ES2365409T3 - DIHYDROPIRIDINE COMPOUNDS FOR NEURODEGENERATIVE DISEASES AND DEMENTIA. - Google Patents

Abstract

A compound for use in combination with donepezil or a pharmaceutically acceptable salt thereof in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient; wherein the compound is 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of a of its pharmaceutically acceptable salts.

Description

The invention relates to dihydropyridine compounds for use in combination with a cholinesterase inhibitor for the treatment of a variety of diseases and disorders. The invention also relates to a cholinesterase inhibitor for use in combination with a dihydropyridine compound for the treatment of a variety of diseases and disorders.

Background of the Invention

Receptor amino acid receptors are classified into two general types. Receptors that attach directly to the opening of cationic channels in the cell membrane of neurons are called "ionotropic." This type of receptor has been subdivided into at least three subtypes, which are defined by the depolarizing actions of the selective agonist N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazol-4propionic acid ( AMPA), and kainic acid. The second general type is the G protein or "metabotropic" excitatory amino acid receptor coupled to second messengers. This second type, when activated by the agonists quisqualate, ibotenate, or trans-1-aminocyclopentane-1,3-dicarboxylic acid, leads to the hydrolysis of phosphoinositide in the postsynaptic cell. Both types of receptors seem not only to mediate normal synaptic connections during development, but also to change the effectiveness of synaptic transmission throughout life. Schoepp et al, Trends Pharm Sci., 11: 508 (1990); McDonald et al, Brain Res. Rev., 15:41 (1990).

Excessive excitation by neurotransmitters can cause degeneration and death of neurons. It is believed that this degeneration is mediated in part by the excitotoxic actions of excitatory amino acids, glutamate and aspartate, at the NMDA receptor, the AMPA receptor, and the kainate receptor. This excitotoxic action is responsible for the loss of neurons in cerebrovascular disorders such as cerebral ischemia or cerebral infarction resulting from a range of conditions, such as thromboembolic or hemorrhagic stroke, cerebral vasospasm, hypoglycemia, cardiac arrest, status epileptic, perinatal asphyxia, anoxia such as that due to almost drowning, lung surgery and brain trauma as well as beating, Alzheimer's disease, and Huntington. United States Patents Nos. 6,765,006 and 5,843,945.

US 2004/0023973 describes 1,2-dihydropyridine compounds and their use as non-NMDA receptor inhibitors, specifically an AMPA receptor.

Shigeta et al. (CNS Drug Reviews, Branford, CT, US, vol. 7, No. 4, December 21, 2001, 353-368) describe the use of donepezil for the treatment of Alzheimer's disease and describes pharmacodynamic, pharmacokinetic and Donepezil clinics.

US 2006/0100249 describes compositions comprising 1,2-dihydropyridin-2-one compounds and an immunoregulatory or anti-inflammatory agent, and their use in the treatment of neurodegenerative diseases.

US 2003/0144255 discloses a pharmaceutical composition comprising a compound of 4,4'-diaminodiphenylsulfone combined with a compound having cholinesterase inhibitory activity, and its use in the treatment of dementia.

There is a need in the art for new compounds and new methods for the treatment of diseases and disorders that may be mediated to some extent by NMDA receptors, AMPA receptors and / or kainate receptors. The invention relates to this, as well as others, important purposes.

Compendium of the Invention

The invention provides a compound for use in combination with donepezil or a pharmaceutically acceptable salt thereof in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia.

or one or more cognitive impairments in a patient; wherein the compound is 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts.

The present invention also provides a compound for use in combination with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient

or (ii) dementia or one or more cognitive impairments in a patient; where the compound is donepezil or one of its pharmaceutically acceptable salts.

The present invention also provides the use of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the manufacture of a medicament for use in combination with donepezil or one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient.

The present invention also provides the use of donepezil or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in combination with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine -2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient.

Donepezil or its pharmaceutically acceptable salt and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine, its pharmaceutically acceptable salt, its hydrate or the hydrate of one of its salts Pharmaceutically acceptable for use in the present invention can be (a) administered separately to the patient or (b) administered to the patient in the form of a pharmaceutical composition.

Dementia can be caused by Alzheimer's disease, Parkinson's disease, or Huntington's disease.

The combination of the 1,2-dihydropyridine and donepezil compound unexpectedly produces synergistic effects in the treatment and / or prophylaxis of dementia or cognitive impairments.

The combination of the 1,2-dihydropyridine and donepezil compound also unexpectedly produces synergistic effects in the treatment of neurodegenerative diseases.

Brief Description of the Figures

Figure 1 shows the effect of Compound A (that is, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one) on the content of dopamine, DOPAC, and HVA in the striated nuclei of mice. Figure 2 shows the effects of donepezil on the turnover of dopamine in the striated nuclei of mice. Figures 3A-E show the effects of a combination of Compound A (that is, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one) and donepezil on the Dopamine replacement, DOPAC, HVA, DOPAC / DA and HVA / DA, respectively, in the striatum nuclei of mice. Figure 4 shows the effects of Compound A (that is, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2one) and the combination of Compound A (that is , 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one) and Donepezil on kainate-induced neuronal toxicity in cortical neurons. The * indicates that p <0.05 vs. control, and # indicates that p <0.05 vs. Donepezil alone. Figure 5 shows the effect of Compound A (that is, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one) and the combined treatment of Compound A (i.e., 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one) and Donepezil on NMDA-induced neural toxicity in cortical neurons. The * indicates that p <0.05 vs. control.

Detailed description of the invention

"Patient" refers to animals, preferably mammals, more preferably humans. The term "patient" includes men and women; and includes adults, children and neonates. In one embodiment, the patient may be a companion animal, such as a dog or a cat.

"Cognitive impairments" refers to a deficit acquired in one or more of the memory function, problem solving, orientation, and abstraction that affects a patient's ability to function independently.

"Dementia" refers to a global deterioration of intellectual function in clear unconsciousness, and is characterized by one or more symptoms of disorientation, memory impairment, impairment of judgment, and deterioration of intellect.

"Administered separately" with reference to the administration of two or more compounds for the treatment and / or prevention of the diseases and disorders described herein includes, for example, the successive administration of the compounds in any order or simultaneous administration. of the compounds. Simultaneous administration of the compounds means that the compounds are administered to the patient essentially at the same time or at exactly the same time, depending on the mode of administration. Successive administration of the compounds may occur in any order and may occur during any amount of time between the administration of the compounds. Successive administration may be based on factors that may influence the compounds to be administered first and the compounds to be administered secondly, and how much time elapses between the administration of the compounds. For example, when two or more compounds are administered separately and successively, the factors that influence when the compounds are administered to the patient include, for example, (a) the time or times that provide the best efficacy for administering the compound, (b ) the time or times that provide the least side effects to administer the compound, (c) the dosage of the compound, (d) the route of administration of the compound, (e) the disease or disorder being treated, (f) the patient being treated, (g) the in vivo relationships of the compounds being administered, and other similar factors known in the art. Preferably, the time intervals for successive administration are chosen so that the effect on the disease or disorder being treated in the combined use of the active ingredients is greater than the additive when compared with the effect that could be obtained by means of using only one of the active ingredients.

The term "combination" refers to the 1,2-dihydropyridine compound and the second active ingredient (donepezil or a pharmaceutically acceptable salt thereof) that are being administered separately in the form of different pharmaceutical compositions or formulations. Pharmaceutical compositions or formulations may have the same or different modes of administration.

"Active ingredient" refers to the 1,2-dihydropyridine compound described herein and donepezil or one of its pharmaceutically acceptable salts described herein that are responsible for the treatment and / or prophylaxis of a disease or disorder.

"Monotherapy" is a therapy that uses only one active ingredient for the treatment and / or prophylaxis of a disease or disorder.

"Combined therapy" is a therapy in which two or more active ingredients are administered separately or administered in the form of a pharmaceutical composition for the treatment and / or prophylaxis of a disease.

"Therapeutically effective amount" refers to the amount of active ingredient that is necessary for the treatment and / or prophylaxis of a disease. When two or more active ingredients are administered for a combination therapy, the term "therapeutically effective amount" refers to the amount of active ingredients that are necessary for the treatment and / or prophylaxis of a disease and includes, for example: (a ) a therapeutically effective amount of a first active ingredient and a therapeutically effective amount of a second active ingredient (that is, for the combination therapy the amount of each active ingredient that would be used in monotherapy for treatment and / or prophylaxis is used of a disease); (b) a therapeutically effective amount of a first active ingredient and a subtherapeutic amount of a second active ingredient, which effectively combined provide the treatment and / or prophylaxis of a disease (e.g., the subtherapeutic amount of a second active ingredient in a combination therapy to achieve a result that would be equal to or greater than the result that the second active ingredient would achieve if it were used for monotherapy); (b) a subtherapeutic amount of a first active ingredient and a therapeutically effective amount of a second active ingredient, which effectively combined provide the treatment and / or prophylaxis of a disease (e.g., the subtherapeutic amount of the first active ingredient is it can be used in combination therapy to achieve a result that would be equal to or greater than the result that the first active ingredient would achieve if it were used for monotherapy); and (d) a subtherapeutic amount of a first active ingredient and a subtherapeutic amount of a second active ingredient, whose combined therapy provides the treatment and / or prophylaxis of a disease or disorder (e.g., the subtherapeutic amount of the first ingredient active can be used in combination therapy to achieve a result that would be equal to or greater than the result that the first active ingredient would achieve if it were used for monotherapy; and the subtherapeutic amount of the second active ingredient can be used in combined therapy to achieve a result that would be equal to or greater than the result that the second active ingredient would achieve if it were used for monotherapy).

"Commercial packages", also known as kits, may include a combination of (i) a first pharmaceutical composition or formulation comprising the 1,2-dihydropyridine compound); (ii) a second pharmaceutical composition or formulation comprising the second active ingredient (donepezil or a pharmaceutically acceptable salt thereof); (iii) FDA-approved instructions for the use of pharmaceutical compositions or formulations for the treatment of or prevention of the disease; and (iv) optionally other materials for the administration of pharmaceutical compositions or formulations (eg, syringes, diluents, medical gloves, hand sanitizers, and the like); to verify drug levels in the body; to assist in the observance of the dosage of the medication by the patient; or to verify the disease status. The commercial package can supply enough medication and materials for days, weeks or months. Also described herein are "commercial packages" that may include (i) the pharmaceutical composition or formulation comprising both the 1,2-dihydropyridine compound and the second active ingredient (donepezil or a pharmaceutically acceptable salt thereof); (ii) the instructions approved by the FDA for the use of the pharmaceutical composition or formulation for the treatment or prevention of the disease; and (iii) optionally other materials for the administration of pharmaceutical compositions or formulations (eg, syringes, diluents, medical gloves, hand sanitizers, and the like); to verify drug levels in the body; to assist in the observance of the dosage of the medication by the patient, or to verify the condition of the disease. The commercial package can supply enough medication and materials for days, weeks or months.

"Hydrate" refers to a compound that contains a molecule of water of crystallization. The crystallization water molecule may be an integer of 1 or more, for example 1 to 10; or it can be any fraction greater than 0 or a fraction of an integer from 1 to 10. For example, the hydrate can be represented as a compound · ¼H2O; compound · 1/2 H2O; compound · 2H2O; compound · 2H2O; compound · 5½H2O; compound · 6H2O; and the like The "compound" can be any of those described herein, such as 3- (2-cyanophenyl) -5

10 (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one.

"Pharmaceutically acceptable salts" are well known in the art and include those of inorganic acids, such as hydrochloride, sulfate, hydrobromide and phosphate; and those of organic acids, such as formate, acetate, trifluoroacetate, methanesulfonate, benzenesulfonate and toluenesulfonate. When certain are selected

15 substituents, the compounds of the invention can form, for example, alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; salts of organic amines, such as a salt with trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine or N, N'-dibenzylethylenediamine. One skilled in the art will recognize that the compounds of the invention can be made in the form of any other pharmaceutically acceptable salt.

The term "1,2-dihydropyridine compound" used herein includes 1,2-dihydropyridine compounds, pharmaceutically acceptable salts of 1,2-dihydropyridine compound, stereoisomers of 1,2-dihydropyridine compound, salts Pharmaceutically acceptable stereoisomers of 1,2-dihydropyridine compound, hydrates of 1,2-dihydropyridine compounds, hydrates of pharmaceutically salts

Acceptable compounds of 1,2-dihydropyridine compound, stereoisomers of hydrates of 1,2-dihydropyridine compound, and stereoisomers of hydrates of pharmaceutically acceptable salts of 1,2-dihydropyridine compound.

The 1,2-dihydropyridine compound used in the methods and compositions described herein is Compound A:

image 1

The IUPAC name for Compound A is 2- (2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile. Compound A may also be referred to as 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one.

Throughout the specification, the terms "Compound A", "2- (2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile", and "3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one" include its pharmaceutically acceptable salts, its stereoisomers, the pharmaceutically acceptable salts of its stereoisomers, its hydrates , the hydrates of their pharmaceutically acceptable salts, the stereoisomers of their hydrates, and the stereoisomers of their pharmaceutically acceptable salts. In another embodiment, it

40 claims that the terms "Compound A," "2- (2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile", and "3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one "include its pharmaceutically acceptable salts, its hydrates, and the hydrates of its pharmaceutically acceptable salts.

The 1,2-dihydropyridine compound and the methods for making the 1,2-dihydropyridine compound used

45 in the present invention are described in United States Patent No. 6,949,571, United States Publication No. 2004/0023973, and PCT Publication No. WO 03/047577, WO 04/009553, WO 06/004100, and WO 06/004107, whose descriptions are incorporated herein by reference in their entirety. Methods for making other AMPA receptor antagonists are described in WO 2005/094797.

The present invention preferably employs 1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2-yl) methylpiperidine; which is represented by formula (B):

image 1

5 The compound of Formula (B), known as donepezil, can exist as a polymorphic form or as a polymorphic crystal. For example, donepezil can be in a polymorphic form (II), (III), (IV), or (V); preferably a polymorphic form (III). Donepezil can be in the form of polymorphic crystals (A), (B), or (C). Polymorphic forms, polymorphic crystals, and methods for the preparation of polymorphic forms and polymorphic crystals are described in US Pat. Nos. 5,985,864, 6,140,321 and 6,245,911, whose

10 descriptions are incorporated herein by reference in their entirety.

In another embodiment, the present invention employs 1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2yl) methylpiperidine hydrochloride, which is also known as donepezil hydrochloride, and which is represented by the formula

image2

The compounds of the invention may have one or more asymmetric carbon atoms, depending on the substituents, and may have stereoisomers, which are within the scope of the invention. For example, donepezil or its pharmaceutically acceptable salts may be in the forms described in the Requests for

20 Japanese Patent Nos. 4-187674 and 4-21670.

which may be in the form of a pharmaceutically acceptable salt, such as a hydrochloride salt. Japanese Patent Application No. 4-21670 describes compounds of formula (B3):

image3

image 1

which may be in the form of a pharmaceutically acceptable salt, such as a hydrochloride salt; and compounds of formula (B4):

image 1

which may be in the form of a pharmaceutically acceptable salt, such as a hydrochloride salt; and compounds of formula (B5):

image 1

5 Throughout the report, the terms "donepezil" and "1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2il) methylpiperidine" are intended to include one or more of the following (p eg, combinations of two or more thereof): pharmaceutically acceptable salts; stereoisomers; polymorphic forms and polymorphic crystals.

The donepezil described herein is commercially available or can be prepared by methods known in the art, such as those described, for example, in United States Patents Nos. 4,895,841, 5,985,864, 6,140,321 and 6,245,911; WO 98/39000, and Japanese Patent Applications Nos. 4-187674 and 4-21670.

Pharmaceutical compositions comprising a therapeutically effective amount of: (i) at least one AMPA receptor antagonist are also described herein; (ii) at least one nootropic; and (iii) a pharmaceutically acceptable excipient. The AMPA receptor antagonist is the 1,2-dihydropyridine compound described herein. The nootropic is the donepezil cholinesterase inhibitor or one of its pharmaceutically acceptable salts described herein.

Use combinations comprising a therapeutically effective amount of (i) at least one AMPA receptor antagonist and (ii) at least one nootropic are also described herein. The combination can be administered separately (e.g., simultaneously, successively) to a patient for the treatment of the diseases or disorders described herein (e.g., cognitive impairments,

25 dementia and the like). The AMPA receptor antagonist is the 1,2-dihydropyridine compound described herein. The nootropic is the donepezil cholinesterase inhibitor or one of its pharmaceutically acceptable salts described herein.

Commercial packages (eg, kits) comprising an amount are also described herein.

Therapeutically effective of: (i) at least one AMPA receptor antagonist and (ii) at least one nootropic; and (iii) instructions for simultaneous, separate or successive use in the treatment of the diseases and disorders described herein. The AMPA receptor antagonist is the 1,2-dihydropyridine compound described herein. The nootropic is the donepezil cholinesterase inhibitor or one of its pharmaceutically acceptable salts described herein.

Also described herein are pharmaceutical compositions comprising a therapeutically effective amount of: (i) the 1,2-dihydropyridine compound described herein (ii) donepezil or a pharmaceutically acceptable inhibitor salt thereof, and (iii) ) at least one pharmaceutically acceptable excipient. Combinations comprising an amount are further described herein.

Therapeutically effective of: (i) the 1,2-dihydropyridine compound described herein and (ii) donepezil or a pharmaceutically acceptable salt thereof; where the compounds can be administered separately (e.g., simultaneously, successively) to a patient for the treatment of the diseases or disorders described. Commercial packages (eg, kits) comprising a therapeutically effective amount of: (i) the 1,2-dihydropyridine compound described herein (ii) donepezil or are also described herein.

One of its pharmaceutically acceptable salts; and (iii) instructions for the simultaneous, separate or successive use of

(i) and (ii) in the treatment of the diseases and disorders described herein. The 1,2-dihydropyridine compound may be any of those described herein. The pharmaceutical compositions may comprise a therapeutically effective amount of: (i) donepezil; (ii) 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl1,2-dihydropyridin-2-one; and (iii) at least one pharmaceutically acceptable excipient.

Methods for the treatment of one or more cognitive impairments in a patient in need thereof are further described herein; (ii) methods for the treatment of dementia in a patient in need; and (iii) methods to delay the onset of dementia or one more cognitive impairment in a patient in need by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b ) donepezil or one of its pharmaceutically acceptable salts. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof may be administered separately or may be administered in the form of a composition.

Methods for the treatment of dementia include methods for the treatment of behavioral disorders associated with dementia. Illustrative behavioral alterations include sexual disinhibition, changes in activity, changes in interpersonal relationships, physical aggressiveness, physical non-aggressiveness (e.g., erratic ambulation), verbal aggressiveness, and verbal non-aggressiveness ( e.g. repetitive vocalization).

The cause or causes of impairment or cognitive impairment or dementia may be known or unknown. For example, dementia and deterioration or cognitive impairment may be caused by Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Lewy body disease, vascular diseases (e.g. ., cerebrovascular diseases), HIV, AIDS, epilepsy, brain tumors, brain lesions, multiple sclerosis, Down syndrome, Rett syndrome, progressive supranuclear paralysis, frontal lobe syndrome, schizophrenia , traumatic brain injuries (eg, closed head injuries), post-coronary artery bypass graft surgery, electroconvulsive shock therapy, chemotherapy, radiation therapy, radiation exposure, encephalitis , meningitis, fetal alcohol syndrome, Korsakoff syndrome, anoxic brain injury, cardiopulmonary resuscitation, diabetes, menopause, stroke, a high cholesterol levels, or spinal cord disorders (e.g. eg, spinal cord injury, spinal cord ischemia, spinal cord infarction, spinal cord seizures). In one embodiment, cognitive impairments may be mild cognitive impairments or cognitive impairments associated with age. For additional descriptions of cognitive impairments, dementia, and the causes of cognitive impairments and dementia, the descriptions of US Patent No. 6,458,807, United States Publication No. 2006/0018839, and WO 2005/074535 is incorporated herein by reference in its entirety.

Cognitive impairments can be cognitive impairments or canine dysfunctions (CCD), which are impairments of cognitive abilities related to the age of dogs characterized by changes in behavior that cannot be fully attributed to general medical conditions. When veterinary compositions are prepared, one skilled in the art would appreciate that the composition could contain a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein (eg, 3- (2-cyanophenyl)) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one), (b) donepezil or a pharmaceutically acceptable salt thereof, and (c) a veterinarily acceptable carrier. The dosage amount for a dog can be easily achieved based on the dosages that are acceptable to humans and the size and weight of the dog.

Methods for the treatment of a neurodegenerative disease are also described herein; and (ii) methods to delay the onset of a neurodegenerative disease in a patient in need thereof by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b) donepezil or a of its pharmaceutically acceptable salts. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof can be administered separately or can be administered in the form of a composition. The neurodegenerative disease can be any known in the art. Illustrative neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, Lewy body disease, prion diseases (eg, Creutzfeldt-Jakob disease ), multiple sclerosis, epilepsy, strokes, and the like.

Methods for the treatment of Alzheimer's disease are also described herein; and (ii) methods to delay the onset of Alzheimer's disease in a patient in need thereof by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b) Donepezil or one of its pharmaceutically acceptable salts. Alzheimer's disease can be cam, moderate, or severe. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof can be administered separately or can be administered in the form of a composition.

Methods for the treatment of Parkinson's disease are also described herein; and (ii) methods to delay the onset of Parkinson's disease in a patient in need thereof by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b) Donepezil or one of its pharmaceutically acceptable salts. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof can be administered separately or can be administered in the form of a composition.

The use of (i) methods for the treatment of Huntington's disease is also described herein; and (ii) methods to delay the onset of Huntington's disease in a patient in need thereof by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b) Donepezil or one of its pharmaceutically acceptable salts. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof can be administered separately or can be administered in the form of a composition.

Methods for the treatment of amyotrophic lateral sclerosis are also described herein; Y

(ii) methods for delaying the onset of amyotrophic lateral sclerosis in a patient in need thereof by administering a therapeutically effective amount of: (a) the 1,2-dihydropyridine compound described herein and (b) donepezil or one of its pharmaceutically acceptable salts. The 1,2-dihydropyridine and donepezil compound or a pharmaceutically acceptable salt thereof can be administered separately or can be administered in the form of a composition.

The 1,2-dihydropyridine compounds described herein, and donepezil or a pharmaceutically acceptable salt thereof can be administered orally, topically, parenterally, by inhalation (nasal or oral), or rectally in unit dosage formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral includes subcutaneous, intravenous, intramuscular, intrathecal, intrasternal injection, or infusion techniques.

The daily dose of the 1,2-dihydropyridine compounds of the invention (3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one) is usually about 30 µg to 10 grams, preferably, 100 µg to 5 grams or, more preferably, 100 µg to 100 mg in the case of oral administration. For administration by injection, the daily dose is usually about 30 µg to 1 gram, preferably 100 µg to 500 mg or, more preferably, 100 µg to 30 mg. The compound is administered once a day or in several servings a day. When used in the context of a dosage amount, the numerical weight refers to the weight of the 1,2-dihydropyridine compound of the invention, excluding any salt, counterion, hydrate, and the like. Therefore, to obtain the equivalent of 500 milligrams of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, it would be necessary to use more than 500 milligrams of one and / or pharmaceutically acceptable hydrate salt of the compound, due to the additional weight of the pharmaceutically acceptable salt and / or hydrate.

The daily dose of donepezil is usually about 0.1 milligrams to 100 milligrams, preferably 1 milligram to 50 milligrams, more preferably 5 milligrams to 25 milligrams. In another embodiment, the daily dose is 10 milligrams to 20 milligrams; or from 5 milligrams to 10 milligrams. The compounds are administered once a day or in several portions per day. When used in the context of a dosage amount, the numerical weight refers to the weight of donepezil, excluding any salt, counterion, and the like. Therefore, to obtain the equivalent of 10 milligrams of donepezil, it would be necessary to use more than 10 milligrams of donepezil hydrochloride, due to the additional weight of the hydrochloride.

In one embodiment, the mode of administration is by injection, such as subcutaneous injection, intramuscular injection, intravenous injection, or intra-arterial injection. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the technique using suitable dispersing or wetting agents, suspending agents (e.g., methylcellulose, Polysorbate 80, hydroxymethyl cellulose, acacia, tragacanth in powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate and the like), pH modifiers, buffers, solubilizing agents (e.g., polyoxyethylene hydrogenated castor oil, Polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, Macrogol, ethyl ester grade acid of castor, and the like) and preservatives. The sterile injectable preparation may also be sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, in the form of a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvent or suspending medium. For this purpose any soft fixed oil including synthetic mono- or diglycerides can be used, in addition, fatty acids, such as oleic acid, can be used in the preparation of injectables. The preparations can be lyophilized by means of methods known in the art.

Solid dosage forms for oral administration may include chewing gum, capsules, tablets, sublingual tablets, powders, granules, and gels. In such solid dosage forms, the active compound can be mixed with one or more inert diluents such as lactose or starch. As is normal practice, such dosage forms may also comprise other substances including lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. The tablets can be prepared with enteric or film coatings, preferably film coatings.

To make tablets, the compounds may be mixed with pharmaceutically acceptable carriers known in the art such as, for example, vehicles (e.g., lactose, white sugar, mannitol, glucose, starches, calcium carbonate, crystalline cellulose, silicic acid , and the like), binders (e.g., water, ethanol, miranol, glucose solution, starch solution, gelatin solution, polyvinylpyrrolidone, and the like), disintegrants

(e.g., dry starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, fatty acid esters and polyoxyethylene sorbitan, sodium lauryl sulfate, stearic acid monoglyceride, starches, lactose, and the like), absorption promoters ( eg, quaternary ammonium base, sodium lauryl sulfate, and the like), wetting agents (eg glycerin, starches, and the like), lubricants (eg, stearates, polyethylene glycol, and the like), and agents flavorings (eg, sweeteners). The tablets may be in the form of a conventional tablet, a molded tablet, a wafer and the like.

Sublingual administration refers to administration in the mouth (e.g., under the tongue, between the cheek and gum, between the tongue and the palate). The lining of the highly vascular mucosa in the mouth is a convenient location for administering the compounds in the body.

In other embodiments, the solid dosage form may be packaged in the form of granules or powder in a pharmaceutically acceptable carrier, where the granules or powder are removed from the container and sprinkled on the food or mixed with a liquid, such as water. or juice, or in which the granules are inserted into capsules. In this embodiment, the compounds described herein can be mixed with flavoring or sweetening agents. The packaging material can be plastic, coated paper, or any material that prevents water

or moisture reach the granules and / or dust.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, sublingual solutions, suspensions, and syrups containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifiers and suspending agents, and sweetening, flavoring, and perfuming agents. To make sublingual solutions, the compounds can be mixed with various carriers, excipients, pH adjustment agents, and the like (e.g., water, sugar, lactic acid, acetic acid, fructose, glucose, saccharin, polyethylene glycol, propylene glycol , alcohol, bentonite, tragacanth, gelatin, alginates, aspartame, sorbitol, methylparaben, propylparaben, sodium benzoate, artificial flavors and coloring agents).

For administration by inhalation, the compounds may be released from an insufflator, a nebulizer or a pressurized container or other convenient means of releasing a spray spray. Pressurized containers may include a suitable propellant. Alternatively, for administration by inhalation, the compounds may be administered in the form of a dry powder composition or in the form of a liquid spray.

Suppositories for rectal administration can be prepared by mixing the active compounds with suitable non-irritating excipients such as cocoa butter and polyethylene glycols that are solid at room temperature and liquid at body temperature. Alternatively, an enema can be prepared for rectal administration of the compounds described herein.

For topical administration to the epidermis, the compounds may be formulated in the form of ointments, creams or lotions, or in the form of the active ingredient of a transdermal patch. The compounds can also be administered via iontophoresis or osmotic pump. Ointments, creams and lotions can be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Alternatively, ointments, creams and lotions may be formulated with an aqueous or oily base and may also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, and / or coloring agents. As for creams or lotions, the compounds may be mixed to form a homogeneous cream or lotion, mild, for example, with one or more preservatives (e.g., 1% or 2% benzyl alcohol (w / w) ), emulsifying waxes, glycerin, isopropyl palmitate, lactic acid, purified water, sorbitol solution. Such topically administrable compositions may contain polyethylene glycol 400. To form ointments, the compounds may be mixed with one or more preservatives (eg, 2% benzyl alcohol (w / w)), petrolatum, emulsifying wax, and Tenox (II ) (e.g., butylated hydroxyanisole, propyl gallate, citric acid, propylene glycol). Woven pads or rolls of bandage material, e.g. eg, gauze, can be impregnated with transdermally administrable compositions for topical application.

The compounds may also be applied topically using a transdermal system, such as one of an adhesive polymer with an acrylic base with a resinous crosslinking agent impregnated with the compounds described herein and laminated to an impermeable support. For example, the compounds can be administered in the form of a transdermal patch, such as a transdermal sustained release patch. The transdermal patches may include any conventional form such as, for example, an adhesive matrix, a polymer matrix, a reservoir patch, a laminated matrix or monolithic structure, and generally comprise one or more support layers, adhesives, boosters penetration, and / or membranes for speed control. Transdermal patches generally have a release liner that is removed to expose the active adhesive / ingredient or ingredients before application. Transdermal patches are described, for example, in United States Patents Nos. 5,262,165, 5,948,433,

6,010,715 and 6,071,531, whose descriptions are incorporated herein by reference in their entirety.

The invention provides the compounds that are to be administered nasally to a patient for the treatment of the diseases and disorders described herein. "Nasally administered" or "nasal administration" is intended to mean that at least one compound is combined with a release system suitable for absorption through a patient's nasal mucosa. Generally, smaller doses of the compound can be used for nasal administration when compared, for example, with the dose required for oral administration.

The compounds can be administered, for example, in the form of nasal sprays, nasal drops, nasal suspensions, nasal gels, nasal ointments, nasal creams or nasal powders. The compounds can also be administered using nasal buffers or nasal sponges. The compounds can be carried to a viscous base through conventionally used systems, for example, natural gums, methylcellulose and derivatives, acrylic polymers (carbopol) and vinyl polymers (polyvinylpyrrolidone). To the compositions, many other excipients known in the art such as water, preservatives, surfactants, solvents, adhesives, antioxidants, buffers, bio-adhesives, viscosity enhancing agents and agents for adjusting pH and osmolarity can be added .

Nasal release systems can take various forms including aqueous solutions, non-aqueous solutions and combinations thereof. Aqueous solutions include, for example, aqueous gels, aqueous suspensions, aqueous liposomal dispersions, aqueous emulsions, aqueous microemulsions and combinations thereof. Non-aqueous solutions include, for example, non-aqueous gels, non-aqueous suspensions, non-aqueous liposomal dispersions, non-aqueous emulsions, non-aqueous microemulsions and combinations thereof.

In other embodiments, the nasal release system may be a powder formulation. Powder formulations include, for example, powder mixtures, powder microspheres, coated powder microspheres, liposomal dispersions and combinations thereof. Preferably, the powder formulation is powder microspheres. The powder microspheres are preferably formed from various polysaccharides and celluloses selected from starch, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, alginate, polyvinyl alcohol, acacia, chitosan, and mixtures of two or more thereof .

In certain embodiments, the particle size of the droplets of the aqueous and / or non-aqueous solution or of the powders released to the nasal mucosa may be, for example, from about 0.1 microns to about 100 microns; from about 1 micron to about 70 microns; from about 5 microns to about 50 microns; or from about 10 microns to about 20 microns. Particle sizes can be obtained using suitable containers or measuring devices known in the art. Illustrative devices include mechanical pumps in which the release is performed by movement of a piston; compressed air mechanisms in which the release is carried out by means of hand-pumped air in the container, techniques with compressed gas (e.g., nitrogen) in which the release is carried out by means of the controlled release of a gas compressed in the sealed container; techniques with liquefied propellants in which a low boiling liquid hydrocarbon

(eg, butane) is vaporized to exert pressure and force the composition through a fixed dose valve; and the like The powders can be administered, for example, in such a way that they are encapsulated in a capsule which is then placed in an inhalation or insufflation device. A needle penetrates through the capsule to make pores in the upper and lower part of the capsule and air is sent to expel dust particles. The powder formulation can also be administered in a jet spray of an inert gas or suspended in liquid organic fluids.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound dispersed in a nasal release system that improves the solubility of the compound. The nasal release system that improves solubility may include one of the following or combinations thereof: (i) a glycol derivative (eg, propylene glycol, polyethylene glycol, mixtures thereof);

(ii) a sugar alcohol (eg, mannitol, xylitol, mixtures thereof); (iii) glycerin; (iv) a glycol derivative (e.g.,

propylene glycol, polyethylene glycol or mixtures thereof) and glycerin; (v) ascorbic acid and water, (vi) sodium ascorbate and water; or (vii) sodium metabisulfite and water.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound described herein and a nasal release system, wherein the nasal release system comprises at least one buffer to maintain the pH of the compound described in the herein, at least one pharmaceutically acceptable thickening agent and at least one humectant. The nasal release system may optionally further comprise surfactants, preservatives, antioxidants, bio-adhesives, pH adjusting agents, isotonic agents, solubilizing agents, and / or other pharmaceutically acceptable excipients. The compound described herein may optionally be dispersed in a nasal release system that improves its solubility.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound described herein and a nasal release system, wherein the nasal release system comprises at least one solubilizing agent, at least one pharmaceutically acceptable thickening agent. and at least one humectant. The nasal release system may additionally comprise buffers, pH adjusting agents, isotonic agents, surfactants, preservatives, antioxidants, bio-adhesives, and / or other pharmaceutically acceptable excipients. The compound described herein may optionally be dispersed in a nasal release system that improves its solubility.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound described herein and a nasal release system, wherein the nasal release system comprises at least one buffer to maintain the pH of the compound, at least a pharmaceutically acceptable thickening agent, at least one humectant, and at least one surfactant The nasal release system may optionally further comprise pH adjusting agents, isotonic agents, solubilizing agents, preservatives, antioxidants, bio-adhesives, and / or other pharmaceutically acceptable excipients. The compound described herein may optionally be dispersed in a nasal release system that improves its solubility.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound described herein and a nasal release system, wherein the nasal release system comprises at least one pharmaceutically acceptable thickening agent, at least one humectant. , at least one surfactant, and at least one solubilizing agent. The nasal release system may additionally comprise buffers, pH adjusting agents, isotonic agents, preservatives, antioxidants, bio-adhesives, and / or other pharmaceutically acceptable excipients. The compound can optionally be dispersed in a nasal release system that improves its solubility.

Also described herein is a nasally administrable pharmaceutical composition comprising at least one compound described herein and a nasal release system, wherein the nasal release system comprises at least one buffer to maintain the pH of the compound, at less a pharmaceutically acceptable thickening agent, at least one humectant, at least one surfactant, and at least one solubilizing agent The nasal release system may additionally comprise buffers, pH adjusting agents, isotonic agents, preservatives, antioxidants, bio-adhesives, and / or other pharmaceutically acceptable excipients. The compound described herein may optionally be dispersed in a nasal release system that improves its solubility.

The buffer has a pH that is selected to optimize the absorption of the 1,2-dihydropyridine compound through the nasal mucosa. The specific pH of the buffer may vary depending on the specific nasal release formulation as well as the specific compound selected. Buffers that are suitable for use in the invention include acetate (e.g., sodium acetate), citrate (e.g., sodium citrate dihydrate), phthalate, borate, prolamine, trolamine, carbonate, phosphate (p eg, monopotassium phosphate, disodium phosphate), and mixtures of two or more thereof.

The pH of the compositions should be maintained from about 3.0 to about 10.0. Compositions that have a pH of less than about 3.0 or more than about 10.0 may increase the risk of irritation of the patient's nasal mucosa. Additionally, it is preferable that the pH of the compositions be maintained at about 3.0 to about 9.0. With respect to non-aqueous nasal formulations, suitable forms of buffering agents can be selected such that when the formulation is released into the nasal cavity of a mammal, selected pH ranges are reached there after contact with, e.g. eg, a nasal mucosa.

The solubilizing agent for use in the compositions of the invention may be any of those known in the art, such as carboxylic acids and salts thereof. Exemplary carboxylic acid salts include acetate, gluconate, ascorbate, citrate, fumarate, lactate, tartrate, maleate, maleate, succinate, or mixtures of two or more thereof.

The viscosity of the compositions of the present invention can be maintained at a desired level using a pharmaceutically acceptable thickening agent. For example, the viscosity can be at least 1000 cps; from about 1000 to about 10,000 cps; from about 2000 cps to about 6500 cps; or from about 2500 cps to about 5000 cps. Thickening agents that can be used in accordance with the present invention include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, polyvinyl alcohol, alginates, acacia, chitosan, and mixtures of two or more thereof . The concentration of the thickening agent will depend on the agent selected and the desired viscosity. Such agents can also be used in a powder formulation.

Nasally administrable compositions may also include a humectant to reduce or prevent dryness of mucous membranes and to prevent irritation. Suitable humectants that can be used include, for example, sorbitol, mineral oil, vegetable oil and glycerol; analgesic agents; membrane conditioners; sweeteners; and mixtures of two or more thereof. The concentration of the humectant will vary depending on the agent selected. In one embodiment, the humectant may be present in the nasal release system at a concentration ranging from about 0.01% to about 20% by weight of the compositions.

The nasal release system may additionally comprise surfactants that increase the absorption of the compound. Suitable surfactants include nonionic, anionic and cationic surfactants. Illustrative surfactants include oleic acid, polyoxyethylene fatty acid derivatives, partial esters of sorbitol anhydride, such as, for example, Tweens (e.g., Tween 80, Tween 40, Tween 20), Spans (e.g., Span 40, Span 80, Span 20), polyoxyl stearate 40, polyoxyethylene stearate 50, fusyanates, bile salts, octoxynol, and mixtures of two or more thereof. Illustrative anionic surfactants include long chain hydrocarbon salts

(eg, C6-C30 or C10-C20) having one or more of the following functional groups: carboxylates; sulfonates; and sulfates. Long chain hydrocarbon salts having sulfate functional groups, such as sodium ketostearylsulfate, sodium dodecyl sulfate and sodium tetradecyl sulfate, are preferred. A particularly preferred anionic surfactant is sodium lauryl sulfate (ie, sodium dodecyl sulfate). Surfactants may be present in an amount of about 0.001% to about 50% by weight, or about 0.001% to about 20% by weight

The pharmaceutical compositions of the invention may further comprise an isotonic agent, such as sodium chloride, dextrose, boric acid, sodium tartrate or other inorganic or organic solutes.

The nasal pharmaceutical compositions described herein may optionally be used in combination with an agent for pH adjustment. Illustrative pH adjustment agents include sulfuric acid, sodium hydroxide, hydrochloric acid, and the like.

To prolong its shelf life, preservatives can be added to nasally administrable compositions. Suitable preservatives that can be used include benzyl alcohol, parabens, thimerosal, chlorobutanol, benzalkonium chloride, or mixtures of two or more thereof. Benzalkonium chloride is preferably used. Generally, the preservative will be present at a concentration of up to about 2% by weight. The exact concentration of the preservative, however, will vary depending on the intended use and can easily be determined by one skilled in the art.

Other ingredients that prolong the shelf life such as, for example, antioxidants can be added. Some examples of the antioxidants include sodium metabisulfite, potassium metabisulfite, ascorbyl palmitate and the like. Generally, the antioxidant will be present in the compositions at a concentration of about 0.001% to about 5% by weight of the total composition.

Nasal release systems can be made following the procedures described, for example, in United States Patents Nos. 6,451,848, 6,436,950, and 5,874,450, and WO 00/00199, whose descriptions are incorporated herein by reference in their entirety.

Examples

The following examples are for illustrative purposes only and are not intended to limit the spirit or scope of the claims.

Example 1

The role of dopamine in cognition, emotion and motor functioning has been studied. It was reported that there was a link between age-related nigrostriatal dopaminergic deficit and age-related cognitive function deficit. Erixon-Lindrotha et al, Psychiatry Research: Neuroimaging, 138: 1-12 (2005). It was also reported that pharmacological manipulation of dopamine systems could modify cognitive performance in humans. Luciana et al, Cereb. Cortex, 8: 218-226 (1998); Kimberg et al, Neuropsychologia, 41: 1020-1027 (2003). The facilitation of dopaminergic activity could improve cognitive function in humans.

Acetylcholinesterase inhibitors are often used to treat dementia. It was reported that donepezil may increase the release of dopamine in the brain, and it was also reported that simultaneous administration with the MAO-B inhibitor selegiline increased learning ability. Giacobini et all Neuropharmacology, 35: 205-211 (1996); Takahata et al, Eur. J. Pharmacol., 518: 140-144 (2005). It was also reported that other drugs for Alzheimer's disease (eg, memantine) have some effect on dopamine replacement and are effective in combination with donepezil. Bubser et al, Eur. J. Pharmacol., 229: 75-82 (1992); Tariot et al, JAMA, 291: 317-324 (2004). These results indicated that the improvement of cognitive function by acetylcholinesterase inhibitors can be increased by compounds that have positive effects on dopaminergic activity.

C57BL / 6 mice (SLC, Shizuoka, Japan) were used for the experiment. 3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil were suspended or dissolved in a 0.5% methylcellulose solution. The two drugs were administered orally by means of gavage 1 hour before sampling. At the time of sacrificing the mice, both striated bodies were rapidly dissected and frozen with liquid nitrogen. The samples were then sonicated in 0.5 ml of frozen 0.5 M perchloric acid containing 0.1 M EDTA, and centrifuged at 13000 rpm for 15 min at 4 ° C. Monoamine levels were measured by means of HPLC with electrochemical detection. 20 µl aliquots of supernatant were separated on an Eicompak SC5-ODS column (3 x 150 mm; Eicom, Japan) with a mobile phase of 17% methanol, 83% 0.1M acetate / citrate buffer (pH 3.5 ) containing 190 mg / ml of sodium 1-octanesulfonate and 50 mg / ml of EDTA, and a flow rate of 0.65 ml / min. The peaks of dopamine, DOPAC, HVA, 5-HT, and 5-HIAA were quantified by integrating the area with PowerChrom (eDAQ, Australia), and the concentrations were expressed as nanograms per milligram of striatal tissue.

As shown in Figure 1, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (that is, Compound A) raised the HVA content , which is a metabolite of dopamine in the striatum at 1 mg / kg.

Example 2

Donepezil reduced the dopamine content significantly but the elevation of the dopamine metabolite did not reach significance. Figure 2 shows that DPAC / DA and HVA / DA, which also indicated the increase in dopamine turnover, increased significantly. Symptoms on the behavior of donepezil were observed in the experiment. Mice treated with donepezil showed peripheral side effects at 7.5 mg / kg and higher. These results indicate that monotherapy with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil monotherapy modulated dopamine replacement but none reached significance , suggesting a modest effect on the replacement of dopamine. Figure 3, however, shows that the combination of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil unexpectedly facilitated a higher dopamine turnover in mice compared to monotherapy with individual compounds. This experiment demonstrates that the combined therapy of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil (when compared to monotherapy with 3- ( 2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one or donepezil) can be used to treat cognitive dysfunctions, dementia, and neurodegenerative diseases, such as Alzheimer's disease .

Example 3

Example 5 shows the neuroprotective effects of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil. A combination of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil shows clear neuroprotective effects of excitotoxicity; However, the profile of both drugs is different. Therefore, complementary approaches may be required to maximize neuroprotective effects by combining drugs to stop the progress of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and the like. .

It has been described that neurodegenerative diseases exhibit excitotoxicity in the neurodegeneration process. Rego et al, Neurochemical Research, 28: 1563-1574 (2003). 3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one is an AMPA antagonist. AMPA antagonists have an effect on the endocytotoxic conditions induced by kainate and metabolic stress. Ohno et al, JPET, 306: 66-72 (2003); Matsuoka et al, Neuroreport, 6: 2205-2208 (1995). On the other hand, it is known that AMPA antagonists do not show protective effects or show them in a limited way against NMDA-induced toxicity. Kristensen, Brain Res., 917: 21-44 (2001).

Donepezil, commercially available as ARICEPT® (Eisai, Inc., Teaneck, NJ) is prescribed for Alzheimer's disease. Donepezil improves cognition in patients with dementia, which is caused by the inhibition of acetylcholinesterase. Donepezil exhibits neuroprotective effects against excitotoxicity induced by NMDA, but not by kainate. Akasofu et al, Eur. J. Pharmacol., 530: 215-222 (2006). The neuroprotective effects of donepezil are supported by clinical findings in cerebral atrophy of patients with Alzheimer's disease. Hashimoto et al, Am J. Psychiatry, 162: 676-682 (2005). Donepezil could be a modified disease drug for neurodegenerative diseases; however, the neuroprotective effects of donepezil are insufficient to completely stop the progression of Alzheimer's disease.

Combined therapy is sometimes beneficial but sometimes it is harmful. Therefore it is very important to confirm the safety and effectiveness of the combination. The combined therapy of the two drugs (3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil) in cultured neurons potentiated the beneficial effects against neurodegeneration induced by NMDA and kainate. Neurodegeneration was not observed due to the combination of drugs. Therefore, the combination of drugs may be a favorable option for the treatment of neurodegeneration, which leads to diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the like.

Cortical cell cultures were prepared from fetal rats of the Wistar strain (gestational age 17-19 days, Charles River Japan, Kanagawa, Japan). The cortex was dissected and kept in Neurobasal * medium ((ice-cold Neurobasal medium (Gibco BRL) containing a 2% B27 supplement ((Gibco BRL), 0.5 mM L-glutamine (Gibco BRL), liquid 2% Antibiotic-Antifungal (Gibco BRL), 25 µM 2-mercaptoethanol)) and then incubated at 37 ° C for 30 min in HBSS without Ca2 + / Mg2 + containing 0.25% trypsin (Gibco BRL) and 0, 2 mg / ml deoxyribonuclease (DNase) (Sigma, St. Louis, MO, USA) Cortical tissues were dissociated to individual cells by gentle crushing.The cell suspension was mixed with Neurobasal medium with inactivated fetal calf serum with 10% heat (Gibco BRL) The cell suspension was centrifuged at 3-10 xg for 3 min and the resulting sediments were resuspended in the medium described above.

The cells were plated at an initial cell density of 2.0 x 105 cells / cm 2 in plates with wells coated with poly-L-lysine. The cells were cultured in an incubator with 5% CO2 (v / v), at 37 ° C in Neurobasal medium containing 2% B27 supplement for 7 days.

The cultures were exposed to 200 µM NMDA (Sigma) or 200 µM kainite in neurobasal medium containing 2% B27 supplement (-AO) over 24 h at 37 ° C. The cells were treated with donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one over 24 hours before and during the exposure of these agonists.

The efflux of LDH in the culture medium was measured after the attack with NMDA or kainate. The remaining LDH in the cells was measured after lysing the cells with 0.1 M phosphate buffer (0.1 M Na2HPO4 and 0.1 M KH2PO4: pH 7.4) containing 0.5% Triton X-100 . The release of LDH as an indicator of neuronal injury was presented as the percentage loss of LDH in the culture medium, with respect to the total LDH activity per culture (measured total LDH / LDH efflux). The decrease in NADH was spectrophotometrically verified at 340 nm as a measure of LDH activity.

In the neuronal toxicity induced by kainate, donepezil showed a modest effect on neuronal survival. Figure 4 shows that the simultaneous administration of donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl1,2-dihydropyridin-2-one significantly and unexpectedly increased neuronal survival compared to donepezil alone.

In the experiment with NMDA, 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one showed effects and reached a 0.3 mM plateau. As shown in Figure 5, the simultaneous administration of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil unexpectedly and significantly increased neuronal survival.

Example 4

Cultures of organotypic hippocampal sections were prepared using the basic method of Pringle et al, Brain Research, 755: 36-46 (1997) modified as follows. Wistar rat pups (8-11 days old) were decapitated and the hippocampus was rapidly dissected in ice-cold Gey balanced salt solution with a supplement of 4.5 mg / ml glucose. Cross sections (400 µm) were cut with a McIlwain tissue cutter and placed back in ice-cold Gey balanced salt solution. Sections were separated and placed in Millicell CM culture inserts (4 per well) and kept at 37 ° C / 5% CO2 for 14 days. The maintenance medium consisted of 25% heat inactivated bone serum, 25% Hank balanced saline solution (HBSS) and 50% minimum essential medium with added Earle salts (MEM) with a 1 mM and 4 glutamine supplement , 5 mg / ml glucose. The medium was changed every 3-4 days.

The cultures were transferred to serum-free medium (75% SFM-MEM, 25% HBSS) with a 1 mM glutamine supplement and 4.5 mg / ml glucose) containing 5 µg / ml of the iodide fluorescent exclusion dye of propidium (PI), and allowed to equilibrate for 60 minutes before image formation. PI fluorescence was detected using a DMIL Leica inverted microscope equipped with a set of rhodamine filters. Any culture in which PI fluorescence was detected in this phase was excluded from further study. 3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and / or donepezil HCl was added to the SFM and the cultures were returned to the incubator for 24 hours. After this time, the cultures were exposed to a neurotoxic attack (see below for protocols) in the presence of PI and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2- dihydropyridin-2-one and / or donepezil HCl before returning them to the SFM containing 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and / or donepezil HCl Neural impairment was assessed by imaging using PI fluorescence 24 hours after the onset of the neurotoxic attack. Neurotoxicity protocols are outlined below.

(one)

 AMPA: The cultures were transferred to SFM (+ PI) of new contribution containing 30 J.1M S-AMPA for 3 hours under normal growing conditions. At the end of this period, the cultures were returned to SFM of new contribution for 21 hours before the determination of neuronal death by means of imaging with PI. GYKI52466 (30 µM) was used as a positive neuroprotective control.

(2)

 Oxygen-Glucose Deprivation (OGD): An experimental ischemia was performed by exposing the cultures to oxygen-glucose deprivation (OGD) as previously described. Pringle et al, Brain Research, 755: 36-46 (1997); Pringle et al, Stroke, 27: 2124-2130 (1996); Morrison et al, Br. J. Pharmacol., 137: 1255-1268 (2002). OGD was induced by transferring cultures to SFM (+ PI) without glucose that had been saturated with 95% N2 / 5% CO2. The culture plates (without lids) were then sealed in an airtight chamber in which the atmosphere was saturated with 95% N2 / 5% CO2 by continuously passing gas at 10 L / min for ten minutes before sealing it and place it in the incubator for 50 min. (OGD total time was therefore 60 mins). At the end of the OGD period the cultures were returned to normoxic SFM containing PI and returned to the incubator for 23 hours.

(3)

 Hypoxia: Hypoxia was performed by exposing the cultures to oxygen deprivation as previously described. Pringle et al, Brain Research, 755: 36-46 (1997); Pringle et al, Stroke, 27: 2124-2130 (1996). Hypoxia was induced by transferring the cultures to SFM (+ PI) that had been saturated with 95% N2 / 5% CO2. The culture plates (without lids) were then sealed in an airtight chamber in which the atmosphere was saturated with 95% N2 / 5% CO2 by continuously passing gas at 10 L / min for ten minutes before sealing it and place it in the incubator for 170 mins (the total hypoxia time was therefore 180 mins). At the end of the hypoxia period, the cultures were returned to normoxic SFM (+ PI) and returned to the incubator for 21 hours. The free radical scavenger MnTBAP (100 µM) was used as a positive neuroprotective control.

(4)

 NMDA: The cultures were transferred to newly contributed SFM (+ PI) containing 10 µM NMDA for 3 hours under normal culture conditions. At the end of this period, the cultures were returned to SFM of new contribution for 21 hours before the determination of neuronal death by means of imaging with PI. MK801 (10 µM) was used as a positive neuroprotective control.

(5)

 Free radical-mediated toxicity (Duroquinone): Duraquinone produces superoxide-mediated neurotoxicity producing a selective neuronal lesion in the CA1 region. Wilde et al, J. Neurochem., 69 (2): 883-886 (1997). The cultures were transferred to fresh SFM (+ PI) containing 100 µM duraquinone for 3 hours under normal culture conditions. At the end of this period, the cultures were returned to SFM of new contribution for 21 hours before the determination of neuronal death by means of imaging with PI. MnTBAP (100 µM) was used as a positive neuroprotective control.

Neural impairment was assessed using ImageJ software run on a PC. The images were captured using a monochrome CCD camera and saved for Otlline analysis. Light transmission images were captured before drug addition, and images by PI fluorescence were recorded at the end of the 24 hour recovery period. The area of region CA1 was determined from the transmission image. The area of PI fluorescence in CA1 was measured using the threshold function in ImageJ, and neuronal deterioration was expressed as the percentage of CA1 where PI fluorescence was detected above the background. Morrison et al, Br. J. Pharmacol., 137: 1255-1268 (2002).

MnTBAP and MK-801 were dissolved in distilled water at 10 mM. The NMDA was dissolved in 100 mM distilled water, and the AMPA was dissolved in 3 mM distilled water. GYKI52466 was dissolved in DMSO at 3 mM. Starting solutions (1 mM) of fresh-made donepezil were prepared each day in distilled water, and then seriously diluted in SFM. Solutions of fresh 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (1 mM) each day in DMSO were prepared and further diluted in SFM .

The materials were obtained from the following suppliers: tissue culture plates - Nunc (Fisher Scientific, Loughborough, UK); all other tissue culture plastics were purchased from - Bibby Sterilin (Stone, UK); culture media - Invitrogen (Paisley, UK); PI - Molecular Probes (Leiden, Holland); S-AMPA, GYKI52466, NMDA, MK801 and MnTBAP-Tocris (Bristol, UK); 3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, donepezil hydrochloride (Eisai Co. Ltd).

Data analysis was performed using GraphPad Prism. Data were initially evaluated to determine normality after which statistical significance was determined using a one-way analysis of variance followed by a post-hoc Dunnett test. All data are expressed as the mean ± e.t.m. The starting data and the analysis of the data sets are not provided together with them.

EC50 was determined for 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one in AMPA toxicity. No PI fluorescence was observed in the sections of cultures maintained in SFM for up to 49 hours (simulated controls). In contrast, significant neurodegeneration was observed in the CA1 region of cultures exposed to 30 µM AMPA for 3 hours (65.1 ± 1.7% deterioration in CA1, n = 104, p <0.001 vs. simulated controls). AMPA-mediated neurodegeneration was significantly attenuated by the non-competitive AMPA receptor antagonist GYKI52466, which reduced PI fluorescence to a deterioration of 14.3 ± 2.3% (n = 60, p <0.001 vs. AMPA). AMPA-induced neurodegeneration was reduced in a concentration dependent manner by means of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one. At 0.1 µM 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, no neuroprotective efficacy was observed (deterioration of 63.9 ± 2.6 %, n = 36, p = NS vs. AMPA). When the concentration of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one was increased to 0.3 µM, significant neuroprotection was observed, with a reduction in deterioration at 50.4 ± 2.5% (n = 60, p <0.01 vs AMPA). At the highest concentration of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (3 µM), the neuroprotection was almost complete (deterioration of 0, 1 ± 0.1%, n = 36, p <0.001 vs AMPA). Based on this series of experiments, it was determined that the EC50 for 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one in this AMPA toxicity model was 0.5 µM.

The effect of donepezil on AMPA-mediated toxicity was determined. Initially, a series of cultures (24 per group) was exposed to SFM or donepezil (0.1 to 10 µM) for 48 hours (the solutions were changed to those containing freshly added donepezil after 24 hours) to evaluate the potential toxicity of the compound. No PI fluorescence was observed in any culture in this experiment indicating that the compound was non-toxic in this paradigm (data not shown).

A set of additional preliminary experiments was performed in order to determine the effect of increasing the pre-incubation time (4 to 48 hours) of donepezil before exposure of the cultures to AMPA. As before, no PI fluorescence was observed in cultures maintained in SFM throughout the duration of the experiment, but a significant toxicity was observed in CA1 after exposure to AMPA (deterioration of 46.5 ± 5.0%, n = 24). This was significantly attenuated by 30 µM GYK152466 (deterioration of 21.7 ± 2.7%, n = 24, p <0.001 vs AMPA). No effect of donepezil was observed in the longer pre-incubation times. PI 55.51 ± 2.6% of CA1 (24 hours pre-incubation, n = 24, p = NS), respectively. However, when the pre-incubation time was reduced to 4 hours, a small but significant increase in AMPA-mediated toxicity was observed, with PI fluorescence observed in 57.4 ± 2.3% of CA1 (n = 24, p <0.05 vs AMPA).

No biologically significant effect was observed due to the alteration of the preincubation period; therefore, a conventional 24-hour preincubation period was chosen for the rest of the experiments based on the published evidence. Akasofu et al, Eur. J. Pharmacol., 472 (1-2): 57-63 (2003); Takada et al, Pharmacol. Exp. Ther., 306 (2): 772-777 (2003).

In a series of additional experiments, exposure of cultures to 30 µM AMPA resulted in a PI fluorescence that appeared in 62.4 ± 2.4% of the CA1 region (n = 72). This was significantly attenuated by 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one 0.5 µM, reducing the area of PI fluorescence to 33.6 ± 4.0% of CA1 (n = 24, p <0.001 vs AMPA). No donepezil neuroprotective effect was observed at any of the concentrations tested (0.1 to 10 µM). As previously observed (Figures 8 and 9), the authors of the present invention observed a small but not significant increase in AMPA-mediated toxicity in the presence of the three concentrations of donepezil (0.1 µM: 69.5 ± 2.4% of CA1; 1 µM: 70.7 ± 2.4% of CA1; 10 µM: 70.9% ± 2.8% of CA1; all groups n = 36, p = NS vs. AMPA). While the 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one 0.5 µM was significantly neuroprotective when present alone, the efficacy of 3 (2- Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one was lost when the compound was incubated simultaneously with donepezil. This loss of efficacy was statistically significant at the three concentrations of donepezil (0.1 µM donepezil + - (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one: 70 , 2 ± 1.6% of CA1; 1 µM: 62.6 ± 3.6% of CA1; 10 µM: 68.7% ± 2.4% of CA1; all groups n = 36, p <0.001 vs AMPA).

Experimental ischemia was induced by exposing the cultures to 60 minutes of oxygen-glucose deprivation (OGD). A set of initial experiments were carried out to determine the neuroprotective efficacy of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one against induced neurotoxicity by OGD. Exposure of the cultures at 60 minutes of OGD produced an important PI fluorescence in the CA1 region (deterioration of 50.6 ± 5.2%, n = 24) and this was avoided by means of the MnTBAP free radical scavenger (100 µM ) (deterioration of 0.1 ± 0.0%, n = 24, p <0.001 vs OGD). No significant neuroprotection was observed in cultures treated with the non-competitive AMPA receptor antagonist GYKI52466 (30 µM) (36.9 ± 3.6%, n = 24, p = NS). In order to evaluate the EC50 for 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, a six-point concentration-response curve was generated. Surprisingly, in cultures treated with the lowest concentration of 3- (2-cyanophenyl) -5 (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (0.1 µM), a significant increase was observed of the PI fluorescence (64.4 ± 3.6%, n = 24, p <0.05 vs OGD). There was no significant effect of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one at any other concentration evaluated (up to 3 µM).

Since a separate EC50 could not be established in this model, similar experiments were performed using 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one 0.5 µM, the EC50 for blocking AMPA toxicity.

In a second series of experiments, the effects of donepezil on OGD-induced neurotoxicity were investigated, and how it was affected by 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2 -dihydropyridin-2-one. In cultures exposed to OGD alone, PI fluorescence occurred in 44.8 ± 3.3% of the CA1 region (n = 48). As before, this was completely attenuated by 100 µM MnTBAP (0.6 ± 0.2% deterioration, n = 48, p <0.001 vs OGD), but not by 3- (2-cyanophenyl) -5- (2- pyridyl) -1-phenyl-1,2-dihydropyridine-2-one 0.5 µM (deterioration of 55.3 ± 3.1%, n = 32, p = NS). Pretreatment with donepezil (0.1 to 10 µM) had no effect on neuronal impairment (0.1 µM: impairment of 49.4 ± 2.7%; 1 µM: impairment 54.1 ± 4.7%; 10 µM: 45.3 ± 3.3%; all n = 48, p = NS vs. OGD). In cultures exposed to OGD after the simultaneous application of donepezil 1 µM and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin2-one 0.5 µM there was a small increase , but significant of the PI fluorescence (deterioration 64.3 ± 4.2%, n = 48, p <0.05 vs OGD). No effect was observed at higher concentrations of donepezil (10 µM) or lower (0.1 µM), suggesting that this effect may not be biologically relevant.

The duraquinone-induced injury was determined. Free radical-induced injury occurred through exposure of the cultures to 100 µM duraquinone over 3 hours followed by a recovery period of 21 hours. Cultures that were exposed to 100 µM duraquinone over 3 hours developed neurodegeneration in the CA1 region, resulting in a significant PI fluorescence (deterioration of 51.0 ± 3.4%, n-72). As expected, the duraquinone-induced lesion was completely abolished by the free radical scavenger MnTBAP (100 µM) (deterioration of 0.1 ± 0.0%, n = 24, p <0.001 vs duraquinone). 3- (2-Cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (0.5 µM) was not neuroprotective in this model (deterioration of 69.1 ± 4.0 %, n = 24, p = NS). Donepezil did not reduce the neuronal deterioration induced by duraquinone (0.1 µM: deterioration of 56.4 ± 4.4%; 1 µM: deterioration of 53.2 ± 4.9%; 10 µM: deterioration of 56.6 ± 4.4%, all n = 36, p = NS). Similarly, simultaneous incubation with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one and donepezil did not affect neuronal impairment.

NMDA-induced neurotoxicity was investigated. The exposure of cultures to 10 µM NMDA over 3 hours produced neurodegeneration in the CA1 region as determined by a significant increase in PI fluorescence (deterioration of 35.1 ± 2.2%, n = 72), and this was completely attenuated by the non-competitive NMDA receptor antagonist MK-801 (10 µM) (deterioration of 0.0 ± 0.0%, n = 36, p <0.001 vs NMDA). In contrast, NMDA toxicity was not affected by 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (0.5 µM) (deterioration of 29.1 ± 2.9%, n = 36, p = NS). Donepezil was significantly neuroprotective at the three concentrations tested in a concentration-dependent manner (0.1 µM: deterioration of 23.0 ± 2.4%, p <0.01 vs NMDA; 1 µM: deterioration of 25 , 2 ± 2.3%, p <0.05 vs NMDA; 10 µM: deterioration of 14.5 ± 2.2% p <0.001 vs NMDA; all groups n = 36). The neuroprotective efficacy of low concentrations of donepezil was attenuated by the inclusion of 0.5 µM 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one ( 0.1 µM + 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one: deterioration of 27.9 ± 2.6%; 1 µM + 3- ( 2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one: deterioration of 29.5 ± 3.0%; both groups n = 36). 10 µM donepezil remained neuroprotective in the presence of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one (deterioration of 24.9 ± 2.4% , n = 36, p <0.05 vs NMDA).

Hypoxia-induced neurodegeneration was examined. Hypoxia-mediated neuronal deterioration occurred by exposing the cultures to oxygen deprivation over a period of 3 hours followed by a recovery period of 21 hours. 3-hour hypoxia resulted in a CA1 lesion, as defined by PI fluorescence (deterioration of 45.1 ± 3.5%, n = 96). This was avoided by the free radical scavenger MnTBAP (100 µM) (0.2 ± 0.1% deterioration, n-48, p <0.001 vs hypoxia). Donepezil (0.1 to 10 µM) was highly neuroprotective in this model producing a significant reduction of neuronal deterioration to 0.1 µM (deterioration of 31.7 ± 4.6%, n = 48, p <0.05 vs hypoxia) The neuroprotective efficacy increased when the concentration of donepezil increased to 1 µM (deterioration of 14.9 ± 3.2%, n = 48, p <0.00 1 vs hypoxia). A significant increase in efficacy was not observed when the donepezil concentration was increased to 10 µM (deterioration of 12.9 ± 2.9%, n = 48, p <0.001 vs hypoxia). A concentration-dependent neuroprotective effect of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one was also observed, with a significant efficacy observed at 0.1 µM ( impairment of 24.8 ± 6.3%, n = 24, p <0.001 vs hypoxia). At the highest concentration tested (3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one 5 µM), the compound was almost as effective as MmTBAP, reducing deterioration at 3.9 ± 1.8% (n = 24, p <0.001 vs hypoxia).

A second group of experiments was conducted to investigate whether the neuroprotective effects of donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one were synergistic or additives In order to test the hypothesis, the lowest effective concentrations (i.e. 0.1 µM) of each of the two compounds were used). Hypoxia alone resulted in a deterioration of 36.4 ± 3.9% (n = 48), and this was avoided by MnTBAP (100 µM) (0.0 ± 0.0%, n = 24, p <0.001 vs hypoxia). Neither donepezil (0.1 µM: deterioration of 30.4 ± 3.9%, n = 48) or 3 (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine -2-one (0.1 µM: deterioration of 36.7 ± 4.2%, n = 48, p = NS) were neuroprotective in this series of experiments. Similarly, no reduction in PI fluorescence was observed in cultures incubated with both compounds together (deterioration of 33.8 ± 4.1%, n = 48, p = NS).

It is likely that 0.1 µM is very close to the threshold concentration of neuroprotective efficacy for donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2- ona. The experiment was repeated using 0.5 µM donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one 0.5 µM. In this experiment, the exposure of the cultures to hypoxia resulted in a deterioration of 36.9 ± 4.4% (n = 48) and this was blocked with 100 µM MnTBAP (deterioration of 0.8 ± 0.4%, n = 24, p <0.001 vs hypoxia). Donepezil (0.5 µM) produced a significant decrease in neuronal deterioration (deterioration of 25.3 ± 3.6%, n = 48, p <0.05 vs hypoxia), an effect of the same nature as 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one (0.5 µM) (deterioration of 27.1 ± 3.5%, n = 48, p <0 , 05 vs hypoxia). A similar neuroprotection level was observed when donepezil and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2-one were used in combination (impairment of 24.7 ± 3 , 5%, n = 48, p <0.05 vs hypoxia).

Each of the patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

It will be apparent to one skilled in the art that various modifications to the invention can be made without departing from the spirit or scope of the appended claims.

Claims (8)

one.

 A compound for use in combination with donepezil or a pharmaceutically acceptable salt thereof in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient; wherein the compound is 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of a of its pharmaceutically acceptable salts.

2.

 A compound for use in combination with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient; where the compound is donepezil or one of its pharmaceutically acceptable salts.

3.

 The compound of claim 1 or 2, wherein donepezil or its pharmaceutically acceptable salt, and 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, its salt Pharmaceutically acceptable, its hydrate or the hydrate of one of its pharmaceutically acceptable salts are (a) administered separately to the patient or (b) administered to the patient in the form of a pharmaceutical composition.

Four.

 The compound of any one of the preceding claims wherein the dementia is caused by Alzheimer's disease, Parkinson's disease, or Huntington's disease.

5.

 The use of 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the manufacture of a medicament for use in combination with donepezil or one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient.

6.

 The use of donepezil or one of its pharmaceutically acceptable salts in the manufacture of a medicament for use in combination with 3- (2-cyanophenyl) -5- (2-pyridyl) -1-phenyl-1,2-dihydropyridine-2- one, one of its pharmaceutically acceptable salts, one of its hydrates or a hydrate of one of its pharmaceutically acceptable salts in the treatment of (i) Alzheimer's disease in a patient or (ii) dementia or one or more cognitive impairments in a patient.

7.

 The use of claim 5 or 6, wherein donepezil or its pharmaceutically acceptable salt, and 3- (2-cyanophenyl) 5- (2-pyridyl) -1-phenyl-1,2-dihydropyridin-2-one, its Pharmaceutically acceptable salt, its hydrate or the hydrate of one of its pharmaceutically acceptable salts are (a) for administration separately to the patient or (b) for administration to the patient in the form of a pharmaceutical composition.

8.

 The use of any one of claims 5 to 7, wherein the dementia is caused by Alzheimer's disease, Parkinson's disease, or Huntington's disease.