θ): 9.1° 15.4°, 19.7° mp 125° C.

[00188] The invention, having been described in detail and illustrated by example, is claimed below.

Claims

We Claim:

1. A pharmaceutical formulation for use in treating and/or preventing a

neuroinflammatory disease, a cardiovascular disease, or pain in a patient in need thereof, said method comprising administering to said patient a therapeutically effective dose of a highly selective COX1 inhibitor of Formula 1, or an active metabolite, prodrug, ester, or salt thereof,

Formula 1

wherein

Y and Z are independently CH or N,

R¹ is lower alkyl which is optionally substituted with halogen,

R is hydrogen, lower alkyl or lower alkoxy, and

R is hydrogen, lower alkyl or lower alkoxy.

2 The pharmaceutical formulation of claim 1, wherein said therapeutically effective dose reduces thromboxane A2 in blood plasma of said patient by at least forty percent (40%) relative to blood plasma thromboxane A2 in said patient prior to said

administration.

3. The pharmaceutical formulation of claim 1, wherein said therapeutically effective dose reduces microglial activation in said patient by at least twenty percent (20%) relative to microglial activation in said patient prior to said administration.

4. The pharmaceutical formulation of claim 1, wherein said therapeutically effective dose is achieved by administering said compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, at a daily dose in a range of from 3 to 400 mg per day.

5. The pharmaceutical formulation of claim 4, wherein said therapeutically effective dose is achieved by administering said compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, QD at a dose in the range of 5 to 200 mg.

6. The pharmaceutical formulation of claim 4, wherein said therapeutically effective dose is achieved by administering said compound of Formula 1 , or an active metabolite, prodrug, ester, or salt thereof, BID at a dose in the range of 5 to 200 mg.

7. The pharmaceutical formulation of claim 1, wherein the compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, is ASP6537 or an active metabolite, prodrug, ester or salt form of such compound.

8. The pharmaceutical formulation of claim 7, wherein the method is for treating a neuroinflammatory disease.

9. The pharmaceutical formulation of claim 8, wherein the neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease, Parkinson's disease dementia.

10. The pharmaceutical formulation of claim 7, wherein the method is for treating cardiovascular disease.

11. The pharmaceutical formulation of claim 7, wherein the method is for treating pain.

12. The pharmaceutical formulation of claim 7, wherein the method is for treating acute pain in combination with a fast onset drug for relief of pain.

13. The pharmaceutical formulation of claim 7, wherein the method is for treating chronic pain from spinal cord injury.

14. The method of any of claims 1 to 13, wherein said therapeutically effective dose is administered via a route selected from transdermal, subcutaneous, intramuscular, inhalation, and oral routes of administration.