WO2012170839A2 - Traitement d'une maladie neuro-inflammatoire par des inhibiteurs sélectifs de cox1 - Google Patents

Traitement d'une maladie neuro-inflammatoire par des inhibiteurs sélectifs de cox1 Download PDF

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WO2012170839A2
WO2012170839A2 PCT/US2012/041586 US2012041586W WO2012170839A2 WO 2012170839 A2 WO2012170839 A2 WO 2012170839A2 US 2012041586 W US2012041586 W US 2012041586W WO 2012170839 A2 WO2012170839 A2 WO 2012170839A2
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asp6537
prodrug
ester
salt
dose
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WO2012170839A3 (fr
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Karen J. Brunke
John CAUFIELD
Keni NII
Makoto OHMIYA
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Astellas Pharma Inc
CARDEUS PHARMACEUTICALS Inc
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Astellas Pharma Inc
CARDEUS PHARMACEUTICALS Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D249/12—Oxygen or sulfur atoms

Definitions

  • 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.
  • AD Alzheimer's Disease
  • PDD Parkinson's Disease Dementia
  • ALS Amyotrophic Lateral Sclerosis
  • HD Huntington's Disease
  • MS Multiple Sclerosis
  • AD Alzheimer's Disease
  • 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.
  • 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.
  • Thromboxane A2 (TXA 2 ), which is synthesized by
  • cyclooxygenase-1 (COXl) enzymatic conversion of arachidonic acid to prostaglandin H2 which is then enzymatically converted to TXA 2 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].
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • 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.
  • 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.
  • glucocorticoids potent anti-inflammatory agents
  • 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).
  • 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.
  • 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.
  • 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.
  • the compound is a compound of Formula 1 :
  • Y and Z are independently CH or N
  • R 1 is lower alkyl which is optionally substituted with halogen
  • R is hydrogen, lower alkyl or lower alkoxy
  • R is hydrogen, lower alkyl or lower alkoxy.
  • 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.
  • 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.
  • the highly selective COX1 inhibitor is a compound of Formula 1 or an active metabolite, prodrug, ester, or salt thereof.
  • 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%.
  • 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%.
  • 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.
  • 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).
  • 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.
  • 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.
  • the methods of the invention provide important new treatments for AD, ALS, HD, MS, PDD, PD, and other dementias and
  • 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.
  • 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.
  • the methods of the invention provide important new treatments for cardiovascular disease.
  • a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof is administered at a dose that inhibits platelet aggregation.
  • 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.
  • 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).
  • 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.
  • the methods of the invention provide important new treatments for the treatment of chronic pain.
  • a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof is administered at a dose that inhibits thromboxane A2 synthesis.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the unit dose form contains ASP6537.
  • Figure 1 shows the results of a study, described in Example 1, of the levels of TXB 2 in the brain and plasma of aged and young rats.
  • Figure 2 shows the results of a study, described in Example 1, demonstrating in Panel A that ASP6537 decreases TXB 2 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 TXB 2 levels in the brains of aged rats.
  • ** indicates a p value ⁇ 0.01 compared to vehicle-treated group.
  • Panel B single doses of either ASP6537 or aspirin in the plasma of aged rats caused dose-dependent decreases in TXB 2 levels.
  • 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 TXA 2 agonist U-46619 decreases spontaneous alternation behavior
  • Part B shows that the addition of the TXA 2 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.
  • icv intracerebroventricular
  • 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).
  • alternation rate degree of arm entries without repetitions.
  • 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.
  • each value shows the mean ⁇ standard error of mean.
  • ### P ⁇ 0.001 vs. normal group using Student's test.
  • 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.
  • each value shows the mean ⁇ standard error of the mean.
  • ### P ⁇ 0.001 vs. normal group using Student's test.
  • * P ⁇ 0.05 vs. control group using Dunnett's test.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 16 shows the results of a study, described in Example 11, demonstrating the benefit of ASP6537 over aspirin for concomitant dosing with ibuprofen.
  • ** indicates a p value of ⁇ 0.01 (Student t-test vs. vehicle).
  • 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.
  • the highly selective COX1 inhibitor is a compound of Formula
  • Y and Z are independently CH or N
  • R 1 is lower alkyl which is optionally substituted with halogen
  • R is hydrogen, lower alkyl or lower alkoxy
  • R is hydrogen, lower alkyl or lower alkoxy.
  • 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.
  • R 1 is lower alkyl. In some embodiments, R 1 is chosen from -CH 3 , -CH 2 F, -CF 3 , -CH 2 CH 3 , -CH 2 CF 3 , and -CH(CH 3 ) 2 . In some embodiments, R 1 is chosen from -CH3.
  • R is lower alkoxy. In some embodiments,
  • R is methoxy
  • R is lower alkoxy. In some embodiments,
  • R is methoxy
  • R is methoxy
  • R is methoxy
  • Y is CH
  • the compound of Formula 1 is chosen from
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • esters 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.
  • active metabolite refers 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
  • 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.
  • pharmaceutically acceptable carrier 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.
  • a pharmaceutically-acceptable material 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.
  • 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 hydrox
  • compositions include excipients and diluents.
  • the highly selective COX1 inhibitors described herein can be enriched isotopic forms, e.g., enriched in the content of 2 H, 3 H, n C, 13 C and/or 14 C.
  • the compound contains at least one deuterium atom.
  • deuterated forms can be made, for example, by the procedure described in U.S. Patent Nos.
  • 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
  • thromboxane A2 thromboxane A2
  • TXB 2 inactive metabolite
  • TXA 2 thromboxane B2
  • 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.
  • TXB 2 The elevated level of TXB 2 observed in the plasma correlated with elevated levels in the brain in the AD patients and also in aged rats (Example 1, Figure 1).
  • aged rats make a good model for studying effects on plasma and brain levels of TXB 2 in dementias.
  • ASP6537 can suppress brain thromboxane B2 (TXB 2 ) levels in aged rats by suppressing brain TXA 2 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.
  • the level of TXB 2 brain thromboxane B2
  • 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).
  • 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.
  • 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 TXA 2 reduction in the plasma does not reduce the level of TXA 2 in the brain. In Example 1 , Figure 2A, aspirin dosed daily for 7 days did not reduce TXA 2 in the brain while a single dose of ASP6537 dose-dependently reduced TXB 2 . As mentioned earlier, TXB 2 is the inactive metabolite of TXA 2 . TXB 2 is measured in the experiments in Figures 1 and 2.
  • TXA 2 in the brain reduction of TXA 2 in the plasma (in the absence of drugs such as aspirin which reduce TXA 2 levels in the plasma) is a surrogate for reduction of TXA 2 in the brain and provides a clinically relevant marker for ASP6537 action in the brain.
  • TXA 2 is measured by measurement of its inactive metabolite, TXB 2 .
  • 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 TXA 2 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 TXA 2 levels, as indicated by measuring thromboxane B2 levels, on cognition.
  • U-46619 is an agonist of TXA 2 .
  • 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.
  • 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.
  • TXA 2 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 TXA 2 by thromboxane synthase.
  • Inhibition of COX1 activity results in decreased TXA 2 synthesis and TXA 2 levels in neurons.
  • the suppression of brain TXA 2 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.
  • 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 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.
  • 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.
  • NMD A N-methyl-D-aspartate
  • a rat 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.
  • 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.
  • the ability of donepezil hydrochloride, an acetylcholinesterase inhibitor, to enhance spatial memory was assessed in aged rats using the Morris water maze.
  • 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.
  • APP amyloid precursor protein
  • Prostaglandins 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.
  • ASP6537 and other compounds of Formula 1, or an active metabolite, prodrug, ester, or salt thereof, are 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
  • 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].
  • 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 TXA 2 (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 TXA 2 ) as described above for neuroinflammatory diseases.
  • 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.
  • 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.
  • 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.
  • symptoms in patients such as evaluation of cognition, disease specific neuronal tests, daily living activities, and overall clinical response.
  • the treatment methods of the invention are disease modifying in nature.
  • COXl inhibition also lessens or arrests the neuronal damage in AD in response to amyloid beta and tau through overall decreased neuroinflammation
  • a disease modifying effect occurs when the pharmacologic treatment delays the underlying pathological or
  • 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
  • ASP6537 In human clinical studies, single doses of ASP6537 have been demonstrated to reduce the levels of plasma thromboxane B2 (TXB 2 ), as described in Example 7 and shown in Figures 12-15. Multiple doses of ASP6537 predictably and effectively reduced TXB 2 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 TXA 2 synthesis (as measured by its inactive metabolite, TXB 2 ) in the brain is through once daily (QD) or twice daily (BID) dosing.
  • QD once daily
  • BID twice daily
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • Nonsteroidal anti-inflammatory drugs 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.
  • COXl cyclooxygenases-1 and 2
  • 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
  • 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.
  • GI gastrointestinal
  • 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.
  • DCIs drug-drug interactions
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Inflammation is a neuropathological hallmark of amyotrophic lateral sclerosis (ALS), a rapidly progressing neurodegenerative disease.
  • ALS amyotrophic lateral sclerosis
  • 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.
  • ASP6537 inhibits microglia activation about 75% at the dose administered in this animal study.
  • 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.
  • 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.
  • 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.
  • 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.
  • Parkinson's Disease is characterized by loss of dopaminergic neurons from the substantia nigra.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Parkinson's Disease frequently progresses to Parkinson's Disease
  • PDD Dementia
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Huntington's Disease is characterized by the progressive death of medium spiny dopamine receptor bearing striatal GABAergic neurons.
  • 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.
  • ASP6537 and other compounds of Formula 1 are useful in the treatment of the dementia and cognitive impairment associated with HD.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, spino
  • frontotemporal dementia frontotemporal dementia
  • Gaucher's disease 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.
  • 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.
  • 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.
  • 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.
  • ASP6537 and 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.
  • ASP6537 and other drugs commonly used for the treatment of MS such as interferon beta, glatiramer, mitox
  • combination therapies 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.
  • 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.
  • 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.
  • 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.
  • 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-HT 6 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • riluzole riluzole, baclofen and antidepressants or other drugs used to treat
  • 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.
  • a cholinesterase inhibitor is used in one embodiment.
  • donepezil is a cholinesterase inhibitor suitable for such use and can be administered at the recommended doses of 5 or 10 mg QD.
  • 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.
  • 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.
  • 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.
  • 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.
  • the COX1 inhibitor is ASP6537 and the other drug is donepezil.
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods of the invention provide important new treatments for cardiovascular disease.
  • a compound of Formula 1, or an active metabolite, prodrug, ester, or salt thereof is administered at a dose that inhibits platelet aggregation.
  • 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.
  • 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).
  • 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.
  • Acetylsalicylic acid inhibits both the COX- 1 -dependent production of thromboxane A2 (TXA 2 ) in platelets as measured with its inactivate metabolite thromboxane B2 (TXB 2 ) and COX2-dependent production of anti-aggregatory prostaglandin 12 (PGI 2 ) in vessel walls, resulting in the "aspirin dilemma.”
  • TXA 2 thromboxane A2
  • TXB 2 inactivate metabolite thromboxane B2
  • PKI 2 anti-aggregatory prostaglandin 12
  • 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.
  • 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.
  • 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.
  • ACS acute coronary syndrome
  • MI myocardial infarction
  • transient ischemic attacks and is considered standard drug for treating those conditions.
  • 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.
  • 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.
  • TXA 2 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 ⁇ g/mL collagen.
  • indomethacin (10 ⁇ 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 TXA 2 , TXB 2 , in the supernatant was measured according to a standard procedure using a TXB 2 EIA kit (Cayman Chemical) following the manufacturer's guidelines.
  • PGI 2 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 PGI 2 production. After replacement of the buffer, the slices were incubated with vehicle or test drug at 37 °C for 15 min. AA (20 ⁇ ) was then added to the mixture, which was further incubated at 37 °C for 15 min.
  • TXA 2 and PGI 2 in vitro in tissue were as follows.
  • ASP6537 inhibited platelet TXB 2 production with a geometric mean IC 5 o value of 0.00358 ⁇ , which was 5,000-fold higher than the value determined for aspirin (18.3 ⁇ ).
  • ASP6537 and aspirin inhibited PA with a geometric mean IC 50 value of 0.00835 and 42.3 ⁇ , respectively.
  • the effects of these two drugs on TXB 2 production correlated well with those on PA.
  • ASP6537 also inhibited vessel 6-keto-PGFla production, the IC 50 ratios of TXB 2 to 6-keto-PGFla production for ASP6537 and aspirin were 20.8 and 0.738, respectively.
  • ASP6537 preferentially inhibited TXA 2 (as measured by measuring TXB 2 ) synthesis over that of 6-keto-PGFla, and this inhibition was markedly more selective than that of aspirin.
  • 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.
  • MC methylcellulose
  • 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 PGI 2 , 2,3-dinor-6-keto PGFla, in the urine samples was measured using an EIA kit (Cayman Chemical).
  • 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 PGI 2 metabolite concentrations at all examined doses.
  • ASP6537 reduced urinary PGI 2 metabolite excretion in normal rats, indicating that COXl is also involved in PGI 2 production under normal conditions in vivo.
  • ASP6537 and aspirin inhibit PA in normal rats at doses of 3 and 100 mg/kg, respectively.
  • a 33 -fold greater dose of ASP6537 was required for an inhibitory effect on urinary PGI 2 metabolite excretion to be observed compared with that necessary for the disruption of PA, whereas aspirin inhibited both urinary PGI 2 metabolite excretion and PA at an identical dose.
  • the selective inhibition of ASP6537 on TXA 2 and PA over PGI 2 is attributable to the observed COX1/COX2 selectivity. Both COX1 and COX2 appear to be involved in vascular PGI 2 production under normal physiological conditions, and ASP6537 has a superior ability to aspirin for maintaining TXA 2 /PGI 2 balance.
  • ASP6537 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 ⁇ final concentration). Plasma was then prepared, and the amount of TXB 2 and 6-keto PGFla was measured according to standard procedures using EIA kits (Cayman Chemical).
  • ASP6537 and aspirin 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.
  • induced COX2 dominates over COX1 in catalyzing PGI 2 biosynthesis due to its preferential coupling with PGI 2 synthase.
  • ASP6537 exerted greater selective inhibition of TXA 2 over PGI 2 .
  • ASP6537 demonstrated superior anti-thrombotic properties to aspirin.
  • 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.
  • DPF-10R Doppler flow probe
  • 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.
  • 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.
  • 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.
  • 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.
  • ASP6537 may be taken in combination with an NSAID as described above at standard doses and intervals.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the methods of the invention provide important new treatments for the treatment of chronic or acute pain.
  • a compound of Formula 1 or an active metabolite, prodrug, ester, or salt thereof, is administered at a dose that inhibits thromboxane A2 synthesis.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • Nonsteroidal anti-inflammatory drugs currently fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both
  • COXl cyclooxygenases-1 and 2
  • PGs prostaglandins
  • COXl cyclooxygenases-1 and 2
  • PGs prostaglandins
  • 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.
  • GI gastrointestinal
  • 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.
  • COXl a second isoform of COX was identified and named COX2, with the previously known COX denoted as 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.
  • COX2 mRNA and proteins are predominantly expressed in inflamed tissues, rapidly producing
  • COXl -derived prostaglandin E2 PGE2
  • PGE2 prostaglandin E2
  • coxibs 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.
  • 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,.
  • 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.
  • 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.
  • 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.
  • COX2 inhibitors used in clinical settings such as rofecoxib
  • rofecoxib used to treat chronic pain
  • 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.
  • 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.
  • 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.
  • 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.
  • these pharmaceutical formulations of the invention are provided as a sustained release formulation in a unit dose form that is a tablet or capsule.
  • 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.
  • 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.
  • the highly selective COX1 inhibitor is formulated as an oral disintegrating tablet or oral dispersable tablet.
  • 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.
  • delivery directly to the CSF through nasal passages for lower doses, delivery directly to the CSF through nasal passages, transdermal patch delivery, and subcutaneous or IM
  • a depot formulation are particularly useful.
  • direct delivery to the CSF for example, through direct injection via pump or depot
  • 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.
  • 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.
  • 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.
  • PEG 400 polyethylene glycol 400
  • ASP6537 also known as FK881
  • ASP6537 is practically insoluble in water or isotonic saline solutions. It is sparingly soluble in polyethylene glycol 400.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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 drying/melting step; and/or (c) using a Fitzmill to size the meted cake.
  • a suitable process for film coating the tablets in accordance with the invention can involve sequential steps, such as: 1.
  • therapeutically effective doses reduce brain levels of TXA 2 (as can be measured by measuring reduced levels of TXB 2 ) to normal or near normal levels and result in improved cognition in patients in need of treatment.
  • 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.
  • 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).
  • 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.
  • 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.
  • the formulation will also comprise one or more pharmaceutically acceptable carriers.
  • the compounds of this invention can be prepared from readily available starting materials using, for example, the following general methods and procedures.
  • 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).
  • 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.
  • compound 1-1 is reacted with potassium cyanate to provide compound 1-2.
  • 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.
  • compound 1-2 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.
  • 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.
  • 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.
  • a tertiary amine e.g., pyridine
  • a suitable solvent e.g. toluene
  • compound 1-4 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.
  • 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.
  • 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, ⁇ , ⁇ -dimethylaniline, etc.), an alkali metal hydroxide (e.g.
  • the base can be used as a solvent.
  • the reaction is continued until substantially complete which typically occurs within about 1 to 16 hours.
  • the compound of formula 1 can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.
  • Example 1 Higher Plasma and Brain TXB 2 Levels Correlate with Aging and AD, and ASP6537 Reduces TXB 2 Levels Where Aspirin is Ineffective in Reducing Brain TXB2 Levels
  • ASP6537 (administered at doses of 1, 3 and 10 mg/kg, po) significantly decreased TXB 2 levels both in plasma and brain tissue from aged rats, as evidenced by Figure 2. Aspirin decreased TXB 2 levels in plasma but had no effect on TXB 2 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 TXB 2 levels in plasma as expressed in pg/mL. Statistical analyses performed using the Dunnett's multiple comparison test.
  • Thromboxane B2 is the stable metabolite of TXA 2 .
  • 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.
  • mice (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.
  • ASP6537 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.
  • 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
  • MK-801 is an antagonist of N- methyl-D-aspartate (NMD A), a glutamate receptor.
  • NMD A N- methyl-D-aspartate
  • 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.
  • 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.
  • 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.
  • 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.
  • the same location was not used on 2 consecutive days.
  • 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).
  • 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.
  • the average velocity was calculated from the average of daily velocity for 4 days.
  • 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).
  • 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.
  • the ability of donepezil hydrochloride, an acetylcholinesterase inhibitor, to enhance spatial memory was assessed in aged rats using the Morris water maze.
  • 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.
  • donepezil hydrochloride exerts no ameliorating effect on the spatial memory deficits seen in aged rats.
  • 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.
  • 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.
  • 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.
  • APP amyloid precursor protein
  • mice 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • brain and plasma concentrations of ASP6537 were measured in 3 conscious male rhesus macaques using [11C]ASP6537 and positron emission tomography
  • [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.
  • ASP6537 is a brain penetrant COX1 inhibitor, especially well-suited for treatment of inflammatory neurological diseases in accordance with the present invention.
  • a therapeutically effective dose can be administered as a dose that achieves at least TXB 2 inhibition to IC25, including but not limited to a dose that achieves reduction of TXB 2 levels to IC50 or to IC75 or higher (in the absence of other drugs which reduce the level of TXB 2 in plasma such as aspirin).
  • 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.
  • hWBA human whole blood assay
  • SC-560 shows only slight selectivity in the same 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.
  • 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.
  • MC methylcellulose
  • Enzyme immunoassay kits for TXB 2 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
  • TXB 2 thromboxane
  • the IC50 value of ASP6537 for inhibiting COX1 activity was 0.0049 ⁇ , while that for inhibiting COX2 activity was 3.2 ⁇ . These results showed that ASP6537 had COX1 selective inhibition activity in human whole blood (650-fold).
  • the IC50 values of rofecoxib were 38 and 0.25 ⁇ for COX1 and COX2 respectively.
  • ASP6537 and aspirin were dissolved in dimethylsulfoxide (DMSO) and diluted with reaction buffer (0.1 M Tris-HCl [pH 7.3] containing 2 ⁇ 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.
  • PGI 2 synthesis was initiated by the addition of 10 ⁇ AA, and after 5 min (rhCOXl) or 10 min (rhCOX2), PGI 2 production was terminated by adding 1 mM indomethacin.
  • concentrations of the stable metabolite of PGI 2 , 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.
  • ASP6537 inhibited AA-induced 6-keto-PGFla production by rhCOXl with a geometric mean IC 50 of 0.000703 ⁇ /L, but had no detectable inhibitory effects on that by rhCOX2, even at concentrations up to 100 ⁇ /L.
  • aspirin inhibited AA-induced 6-keto-PGF la production by both rhCOXl and rhCOX2, with geometric mean IC 50 values of 37.9 and 61.9 ⁇ , respectively.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aspirin had a potent ulcerogenic effect at the antithrombotic dose.
  • 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
  • GABA A (Agonist site) 0.00 6.14 4.25 67.62 (Muscimol: 1x10 " ' mol/L)
  • LeukotrieneB 4 0.00 0.00 3.73 89.25 (Leukotriene B4: 1x10 s mol/L)
  • Muscarinic 2 2.70 0.00 0.00 65.54 (Atropine: 1x10 s mol/L)
  • Neurokinin 0.00 0.00 0.00 53.09 Neurokinin A: 3x10 s mol/L
  • Neurokinin 3 2.22 0.60 1.84 81.65 (Neurokinin B: 1x10 s mol/L)
  • Nicotinic 1.82 9.00 2.12 78.09 (Nicotine: 3x10 s mol/L)
  • Serotonin transporter 1.55 10.46 10.44 77.07 (Imipramime: 1x10 s mol/L)
  • Testosterone 2.00 4.45 11.87 64.70 (Testosterone: 1x10 s mol/L)
  • Vasopressin Vj 1.17 27.55 56.69 76.94 mol/L
  • 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.
  • IC50 ratios of rhCOX2 to rhCOXl for ASP6537 and aspirin were >142,000 and 1.63, respectively, and that ASP6537 inhibited TXA 2 production more selectively than did aspirin in in vitro and in vivo TXA 2 /PGI 2 production studies.
  • TXA 2 /PGI 2 balance by ASP6537 is superior to that of aspirin using guinea pigs and rats.
  • 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 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.,
  • TXA 2 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 ⁇ g/mL collagen.
  • indomethacin (10 ⁇ 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 TXA 2 , TXB 2 , in the supernatant was measured according to a standard procedure using a TXB 2 EIA kit (Cayman Chemical) following the manufacturer's guidelines.
  • TXA 2 and PGI 2 in vitro in tissue were as follows.
  • ASP6537 inhibited platelet TXB 2 production with a geometric mean IC 50 value of 0.00358 ⁇ , which was 5,000-fold higher than the value determined for aspirin (18.3 ⁇ ).
  • ASP6537 and aspirin inhibited PA with a geometric mean IC 50 value of 0.00835 and 42.3 ⁇ , respectively.
  • the effects of these two drugs on TXB 2 production correlated well with those on PA.
  • ASP6537 also inhibited vessel 6-keto-PGF la production, the IC 50 ratios of TXB 2 to 6-keto-PGFla production for ASP6537 and aspirin were 20.8 and 0.738, respectively.
  • ASP6537 preferentially inhibited TXB 2 synthesis over that of 6-keto-PGF la, and this inhibition was markedly more selective than that of aspirin.
  • 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 PGI 2 , 2,3-dinor-6-keto PGFla, in the urine samples was measured using an EIA kit (Cayman Chemical).
  • 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 PGI 2 metabolite concentrations at all examined doses.
  • ASP6537 and aspirin were orally administered 1 h before blood collection. Heparinized blood was immediately transferred into a plastic tube containing indomethacin (10 ⁇ final concentration). Plasma was then prepared, and the amount of TXB 2 and 6-keto PGFla was measured according to standard procedures using EIA kits (Cayman
  • ASP6537 and aspirin 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.
  • ASP6537 demonstrated superior anti-thrombotic properties to aspirin.
  • 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.
  • DPF-10R Doppler flow probe
  • 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.
  • ASP6537 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 TXA 2 /PGI 2 balance and can be used to avoid the aspirin dilemma.
  • 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).
  • ASP6537 shows activity in a model for acute pain equivalent to diclofenac.
  • 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.
  • the analgesic effect of ASP6537 was comparable to that of diclofenac and rofecoxib in a rat model of chronic inflammatory pain.

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Abstract

L'invention concerne des inhibiteurs hautement sélectifs de COX1 pouvant être utilisés pour traiter une maladie neuro-inflammatoire.
PCT/US2012/041586 2011-06-09 2012-06-08 Traitement d'une maladie neuro-inflammatoire par des inhibiteurs sélectifs de cox1 Ceased WO2012170839A2 (fr)

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