Patent Application: US-23561302-A

Abstract:
disclosed is a pharmaceutical composition for the treatment of cognitive symptoms caused by alzheimer &# 39 ; s disease in a mammal . this composition comprising vitamin e , quercitin , caffeic acid , nicotinic acid or derivatives and / or analog thereof , and a pharmaceutically acceptable excipient .

Description:
the present invention relates to a pharmaceutical composition which comprises a combination of antioxidants selected from the group consisting of vitamin e , quercitin , nicotinic acid and caffeic acid . other antioxidants such as selegiline , idebenone , oestrogens , antlinflammatory products , ginkgo bilova , ascorbic acid , beta carotene , melatonin , coenzyme q and phenolic compounds can also be used in the pharmaceutical composition . according to the present invention , it is possible to use from 0 . 1 to 100 mg of vitamin e , quercitin , nicotinic acid and caffeic acid and from 500 to 2500 ui of vitamin e . in a preferred embodiment , the following dose is used : 50 mg of quercitin ; 50 mg of caffeic acid ; 50 mg nicotinic acid and 1000 ui 264 of vitamin e . preferably , rrr - α - tocopherol ( d - alpha tocopherol ) is used as the vitamin e ; quercitin dihydrate ( 3 , 3 ′, 4 ′ 5 , 7 pentahydroxyflavone . is used as the quercitin ; and ( 3 , 4 - dihydroxycinnamic ) acid is used as the caffeic acid . no preferred form of nicotinic acid is used . the pharmaceutical composition of the present invention is best administered to a patient in the solid form such as in a capsule . in the solid form , vitamin e is more stable ( to temperature and light ). in liquid form , vitamin e is less stable and its life time is shorter . furthermore , in this later form , the efficacy of vitamin e cannot be guaranteed for a long period . furthermore , in the liquid form , vitamin e may be insoluble when mixed with other antioxydants or chemical compounds . vitamin e is an essential nutrient that functions as an antioxidant in the human body . it is essential , by definition , because the body cannot manufacture its own vitamin e and thus it must be provided by foods and supplements . vitamin e is a generic term that includes all entities that exhibit the biological activity of α - tocopherol . in nature , eight substances have been found to have vitamin e activity . they are defined by four tocopherols and four tocotrienols . the tocopherols are alpha - tocopherol , beta - t ocopherol , gamma - tocopherol and delta - tocopherol . the tocotrienols are the alpha - tocotrienol , beta - tocotrienol , gamma - tocotrienol and delta - tocotrienol . the vitamin e derivatives or related compound and analogs are potent antioxidants that block the lipid peroxidation by donation of hydrogen to the peroxidated lipids . the central nervous system is especially vulnerable to lipid peroxidation . it has further been observed that vitamin e blocks the neurotoxic effects of the free radicals produced by excitotoxicity and it improves the performance altered by old age . thus according to the present invention , any chemical entities exhibiting the biological activity of α - tocopherol is meant to encompassed by the definition of vitamin e . alternate names for vitamin e include alpha tocopherol , d - alpha - tocopherol , d - beta - tocopherol , d - delta - tocopherol , d - gamma - tocopherol , d - tocopherol , dl - alpha - tocopherol , dl - tocopherol , mixed tocopherols , tocopheryl acetate , tocopheryl succinate . other vitamin e related compounds may also be used in the context of the present invention . they may be selected from the group consisting of vitamin e acetate , dl - α - tocopherol succinate , d - alpha - tocopherol acetate , (+)- alpha - tocopherol , mixed isomers thereof , (+)- alpha - tocopherol and mixed isomers , (+)- alpha - tocopherol acid succinate , (+)- alpha - tocopherol acetate , (+)- alpha - tocopherol , (±)- alpha - tocopherol nicotinate , (±)- alpha - tocopherol acetate , (±)- alpha - tocopherol , dl - α - tocopherol acetate , (±)- alpha - tocopherol phosphat e disodium salt , (+)- α - tocopherol , (±)- 6 - hydroxy - 2 , 5 , 7 , 8 - tetramethylchromane - 2 - carboxylic acid , ( r )- trolox ™ methyl ether , ( s )- trolox ™ methyl ether , 2 -( alpha - d - glucopyranosyl ) methyl - 2 , 5 , 7 , 8 - tetramethylchroman - 6 - ol , 2 , 2 , 5 , 7 , 8 - pentamethyl - 6 - chromanol ( pmc ), 2 , 3 - dihydroxy - 3 , 3 - enono - 1 , 4 - lactone and chromone residues , α - tocophenol hydroquinone , α - tocophenol quinone , vitamin ce ( 5a - tocopheryl ascorbate ) and 2 -( α - d - glucopyranosyl ) methyl - 2 , 5 , 7 , 8 - tetramethyl chroman - 6 - ol . the vitamin e analogs may be selected from the group consisting of : ascorbic acid , beta - caroten , butylated - hydroxy - toluene , butyated - hydroxy - anisol , calcium citrate , canthaxanthin , melatorin , nordihydroguaiaretic acid , propyl gallate , selenium , silymarin , sulfur dioxide , thioctic acid . the acetate and succinate derivatives of the natural tocopherols have vitamin e activity , as do synthetic tocopherols and their acetate and succinate derivatives . of these , d - alpha - tocopherol ( rrr - alpha - tocopherol ) has the highest bioavailability and is the standard against which all the others must be compared . natural and synthetic vitamins e are not equivalent in composition , structure and / or bioavailability . natural vitamin e ( rrr - alpha - tocopherol or di - alpha - tocopherol ) is a single entity . synthetic vitamin e ( al l - rac - alpha - tocopherol or di - alpha tocopherol ) is a mixture of eight stereoisomers in equal amounts . as mentioned above , vitamin e is recognized to be the major antioxidant in lipid body tissues and the primary defence against lipid peroxidation - neutralizing free radicals , terminating chain reactions and limiting free radical / oxidative damage . vitamin e is particularly important in tissues that contain relatively high levels of polyunsaturated fatty acid ( brain and central nervous system ) and in those that are in contact with oxygen ( lung ), providing protection for microsomes and mitochondria . in rats and in cell cultures it slows down damage and neuronal death even in cells with amyloid deposit . in cell cultures and in animals , it reduces neurotoxicity and neuronal death . the hydrogen peroxide and the free radicals produced by beta a4 to cultured neurons and endotelial cells is blocked by vitamin e and other antioxidants . thus , vitamin e reduces beta a4 induced cell death in hippocampal cell cultures . vitamin e also improves cognitive performance in aged animals and reduces degeneration of hippocampal cells following cerebral ischemia . vitamin e status in ea is controversial . a study found increased vitamin e levels suggesting a possible compensatory response to oxidative stress , another found no difference with controls , and another found decrease of vitamin e in ad . the range of daily dose goes between 200 and 3 , 000 ui , with an average range of 400 to 1 , 000 ui . adverse symptoms are uncommon : cataract , haemorrhage risk in patient with vitamin k deficiency , and syncope . it would have protective effects on the immunologic response and cardiac diseases . with high doses ( 3000 iu ), appear indigestion , gastric distress , diarrhea and severe cramps . in a multicenter comparative double blind study of 2 years of duration with selegiline ( 10 mg daily ), vitamin e and their combination , with 341 patients , significantly positive results were obtained with the vitamin e ( 2 , 000 ui / daily ) and the selegiline in single - agent therapy ; but not with their combination . a retarded cognitive deterioration of 25 % was observed for both drugs , as well as the delay in institutionalisation of the patients due to an improvement of the global function . caffeic acid also known as 3 -( 3 , 4 - dihydroxyphenyl )- 2 - propenoic acid , found in many fruits , vegetables , seasonings and beverages consumed by humans , principally in conjugated forms such as chlorogenic acid . its action mechanism is not completely known , but the caffeic acid would have a beneficial effect in atherosclerosis , inflammation , neurodegenerative dysfunctions and acquired immunodeficiency syndromes . equivalents of such a compound may be selected from the group consisting of 5 ( 4 )-( 2 - carboxyethenyl )- 1 , 2 - dihydroxybenzene ; 4 -( 2 ′- carboxyvinyl )- 12 - dihydroxybenzene ; 3 , 4 - dihydroxybenzeneacrylic acid ; 3 , 4 dihydroxycinnamic acid ; 3 -( 3 , 4 - dihydroxyphenyl ) propenoic acid ; 3 , 4 - dihydroxyhydrocinnamic acid ; o - coumaric acid ; p - coumaric acid ; and caffeic acid phenethyl ester . the effects of phenolic compounds like the caffeic acid , present in the olive oil , over the oxidation of the low density protein ( ldl ) have been investigated . this process plays an important role in atherosclerosis , through the alteration of intra - cell signals in the vessel wall . the antioxidant effect of the caffeic acid is protective . when 0 . 025 - 0 . 3 mg / l of caffeic acid is added to isolated ldl , its time of oxidation is prolonged in a dose dependent mechanism . recently the action of the caffeic acid has been studied compared with that of the n - acetylcystein , that of the acetate d - alpha - tocopherol , and that of the ascorbic acid in the modulation of signal transduction related with the apoptosis induced by the ceramide . the ceramide acts as a second messenger in signal transduction produced by the stress or extracellular agents . it was observed that the caffeic acid inhibits significantly , in comparison with the other compounds , the activity of the nf - kappa b binding induced by ceramide and the apoptotic response by its antioxidant effects . another hypothesis on its action mechanism , apart from its antioxidant effect , is the inhibition of the protein tyrosine kinase activity , and this would inhibit the apoptosis . niacin and niacinamide function in the biochemistry of humans and other organisms as components of the two coenzymes : nicotinamide adenine dinucleotide ( nad ) and nicotinamide adenine dinucleotide phosphate ( nadp ). these operate in many enzyme - catalysed oxidation and reduction reactions . the deficiency state in humans causes skin disease , diarrhea , dementia , and ultimately death . lean meats , peanuts and other legumes , and whole - grain or enriched bread and cereal products are among the best sources of niacin . nicotinic acid and nicotinamide , commonly called niacin , are the dietary precursors for nad (+) ( nicotinamide adenine dinucleotide ), which is required for dna synthesis , as well as for the activity of the enzyme poly ( adp - ribose ) polymerase - 1 for which nad (+) is the sole substrate . this enzyme is highly activated by dna strand breaks during the cellular genotoxic stress response and is involved in base excision repair . in vitro as well as animal studies indicate that niacin deficiency increases genomic instability especially in combination with genotoxic and oxidative stress . studies suggest that nicotinamide can be considered as a potent antioxidant capable of protecting the cellular membranes in brain , which is highly susceptible to prooxidants , against oxidative damage induced by reactive oxygen species ( ros ). in animal studies , it had been observed that nicotinamide showed significant inhibition of oxidative damage induced by ros in rat brain mitochondria . in a study with the tertiary butylhydroperoxide -( t - buooh ) treated mouse was used as a model to study the oxidative stress that is associated with various neurodegenerative diseases . the results directly implicate dna damage in apoptosis and necrosis . nicotinamide was able to prevent dna fragmentation induced by low - dose t - buooh . other authors observed that nicotinamide is a robust neuroprotective agent against ischemia / reperfusion - induced brain injury in rats , even when administered up to 2 hours after the onset of stroke : nicotinamide improved both anatomic and functional indices of brain damage . nicotinic acid also known as pyridine 3 - carboxylic acid and analogs thereof may be used in the combination of the present invention for the treatment of ad . to this day , more than 233 different compounds of nicotinic acid are known . each of them may be used in the context of the present invention . more particularly , the nicotinic acid compounds may be selected from the group consisting of : nicotinic acid , isonicotinic acid , 3 - pyridinecarbonitrile , 2 , 4 , 5 , 6 - tetrachloro - 3 - pyridinecarboxylic acid , 2 , 6 ,- dichoro - 5 - fluoro - 3 - pyridinecarboxylic acid , 2 - chloro - 6 - methylnicotinic acid , 5 - bromonicontinic acid , arecoline hydrobromide , no - 711 hydrochloride and nicotinic acid ethyl ester , nicotinic acid ( 1 - 4 - bromo - phenyl )- propylidene )- hydrazide , nicotinic acid ( 1 -( 4 - pentyl - phenyl - ethylidene )- hydrazide , nicotinic acid ( 1 - benzyl - propylidene )- hydrazide , nicotinic acid ( 1 - methyl - 5 - nitro - 2 - oxo - 1 , 2 - dihydro - indol - 3 - ylidene )- hydrazide , nicotinic acid ( 1 - thiophen - 2 - yl - ethylidene )- hydrazide , nicotinic acid ( 2 , 4 - dichloro - benzylidene )- hydrazide , nicotinic acid ( 2 - chloro - benzylidene )- hydrazide , nicotinic acid ( 2 , 4 - dihydroxy - benzylidene )- hydrazide , nicotinic acid ( 2 , 4 - dimethoxy - benzylidene )- hydrazide , nicotinic acid ( 2 - bromo - 3 - phenyl - allylidene )- hydrazide , nicotinic acid ( 2 - hydroxy - benzylidene )- hydrazide , nicotinic acid ( 2 - methyl - 3 - phenyl - allylidene )- hydrazide , nicotinic acid ( 2 - trifluoromethyl - benzylidene )- hydrazide , nicotinic acid ( 3 , 4 , 5 - trimiethoxybenzylidene )- hydrazide , nicotinic acid ( 3 , 5 - di - tert - butyl - 4 - hydroxy - benzylidene )- hydrazide , nicotinic acid ( 3 , 5 - dibromo - 2 - hydroxy - benzylidene )- hydrazide , nicotinic acid ( 3 - ethoxy - hydroxy - benzylidene )- hydrazide , nicotinic acid ( 3 - nitro - benzylidene )- hydrazide , nicotinic acid ( 3 - phenyl - allylidene - hydrazide , nicotinic acid ( 4 - bromo - benzylidene )- hydrazide , nicotinic acid ( 4 - chloro - benzylidene )- hydrazide , nicotinic acid ( 4 - dimethylamino - benzylidene )- hydrazide , nicotinic acid ( 4 - hydroxy - benzylidene )- hydrazide , nicotinic acid ( 4 - isopropyl - benzylidene )- hydrazide , nicotinic acid ( 4 - methyl - benzylidene )- hydrazide , nicotinic acid ( 4 - nitro - benzylidene )- hydrazide , nicotinic acid ( 5 - indanylmethylene )- hydrazide , nicotinic acid n ′-( 1 , 1 - dioxo - tetrahydro - thiophen - 3 - yl )- n ′- phenyl - hydrazide , nicotinic acid n ′-( 4 - methoxy - benzoyl )- hydrazide , nicotinic acid n ′- phenoxyacetyl - hydrazide , nicotinic acid naphthalen - 1 - ylmethylene - hydrazide , nicotinic acid 0 - tolyl ester , nicotinic acid p - tolyl ester , nicotinic acid pyridin - 3 - ylmethylene - hydrazide , nicotinic acid thiophen - 2 - ylmethylene - hydrazide , 1 - bicyclo ( 2 . 2 . 1 ) hept - 2 - yl - ethylamine , nicotinic acid benzo ( 1 , 3 ) dioxol - 5 - ylmethylene - hydrazide , nicotinic acid benzylidene - hydrazide , nicotinic acid fluoren - 9 - ylidene hydrazide , nicotinic acid ( 4 - diethylamino - benzylidene )- hydrazide , nicotinic acid , 1 , 2 - diphenylethylammonium salt , nicotinic acid , 2 - amino 1 -( 4 - nitro - phenyl )- ethanol , nicotinic acid , octadecylamine salt , nicotinic acid ( 2 , 4 - dimethoxy - benzylidene )- hydrazide , nicotinic acid ( 2 - bromo 3 - phenyl - allylidene )- hydrazide , nicotinic acid ( 2 - chloro - benzylidene ) hydrazide , nicotinic acid hydroxamate , nicotinic acid mononucleotide , nicotinic acid n - oxide , 1 - methylnicotinamide chloride salt , 1 - methylnicotinamide iodide salt , and nicotinic acid adenine dinucleotide sodium salt . isonicotinic acid salts may also be used in the combination of the present invention for the treatment of ad . to this day , 86 different compounds of isonicotinic acid salts are known . more particularly , these may be selected from the group consisting of : isonicotinic acid (( 4 - nitro - phenyl )- phenyl - methylene )- hydrazide ; isonicotinic acid ( 1 - allyl - 2 - oxo - 1 , 2 - dihydro - indol - 3 - ylidene ) hydrazide ; isonicotinic acid ( 1 - benyl - 2 - oxo - 1 , 2 - dihydro - indol - 3 - ylidene ) hydrazide ; isonicotinic acid ( 1 - benzyl - propylidene )- hydrazide ; isonicotinic acid ( 1 - methyl - 1h - pyrrol - 2 - ylmethylene )- hydrazide ; isonicotinic acid ( 1 - methyl - 2 - oxo - 1 , 2 , dihydro - indol - 3 - ylidene ) hydrazide ; isonicotinic acid ( 1 - methyl - 2 - oxopropylidene )- hydrazide ; isonicotinic acid ( 1 - p - tolyl - ethylidene )- hydrazide ; isonicotinic acid ( 1 - phenyl - ethylidene )- hydrazide ; isonicotinic acid ( 1h - indol - 2 - ylmethylene )- hydrazide ; isonicotinic acid ( 2 , 3 , 4 , 6 - tetramethyl - benzylidene )- hydrazide ; isonicotinic acid ( 2 , 4 - dihydroxy - benzylidene )- hydrazide ; isonicotinic acid ( 2 , 5 - dimethoxy - benzylidene )- hydrazide ; isonicotinic acid ( 2 - chloro - benzylidene )- hydrazide ; and 1 - oxy - isonicotic acid methyl ester . quercetin is a member of a group of naturally occurring compounds , the flavonoids , which have common flavone nucleus composed of two benzene rings linked through a heterocyclicpyrone ring . quercetin is found in various plants , food products , and dyes of natural origin . the estimated average daily intake of quercetin by an individual in the united states is 25 mg . the food and drug administration nominated quercetin for toxicity and carcinogenicity studies in the rat because it is a chemical that is widely distributed in foods . quercetin was administered to rats by dosed feed since human exposure is by dietary consumption . the quercetin derivatives and / or analogs that may also be used in the combination of the present invention may be selected from the group consisting of flavone , 3 , 3 ′, 4 ′, 5 , 7 - pentahydroxy - 4h - 1 - benzopyran - 4 - one ;. 2 -( 3 , 4 ,- dihydroxypehnyl )- 3 , 5 , 7 - trihydroxy - 4h - 1 - benzopyran - 4 - one ; 3 , 3 ′, 4 ′, 5 , 7 ,- pentahydroxyflavone ; 3 , 5 , 7 , 3 ′, 4 ′- pentahydroxyflavone ; 2 -( 3 , 4 - dihydroxyphenyl )- 3 , 5 , 7 - trihydroxy - 4h - 1 - benzopyran - 4 - one ; 3 , 5 , 7 , 3 ′, 4 ′- pentahydroxyflavone ; 3 ′, 4 ′, 5 , 7 - tetrahydroxyflavan - 3 - ol ; 2 -( 3 , 4 - dihydroxyphenyl )- 4h - 1 - benzopyran - 4 - one ; cyanidelonon 1522 ; c . i . natural yellow 10 ; c . i . natural yellow 10 & amp ; 13 ; c . i . natural red 1 ; c . i . 75670 ; meletin ; quercetine ; quercetol ; quercitin ; quertine ; sophoretin ; t - gelb bzw . grun 1xanthaurine ; and nci - c60106 . quercetin dihydrate analogs may be selected from the group consisting of : quercetin dihydrate ; (±)- taxifolin ; fisetin ; quercetin - 3 - rhamnoside ; quercetin - 3 - d - xyloside ; quercetin - 3 - d - galactoside ; quercitrin ; rutin trihydrate ; morin ; and morin hydrate . as mentioned earlier other antioxydant substances may be used in the context of the present invention . for instance , ginkgo biloba is known for its antioxydant caracteristic for reducing oxidative stress or damage in the brain of ad patients . the ginkgo biloba extract , obtained from the leaf of a tree of the same name , is a vegetable extract used in europe to alleviate the symptoms associated with cognositive disorders . recently its use has been approved in germany for the treatment of dementia . its action mechanism on the central nervous system is only partially known , but its main effects would be related with its antioxidant capacity , diminishing the oxidative stress or damage detected in the brain of patients with alzheimer &# 39 ; s disease . for that purpose , its active principle , characterized as egb 761 , would develop a synergistic action of flavonoids , terpenoids and organic acids that constitute it , that would act synergistically in various processes such as the inflammation homeostasis and the oxidative stress , protecting the membrane and the modulation of the neurotransmission . hofferberth et al . studied 36 patients with typical symptoms of psychotic organic syndromes . these patients were divided in two groups , one treated with placebo and the other with egb 761 , 120 mg / day , during a period of 8 weeks . at the beginning and at the end of the treatment , the patients were subjected to a quantitative electroencephalogram , saccadic eye movements and a psychometric test . four weeks after the beginning of the treatment , a significant difference could already be observed that persisted until the end of it . kanowski et al . performed a multicenter study with 216 patients during 24 weeks divided in a placebo group and a group treated with 240 mg of egb 761 . of the total enrolled patients , 156 completed the protocol and , according to fisher &# 39 ; s statistical test , significant improvement was achieved ( p & lt ; 0 . 005 ) in the treated group . lebars et al . have recently made an important study taking into account the number of patients enrolled and the duration of the treatment . it was a multicenter study with 309 patients of both sexes over 45 years with duration of 52 weeks . in the selection of the cases , dementia was mild to moderate with a score of 9 to 26 carried out according to mini - mental - test - examination ( mmse ) and 3 to 6 points according to the clinical global impression of change , cgic scale . the treatment did not show detectable differences in the cgic , but there was a cognositive significant improvement in the alzheimers disease assessment scale [ adas ]. a meta - analysis comparing the cholinesterase inhibitors of the tacrine , rivastigmine and metrifonate and the egb 761 , based on published double blind studies of at least 6 months of duration , shared similar effectiveness of both types of compounds evaluated with the adas in ad from mild to moderate . future investigations are required to specify these mechanisms in connection with the egb 761 , in order to explore the potential of this vegetable extract . the effectiveness of the following combination referred to as cp o8t was evaluated : it was compared to placebo , according to the following design : a double blind comparative trial with cross - over design was used . patients were divided in 4 groups : group 1 : patients 1 to 5 : they took cpo1p ( which corresponds to a placebo cocktail ) during 180 days . group 2 : patients 6 to 9 took cpo1p during 90 days and cpo8t during 90 days . group 3 : patients 10 to 18 : they took cpo8t during 180 days . group 4 : patients 19 to 22 : they took cpo8t during 90 days and cpo1p during 90 days . 22 patients with probable alzheimer &# 39 ; s disease were included according to the criteria of the dsm iv ( diagnostic and statistical manual mental of disorders ) and of the nincds - ardra national institute of communicative disorders and stroke - alzheimer &# 39 ; s disease and related disorder association ). the selected patients were of both sexes , with ages between 50 and 90 years . they presented a mild to moderate degree of the disease , which was evaluated with the mental mini state of folstein : they had a score from 10 to 24 points . the computed axial tomography ( cat ) or the nuclear magnetic resonance ( nmr ) did not show infection evidence , infarct or other focal lesions previous to the 12 months of the beginning of the study . the patients who presented any of the following items were excluded : all patients and their relatives gave their formal written consent to participate in the study . the cocktail was administered to the patients . the cocktail was given in person to them , and they were reminded by telephone to take the compounds 3 times a week during the 180 days of the study . the cognitive subscale of the scale adas ( alzheimer &# 39 ; s disease assesment scale ) was taken previous to the administration of the drug , and 3 and 6 months after the beginning of its administration to evaluate the changes of the cognitive symptoms . the cognitive subscale was then taken again at 9 months , that is to say , 3 months after the interruption of the administration of the compounds . 1 . mini mental state ( folstein et al , 1975 ): evaluates orientation , memory , attention , concentration , possibility to name objects , repetition , understanding , ability to make a sentence and to copy two polygons in intersection . the lesser cognitive performance , the lower score . the maximum performance is 30 . 2 . alzheimer &# 39 ; s disease assessment scale ( adas ) cognitive subsc ale ( rosen , mohs et al , 1984 ): evaluates memory , attention , reasoning , language , orientation and praxis . the lesser cognitive performance the higher score . maximum severity is 70 . 3 . hamilton depression scale of 21 items : evaluates mood , feeling of guilt , suicide , insomnia , work and activities , inhibition , agitation , anxiety , somatic symptoms , perspicacity , diurne variation , paranoid symptoms and obsessive - compulsive symptoms . clinical interviews were carried out previous to the beginning of the study , and then quarterly during the following 9 months . complete blood and urine tests were made at the beginning of the study and then quarterly during the following 9 months . [ 0161 ] patient division according to medication distribution group 1 : cp01p / cp01p n = 5 group 2 : cp01p / cp08t n = 4 group 3 : cp08t / cp08t n = 9 group 4 : cp08t / cp01p n = 4 evaluation at different periods t 0 m before the treatment t 3 m 3 months t 6 m 6 months t 9 m 9 months ( 3 months after end of treatment ) patients that did not present results from any tests were eliminated from the research . because of this , the groups of treated individuals are not balanced and therefore the means of the groups are used instead of using individual patent date . the statistical study used analysis of variance ( anova ) using randomised block design ( rbd ). the rbd allowed to distinguish possible differences between blocks and columns . the block is the random factor . each block is an individual , or in this case in working with means , a homogeneous group of individuals , who undergo different treatments or different methods or evaluation . therefore , the research consisted in determining if significant differences existed between the means obtained in the different groups under different treatments and between different time periods of evaluation . t 0 m t 3 m t 6 m t 9 m group 1 yij yl . group 2 group 3 group 4 yj . y .. yij is each data , in the case working with means it is considered the mean of each group in each period of time ( box ) s of v f df ss ms f between the blocks i − 1 j . sumyl . 2 − ny .. 2 sc ebl / gl ebl cm ebl / cm d f i − 1 , gld , 0 , 05 between columns j − 1 i . sumyj . 2 − ny .. 2 sc ecol / gl ecol cm ecol / cm d fj − 1 , gld , 0 , 05 error or inside ( i − 1 )( j − 1 ) sc 1 − ( sc ebl + sc ecol ) sc d / gl d total n − 1 sumyij 2 − ny .. 2 the result is significant with 95 % certainty when f & gt ; statistical result of f or , what is the same when p & lt ; 0 , 05 . the rbd is an additive model as long as there is no interaction between the blocks and the columns . to make sure of this , each case was tested for non additivity of tukey . on the other hand , when significant differences are obtained between blocks or between columns in the anova , contract studies must be carried out to determine between which , of the blocks or the columns , these differences are produced . the contrasts oppose the blocks together or the columns together , and establish the origin of the differences . contrasts of scheffe , newman keuls and lsd were used . [ 0173 ] table of means t 0 m t 3 m t 6 m t 9 m mean yl . group 1 41 . 000 44 . 000 42 . 500 45 . 750 43 . 313 group 2 40 . 800 36 . 800 37 . 800 41 . 800 39 . 300 group 3 40 . 444 36 . 667 39 . 889 40 . 444 39 . 361 group 4 40 . 250 36 . 750 38 . 750 39 . 750 38 . 125 mean y . j 40 . 624 38 . 304 39 . 235 41 . 936 40 . 025 [ 0174 ] s of v df ss ms f f groups 3 61 . 5361236 20 . 5120412 7 . 81933039 & gt ; 3 . 88 s ( blocks ) ( p & lt ; 0 . 05 ) periods 3 30 . 3659336 10 . 1286445 3 . 88110857 − 3 . 66 ( p − 0 . 05 ) 9 23 . 6092301 2 . 62324779 total 15 115 . 531287 the anova reveals significant differences ( p & lt ; 0 , 05 ) between the groups or blocks . that is to say between the different treatments as for the average scores obtained for the adas test . however , the differences were not as significant for the different times of evaluation . the different groups were compared to evaluate how their averages differentiate in the adas test group 1 group 2 group 3 group 4 group 1 0 . 12010232 0 . 12742966 0 . 0348689 group 2 0 . 120102316 0 . 99998283 0 . 89121008 group 3 0 . 127429664 0 . 99998283 0 . 8767038 group 4 0 . 034868896 0 . 89121008 0 . 8767038 group 1 group 2 group 3 group 4 group 1 0 . 12010232 0 . 12742966 0 . 0348689 group 2 0 . 049438715 0 . 96941215 0 . 44869167 group 3 0 . 021887839 0 . 96941215 0 . 69663686 group 4 0 . 021364689 0 . 44869167 0 . 69663886 it can be inferred from observing the graph of the means and the contrasts that the existing significant differences between the treatments are mainly found between groups 1 and 4 . group 1 obtained average scores of adas greater than the rest of the groups during the entire time period and moreover , demonstrated a tendency to increase , which indicates a worsening during the treatment . instead , the other groups average scores decreased during the first 3 months of the treatment but started to increase again from that point on until reaching , in general , the values obtained at the beginning of the study . the scheffe test reveals less ( is less sensitive ) and , for that reason , only demonstrates this difference between groups 1 and 4 . on the contrary , the newman test demonstrates significant differences between the scores of the groups 1 and 2 , 1 and 3 , and 1 and 4 , resembling the values obtained by the patients of groups 2 , 3 and 4 with similar behaviors of their averages during the entire time . [ 0182 ] table of means + b287 t 0 m t 3 m t 6 m t 9 m mean yl . group 1 18 . 000 17 . 750 18 . 750 17 . 750 18 . 063 group 2 19 . 000 19 . 200 20 . 000 18 . 000 19 . 050 group 3 19 . 000 20 . 333 19 . 222 18 . 778 19 . 333 group 4 19 . 250 20 . 750 20 . 750 18 . 250 19 . 750 mean y . j 18 . 813 19 . 508 19 . 681 18 . 194 19 . 049 y .. behavior of the average scores during the entire time period for the different groups ( treatments )+ b333 s of v df ss ms f f groups 3 6 . 16135882 2 . 08045287 6 . 08060482 & gt ; 3 . 66 s ( blocks ) ( p & lt ; 0 . 05 ) periods 3 5 . 58373387 1 . 86124482 5 . 49272112 & lt ; 3 . 88 s ( p & lt ; 0 . 5 ) 9 3 . 0497091 0 . 33885857 total 15 14 . 8148016 in this case , the anova reveals significant differences ( p & lt ; 0 , 05 ) between the groups or treatments in their average scores obtained in the mmse test and also in their different evaluation periods . on the one hand , the different groups were compared together and , on the other hand , the different periods of evaluation were compared together , using anova , to evaluate how their averages differentiate in the mmse test . the following tables illustrate the p values of those comparisons : scheffe test group 1 group 2 group 3 group 4 group 1 0 . 46793833 0 . 26549053 0 . 09729582 group 2 0 . 46793833 0 . 97262728 0 . 71923995 group 3 0 . 26549353 0 . 97262728 0 . 92056388 group 4 0 . 09729852 0 . 71923995 0 . 92056388 group 1 group 2 group 3 group 4 group + 0 . 12574399 0 . 12766111 0 . 06535465 b2541 group 2 0 . 12574399 0 . 64535832 0 . 49389362 group 3 0 . 12766111 0 . 64535832 0 . 50054574 group 4 0 . 06535465 0 . 49389362 0 . 50054574 group 1 group 2 group 3 group 4 group 1 0 . 46793833 0 . 05565747 0 . 01564395 group 2 0 . 12558138 0 . 6452055 0 . 26582634 group 3 0 . 05565747 0 . 6452055 0 . 50037628 group 4 0 . 01564395 0 . 26582634 0 . 50037628 t 0 m t 3 m t 6 m t 9 m t 0 m 0 . 74233615 0 . 59628534 0 . 80315143 t 3 m 0 . 74233615 0 . 99412596 0 . 26565802 t 6 m 0 . 59628534 0 . 99412596 0 . 1812412 t 9 m 0 . 80315143 0 . 26565802 0 . 1812412 t 0 m t 3 m t 6 m t 9 m t 0 m 0 . 28400975 0 . 37189716 0 . 33882374 t 3 m 0 . 28400975 0 . 78607857 0 . 12776077 t 6 m 0 . 37189716 0 . 78607857 0 . 13094723 t 9 m 0 . 33882374 0 . 12776077 0 . 13094723 t 0 m t 3 m t 6 m t 9 m t 0 m 0 . 28388375 0 . 18696201 0 . 33869615 t 3 m 0 . 28388375 0 . 78594285 0 . 05570481 t 6 m 0 . 18696201 0 . 76594285 0 . 03376284 t 9 m 0 . 33869615 0 . 05570481 0 . 03376284 in regards to the contrasts between the groups of treatments , the scheffe and newman - keuls tests do not succeed in finding differences between those , except in groups 1 vs 4 , which demonstrate the smallest value of p , although it is not significant ( p & lt ; 0 , 05 ) in either case . however , with a third test ( lsd test ), one can see how groups 1 and 4 present this significant difference amongst each other , which was starting to be seen with prior contrasts . on the other hand , one can also see that a p value very close to 0 , 05 appears when comparing groups 1 vs 3 , which also would have markedly different averages between each other . observing the graph , one can clearly note these differences but also make out the tendencies and the behavior of the means for each of the treatments . note how the means of the groups 2 , 3 and 4 increase during the first 3 months of the treatment while in group 1 one can observe a certain decrease during this same period . then , during the next 3 months , only group 3 shows a decrease in its values and therefore , in the cognitive performance of the patients , while the other groups maintain or increase their scores . it must be emphasized however that the mean of the initial score for group 1 was less than that of the other groups of the study , fact that must be taken into consideration and without which the behaviour of this group would not differ as much from those of the groups 2 and 4 . finally , all the curves start decreasing from a critical point , marked by the end of the treatments , reaching mmse score values similar or even less than those obtained at the beginning of the experiment . in regards so the comparison between the different evaluated time periods , again the scheffe and newman - keuls tests do not show differences between those although they present lesser p values at times t 3m vs t 9m and t 6m vs t 9m . then the lsd test confirms one of the significant differences with p & lt ; 0 , 05 and the other with p = 0 . 0557 . this means that the values of the averages of the mmse are significantly different between 6 and 9 months , and , with a somewhat lower significance , between 3 and 9 months . the graph clearly demonstrates these differences , mostly those which appear between the last two evaluations . [ 0195 ] t 0 m t 3 m t 6 m t 9 m mean yl . group 1 13 . 75 18 . 25 24 . 75 22 . 5 19 . 813 group 2 17 . 39996 15 20 22 . 7999992 18 . 800 group 3 17 . 5555553 18 . 1111107 24 . 4444447 20 . 444447 20 . 139 group 4 17 . 250 18 . 5 22 . 25 22 . 25 20 . 063 mean y . j 16 . 489 17 . 465 22 . 861 21 . 999 19 . 703 y .. s of v df ss ms f f groups 3 4 . 58656281 1 . 52885094 0 . 43474727 & gt ; 3 . 88 ns ( blocks ) periods 3 122 . 32563 40 . 7752098 11 . 5948245 & gt ; 3 . 66 s ( p − 0 . 05 ) 9 31 . 6497869 3 . 51664299 total 15 158 . 581969 in this case , the anova reveals significant differences ( p & lt ; 0 , 05 ) in the average values of the hamilton test for depression between the different periods of evaluation but not between the groups or treatments . the different time periods were compared , given the signification of the anova to evaluate how their averages differentiate in the hamilton test . the following tables demonstrate the p values of these results . t 0 m t 3 m t 6 m t 9 m t 0 m 0 . 8872587 0 . 00216968 0 . 0065899 t 3 m 0 . 88725287 0 . 0076508 0 . 02397896 t 6 m 0 . 002116968 0 . 0076508 0 . 91840172 t 9 m 0 . 0065899 0 . 02397896 0 . 91840172 t 0 m t 3 m t 6 m t 9 m t 0 m 0 . 44252211 0 . 00127918 0 . 0021444 t 3 m 0 . 00127918 0 . 00248516 0 . 00324839 t 6 m 0 . 00127918 0 . 00248516 0 . 49628913 t 9 m 0 . 0021444 0 . 00324839 0 . 49628913 it can be inferred from the contrasts that there are significant differences in the means between the time periods of t 0m vs t 6m , t 0m vs t 9m , t 3m vs t 6m and t 3m vs t 9m . observing the graph of the means , we see how they vary in the time periods mentioned but not between the different groups for each particular period . one can observe how similar the curves are between the groups ( anova ns ), the means increasing in all cases during the entire time period until the end of the treatment . from that point on , only one of the groups demonstrates an increase while the values of the other groups start to decrease . finally , in regards to the adas test , the results of group 1 increased during the entire treatment , which would indicate an increase of the severity of the disease . the rest of the groups demonstrated a marked decrease in the three frist months and then increased , in a small or large measure , but without reaching the severity of group 1 ( group 4 & lt ; group 2 & lt ; group 3 ). as for the results of the mmse , once more , group 1 demonstrates a failure to respond to the treatment because weak values are always obtained , while group 4 obtained the best scores , remaining stable in time during the entire treatment . group 3 improved its scores during the first three months and then they decreased . as for group 2 , the scores increased lightly during the 6 months of the experiment . in this double - blind , controlled study of patients with alzheimer &# 39 ; s disease , treatment with nicotinic acid , caffeic acid , quercitin , and vitamin e combination has proved to be beneficial in delaying disease progression . disease progression was primarily studied using the standardized tests adas and mmse where longer median time reflects the worsening of symptoms . the median time to the primary outcome was longer with each treatment than with placebo alone . there was a trend toward a delay in reaching each of the individual end points making up the primary outcome , with a significant delay in alzheimer &# 39 ; s disease assessment scale ( adas ) in the treatment group # 4 . there were also significant delays in the deterioration of the performance of activities of daily living and the need for care . these findings should be of interest since , to date , no treatment for alzheimer &# 39 ; s disease has shown similar benefits with respect to these outcomes . the possibility that our findings reflect aberrations in the placebo group # 1 is unlikely , since the patients in this group reached the end points at the same rate of significance delay in adas in the treatment groups 2 and 3 . the newman test , demonstrates significant differences between the adas score of the groups 1 and 2 , 1 and 3 , 1 and 4 resembling the values obtained by the patients of the groups 2 , 3 and 4 with similar behaviors of their averages during the entire period . there were no demonstrable differences between the results in the group receiving treatment followed by placebo versus the groups receiving treatment throughout the course of the study ( group 2 vs group 3 , group 2 vs group 4 , and group 3 vs group 4 ). on the other hand , the anova test reveals significant differences ( p & lt ; 0 , 05 ) between the groups or treatments in their average scores obtained in the mmse test and also in their different evaluation periods . the hamilton test , which is a measurement of depression , demonstrates that there are significant differences in the means adas score between the time periods of t 0m vs t 6m , t 0m vs t 9m , t 3m vs t 6m and t 3m vs t 9m . the results of group 1 increased during the entire treatment , which indicate an increase in the severity of the disease ( demonstrate a failure to respond to the treatment ), while group 4 obtained the best adas scores , remaining stable in the time during the entire treatment . group 3 improved its scores during the first three months and then they decreased . as for group2 , the scores increased lightly during the 6 months of the trial . the above findings suggest that the use of nicotinic acid , caffeic acid , quercitin , and vitamin e combination may improve the adas scores and delay clinically important functional deterioration in patients with alzheimer &# 39 ; s disease . nicotinic acid , caffeic acid , quercitin , and vitamin e combination may have enhanced the functioning of nigral neurons or enhanced their survival by inhibiting oxidative stress . in the above study , there was improvement in cognitive portion of the alzheimer &# 39 ; s disease assessment scale and the mini - mental state examination scores in the treatment groups . the role of nicotinic acid , caffeic acid , quercitin , and vitamin e combination in the treatment of neurodegenerative diseases is currently of great interest . previous trials of alpha - tocopherol at much higher doses than used here , have demonstrated benefit in patients with ad . the neuronal populations involved in alzheimer &# 39 ; s disease are more sensitive to oxidative stress than those in other neurodegenerative diseases . also , in elderly populations it has been suggested that antioxidants improve cardiovascular function and the immune response and also reduce the risk of cancer . although it has been found that there is no differences in the frequency of these other types of disease in our study groups , we have no biologic data to evaluate additional possible benefits . nicotinic acid , caffeic acid , quercitin , and vitamin e combination delay functional deterioration , particularly as reflected by the need for institutionalization , and should be considered for use in patients with moderate dementia . statistically significant results were seen in our model that included adjustment for the baseline differences among the groups in the score on the adas test , mini - mental state examination . further studies are needed to evaluate patients with more advanced forms of alzheimer &# 39 ; 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