CN102984938A

CN102984938A - Treatment of MCI and Alzheimer's disease

Info

Publication number: CN102984938A
Application number: CN2010800680043A
Authority: CN
Inventors: M·洛弗尔; B·林恩
Original assignee: University of Kentucky Research Foundation
Current assignee: University of Kentucky Research Foundation
Priority date: 2010-05-13
Filing date: 2010-11-18
Publication date: 2013-03-20
Also published as: WO2011142778A1; AU2010353287A1; CA2799162A1; JP2013526518A; EP2568811A1; EP2568811A4

Abstract

The present invention provides, among other things, therapeutic compositions and methods that can effectively treat, slow or prevent a neurological disease (e.g., a neurodegenerative disease, e.g., mild cognitive impairment (MCI) or Alzheimer's disease (AD)), in particular, based on therapeutically effective amount of nifedipine, oxidized or nitroso nifedipine derivatives, lactam (e.g., a compound of formula (Ic) or (Ic-i), e.g., NFD- L1), thyroxine (T4), triiodothyronine (T3) and combinations thereof.

Description

The methods for the treatment of of mild cognitive impairment (MCI) and Alzheimer's disease

Background of invention

Alzheimer's disease (AD) is as everyone knows a kind of but the carrying out property nerve degenerative diseases do not studied fully yet, and in older population, its impact is increasing individual.Alzheimer's disease affects 4,000,000 Americans at present.Unless state-run aging research statistics show and to find preventative strategies can otherwise may have 14,000,000 Americans to suffer from Alzheimer's disease during to the year two thousand forty.

The earliest period clinical symptoms of Alzheimer's disease is described as being called to the symptom of mild cognitive impairment (MCI).Although the detection to MCI can allow the life style plan and implement necessary correction, there is no at present the therapy that stops MCI to develop into Alzheimer's disease or treatment Alzheimer's disease.

As far back as 2007, United States Senate, official FDA Andrew C.von doctor Eschenbach be prophesy " will increase to while estimating 4,500,000 current Alzheimer's disease cases to the year two thousand fifty approximately 16,000,000 " just.Doctor Eschenbach says that existing five kinds of medicines are approved for treatment AD---Tacrine, profit this bright, galanthamine, donepezil and Memantine hydrochloride---, and wherein first four kinds play a role by the levels of acetylcholine in the rising brain, last a kind of is the N-methyl-D-aspartate receptor antagonist.And doctor Eschenbach points out that the medicine of these five kinds of approved listings does not all demonstrate prevention or slows down the potential nervus retrogression of [AD] patient.He adds: " other countries in we and the world expect jointly, and [] has one day and there will be the new drug that can effect a radical cure this latent property disease and other sacred diseases ... ".

Summary of the invention

The present invention includes and find that nifedipine and oxidation or nitrosoation derivative thereof can effectively suppress in vitro and in vivo A β 1-40 and produce, reduce A β processive enzyme and make relevant biochemical route inactivation.More surprisingly, the present inventor finds that lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) also can effectively suppress in vitro and in vivo A β 1-40 and produce, reduce A β processive enzyme and make relevant biochemical route inactivation.Be not limited to any theory, once estimate that the nitroso nifedipine is likely a kind of prodrug that can be converted into lactam after giving in vivo, described conversion can stoichiometry.Like this, except other aspects, the invention provides a kind of based on nifedipine and oxidation or nitrosoation derivative, and/or lactam and derivative thereof are (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) novel method for the treatment of and composition, it can effectively be treated, slows down or stop mild cognitive impairment (MCI) and/or Alzheimer's disease and delay MCI and develop into AD.

On the one hand, the invention provides a kind of pharmaceutical composition that is suitable for treatment in human subjects, slows down or stop sacred disease, it comprises one or more therapeutic agents and the pharmaceutically acceptable carrier for the treatment of effective dose.In some embodiments, sacred disease is nerve degenerative diseases.In some embodiments, nerve degenerative diseases is mild cognitive impairment (MCI) and/or Alzheimer's disease.In some embodiments, therapeutic agent is the therapeutic agent with structure shown in general formula (Ia) defined herein and that describe.In some embodiments, therapeutic agent is the therapeutic agent with structure shown in general formula (Ib) defined herein and that describe.In some embodiments, therapeutic agent is the therapeutic agent with structure shown in general formula (Ic) defined herein and that describe.In some embodiments, be suitable for therapeutic agent of the present invention and (for example select free nifedipine, oxidized nifedipine, nitroso nifedipine, lactam, formula (Ic) or (Ic-i) compound, as NFD-L1), the group that forms of thyroxine (T4), trilute (T3) and combination thereof.In some embodiments, being suitable for therapeutic agent of the present invention is calcium channel blocker.In some embodiments, being suitable for therapeutic agent of the present invention is not calcium channel blocker.In some embodiments, be suitable for therapeutic agent of the present invention and increase stream in calcium.

In some embodiments, be suitable for therapeutic agent of the present invention and comprise nifedipine.In some embodiments, be suitable for therapeutic agent of the present invention and comprise oxidized nifedipine.In some embodiments, be suitable for therapeutic agent of the present invention and comprise the nitroso nifedipine.In some embodiments, be suitable for therapeutic agent of the present invention and comprise lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1).In some embodiments, be suitable for the mixture that therapeutic agent of the present invention comprises nitroso nifedipine, oxidized nifedipine and nifedipine.In some embodiments, be suitable for the mixture that therapeutic agent of the present invention comprises nitroso nifedipine and lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1).In some embodiments, be suitable for the mixture that therapeutic agent of the present invention comprises lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1), oxidized nifedipine and nifedipine.In some embodiments, be suitable for therapeutic agent of the present invention and comprise 55% nitroso nifedipine, 11% oxidized nifedipine and 34% nifedipine.In some embodiments, be suitable for therapeutic agent of the present invention and comprise that lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1), one or more (for example, two kinds, three kinds, four kinds) in nitroso nifedipine, oxidized nifedipine and nifedipine.In some embodiments, different therapeutic agent described herein further comprises thyroxine (T4) and/or trilute (T3).In some embodiments, be suitable for therapeutic agent of the present invention and comprise nifedipine, oxidized nifedipine, nitroso nifedipine, thyroxine (T4) and/or trilute (T3).

In some embodiments, comprise the therapeutic agent for the treatment of effective dose according to pharmaceutical composition of the present invention, described treatment effective dose is every dosage approximately 0.01 to about 1000mg (for example, approximately 0.01 to about 200mg, approximately 0.01 to about 100mg, approximately 0.1 to about 50mg, approximately 0.01 to about 10mg, approximately 0.01 to about 5mg, approximately 0.01 to about 2.5mg, approximately 0.01 to about 2.0mg, approximately 0.01 to about 1.5mg, approximately 0.01 to about 1.0mg, approximately 0.01 to about 0.5mg, approximately 0.01 to about 0.1mg).In some embodiments, comprise the nitroso nifedipine for the treatment of effective dose according to pharmaceutical composition of the present invention, the treatment effective dose is the about 10mg to 2.5g of every dosage (for example, about 10mg to 2.0g, about 10mg to 1.5g, about 10mg is to about 1000mg, about 10mg to about 500mg).

In some embodiments, comprise the therapeutic agent for the treatment of effective dose according to pharmaceutical composition of the present invention, wherein treat effective dose and be not enough to induce bad reaction in human subjects.In some embodiments, bad reaction is hepatotoxicity wind agitation.In some embodiments, comprise the therapeutic agent for the treatment of effective dose according to pharmaceutical composition of the present invention, wherein treat effective dose and be not enough to induce bad reaction in human subjects, wherein reagent is that nitroso nifedipine and bad reaction are hepatotoxicity wind agitation.

In some embodiments, according to pharmaceutical composition of the present invention be made into preparation in oral, subcutaneous, vein, transdermal, abdominal cavity, intramuscular, the ventricles of the brain, in brain essence, sheath, in encephalic, oral cavity, mucous membrane, nose or rectum give.In some embodiments, being made into preparation according to pharmaceutical composition of the present invention gives for oral.In some embodiments, be made into preparation for quick-release or slowly-releasing according to pharmaceutical composition of the present invention.

In yet another aspect, the invention provides a kind for the treatment of, delay or stop the method for human subjects sacred disease, the method comprises to the object of suffering from or easily suffer from sacred disease and gives therapeutic agent, at least one symptom relevant to sacred disease or feature reduce on abundance, intensity, the order of severity or frequency like this, or delay morbidity.In some embodiments, sacred disease is nerve degenerative diseases.In some embodiments, the invention provides a kind for the treatment of, delay or stop the method for human subjects mild cognitive impairment (MCI) and/or Alzheimer's disease, the method comprises one or more therapeutic agents for the treatment of effective dose to the object of suffering from or easily suffer from MCI or Alzheimer's disease, at least one symptom relevant to MCI or Alzheimer's disease or feature reduce on abundance, intensity, the order of severity or frequency like this, or delay morbidity.In some embodiments, symptom or feature be that cognitive decline, amyloid beta produce, beta-secretase is active, gamma-secretase is active, the Protein tau phosphorylation in double helix silk, brain and/or immunity or the inflammatory situation of central nervous system.In some embodiments, the immunity of central nervous system or inflammatory situation are viral meningitis, viral encephalitis, fungal meningitis, fungoid encephalitis, multiple sclerosis, schizophrenia, myasthenia gravis or Charcot's joint.In some embodiments, amyloid beta produces the generation that comprises A β 1-40.In some embodiments, amyloid beta produces the generation that comprises A β 1-42.In some embodiments, reduce the generation of amyloid beta by the increase of alpha-secretase activity.In some embodiments, alpha-secretase activity is the ADAM-10 activity.In some embodiments, make the activity decreased of gamma-secretase by the activity that suppresses presenilin-1 (PS-1), nicastrin, APH-1 and/or PEN-2.In some embodiments, make the activity decreased of gamma-secretase by suppressing orphan's G albumen-coupled receptor 3 (GPCR-3) activity.In some embodiments, for example, alleviate immunity or inflammatory situation by the level that reduces one or more cell factors (, IL-1, IL-6, TNF-α) in central nervous system.

In some embodiments, the treatment effective dose of reagent of the present invention is enough to increase the level of the glutamate transporter in the human subjects brain.In some embodiments, the glutamate transporter level is neuroglia glutamate transporter EAAT2 level.In some embodiments, the treatment effective dose of reagent of the present invention is not enough to induce the bad reaction of human subjects.In some embodiments, bad reaction is hepatotoxicity wind agitation.

In some embodiments, the therapeutic agent used in the method for the invention has as defined herein and the structure shown in the formula (Ia) of describing.In some embodiments, the therapeutic agent used in the method for the invention has as defined herein and the structure shown in the formula (Ib) of describing.In some embodiments, the therapeutic agent used in the method for the invention has as defined herein and the structure shown in the formula (Ic) of describing.In some embodiments, suitable therapeutic agent (for example is selected from nifedipine, oxidized nifedipine, nitroso nifedipine, lactam, formula (Ic) or (Ic-i) compound, as NFD-L1), thyroxine (T4), trilute (T3) and combination thereof.In some embodiments, suitable therapeutic agent is calcium channel blocker.In some embodiments, suitable therapeutic agent is not calcium channel blocker.In some embodiments, suitable therapeutic agent increases stream in calcium.

In some embodiments, suitable therapeutic agent comprises nifedipine.In some embodiments, suitable therapeutic agent comprises oxidized nifedipine.In some embodiments, suitable therapeutic agent comprises the nitroso nifedipine.In some embodiments, suitable therapeutic agent comprises lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1).In some embodiments, the suitable therapeutic agent that method of the present invention is used comprises the mixture of nitroso nifedipine, oxidized nifedipine and nifedipine.In some embodiments, suitable therapeutic agent comprises the mixture of nitroso nifedipine and lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1).In some embodiments, suitable therapeutic agent comprises the mixture of lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1), oxidized nifedipine and nifedipine.In some embodiments, the suitable therapeutic agent that method of the present invention is used comprises 55% nitroso nifedipine, 11% oxidized nifedipine and 34% nifedipine.In some embodiments, suitable therapeutic agent comprises that lactam (for example, formula (Ic) or (Ic-i) compound, as NFD-L1), one or more (for example, two kinds, three kinds, four kinds) in nitroso nifedipine, oxidized nifedipine and nifedipine.In some embodiments, suitable reagent as herein described further comprises T3/T4.In some embodiments, the suitable reagent that method of the present invention is used comprises nifedipine, oxidized nifedipine and/or nitroso nifedipine, further comprises thyroxine (T4) and/or trilute (T3).

In some embodiments, method of the present invention gives the therapeutic agent of required object treatment effective dose, the treatment effective dose is every dosage approximately 0.01 to about 1000mg (for example, approximately 0.01 to about 200mg, approximately 0.01 to about 100mg, approximately 0.01 to about 50mg, approximately 0.01 to about 10mg, approximately 0.01 to about 5mg, approximately 0.01 to about 2.5mg, approximately 0.01 to about 2.0mg, approximately 0.01 to about 1.5mg, approximately 0.01 to about 1.0mg, approximately 0.01 to about 0.5mg, approximately 0.01 to about 0.1mg).In some embodiments, method of the present invention gives the therapeutic agent nitroso nifedipine that comprises the nitroso nifedipine of required object treatment effective dose, the treatment effective dose is the about 10mg to 2.5g of every dosage (for example, about 10mg to 2.0g, about 10mg to 1.5g, about 10mg is to about 1000mg, about 10mg to about 500mg).In some embodiments, the reagent that method of the present invention is used by oral, subcutaneous, vein, transdermal, abdominal cavity, intramuscular, the ventricles of the brain, in brain essence, sheath, in encephalic, oral cavity, mucous membrane, nose or rectum give.In some embodiments, the reagent that method of the present invention is used gives by oral.

In some embodiments, the method according to this invention, per month once, once every two weeks or, give once in a week reagent.In some embodiments, the method according to this invention, give reagent once a day.In some embodiments, the method according to this invention, twice of every day, every day three times or four times a day give reagent.

In some embodiments, for example, by object level reduction or the rising of biomarker (, protein biology marker complex) compared with the control of method treatment of the present invention.In some embodiments, suitable biomarker is the protein biology marker complex, it comprises at least one in thyroxine transport protein and/or prostaglandin-H2D isomerase albumen, and the different albumen of at least one the second, it is selected from thyroxine transport protein, prostaglandin-H2D isomerase, beta-2-microglobulin, bladder chalone C, superoxide dismutase [Cu--Zn], blood plasma retinol-in conjunction with albumen, phosphatidyl-ethanolamine-in conjunction with albumen, carbonic anhydrase 2 and/or transferrins.In some embodiments, suitable protein biology marker complex comprises prostaglandin-D2-synzyme and thyroxine transport protein (PDS/TTR compound).In some embodiments, suitable biomarker compound comprises the ratio of (i) amyloid beta 40 (A β 40), (ii) amyloid beta 42 (A β 42), (iii) A β 40 and A β 42 and (iv) one or more in the ratio of the tau of phosphorylation and total tau.In some embodiments, be used to humoral sample from object (for example, CSF, serum, whole blood, blood plasma, urine, ascites, saliva, organize diffusate, irrigating solution and combination thereof) and determine biomarker.In some embodiments, the biomarker level in suitable contrast prompting object, object is selected from healthy individual, has determined the Alzheimer's disease patient of disease stage, treats front object and combination thereof.

There is cognitive disorder in the detection scoring indication of the object that in some embodiments, plan is received treatment.In some embodiments, the detection scoring of indication cognitive impairment is marked (for example,, lower than 27 for MMSE, as 21-26). in some embodiments, the detection scoring of indication cognitive impairment is marked (for example,, higher than 0 for clinical dementia grade (CDR), as 0.5, as 1).

In some embodiments, at first method of the present invention further comprises that level based on biomarker and/or cognition detection scoring determines the step of the treatment effective dose of therapeutic agent.

Aspect another, the invention provides a kind of solid orally ingestible that comprises nitroso nifedipine and nifedipine, and wherein the weight ratio of nitroso nifedipine and nifedipine be at least approximately 1: 1 (at least about 2: 1, at least about 4: 1, at least about 8: 1, at least about 16: 1, at least about 32: 1, at least about 64: 1, at least about 100: 1, at least about 200: 1, at least about 500: 1 or at least about 1000: 1).In some embodiments, solid orally ingestible of the present invention further comprises one or more pharmaceutically acceptable excipient (for example, adhesive, buffer, thinner, dispersant, softening agent, film forming agent, glidant, opacifier, preservative, solvent, stabilizing agent, surfactant, supensoid agent and/or tension regulator).In some embodiments, solid pharmaceutical preparation is for controlled release or slowly-releasing.In some embodiments, solid pharmaceutical preparation is for quick-release.

Aspect another, the invention provides benzo [c] [2,7] naphthyridines-5 (6H)-one compound.In some embodiments, provide be the compound of general formula (Ic):

Or its pharmaceutically acceptable salt, wherein:

R ¹and R ²independently for being selected from C _1-6aliphatic, C _1-6the optional substituted radical of assorted aliphatic, aromatic radical, assorted aromatic radical or cyano group;

R ³for being selected from C _1-6aliphatic, C _1-6the optional substituted radical of assorted aliphatic or aromatic radical;

R ⁵the C that is halogen, optionally replaces _1-6aliphatic, hydroxyl, alkoxyl, amino, alkylamino, cyano group, nitro or nitroso; And

N is 0,1,2 or 3.

In some embodiments, provide be the compound of general formula (Ic-i):

Or its pharmaceutically acceptable salt, wherein:

R ¹and R ²be C independently _1-6aliphatic or cyano group;

R ³c _1-6aliphatic;

R ⁵halogen, C _1-6aliphatic, hydroxyl, alkoxyl, amino, alkylamino, cyano group, nitro or nitroso; And

N is 0,1,2 or 3.

In some embodiments, the compounds of this invention is NFD-L1.

Except other aspects, the present invention also provides pharmaceutical composition and the using method thereof that contains compound described herein (for example, the compound shown in formula Ic or Ic-i).In some embodiments, the invention provides a kind of object compound as herein described (for example, the compound shown in formula Ic or Ic-i) by suffering from or easily suffer from sacred disease with treatment, delay or stop the method for human subjects sacred disease.In some embodiments, the invention provides a kind of object compound as herein described (for example, the compound shown in formula Ic or Ic-i) by suffering from or easily suffer from nerve degenerative diseases with treatment, delay or stop the method for human subjects nerve degenerative diseases.In some embodiments, the invention provides a kind of object compound as herein described (for example, the compound shown in formula Ic or Ic-i) by suffering from or easily suffer from MCI or Alzheimer's disease with treatment, delay or stop the method for human subjects mild cognitive impairment (MCI) and/or Alzheimer's disease.In some embodiments, the invention provides the method for a kind of inhibition human subjects beta-secretase (BACE), comprise and give human subjects compound as herein described (for example, the compound shown in formula Ic or Ic-i).In some embodiments, the invention provides a kind of method of mediator's class object inflammation of the central nervous system situation, for example, by giving human subjects compound as herein described (, the compound shown in formula Ic or Ic-i).

In this application, unless otherwise indicated, use "or" mean " and/or ".Term " comprises " and the variant of this term as used in this application, as " containing " and " comprising " and do not mean that and get rid of other additives, component, integer or step.As used in this application, it is identical using the implication of term " about " and " approximately ".The with or without of using in the application approximately/about Any Digit contained normal the floating arbitrarily that those skilled in the relevant art can understand.

Brief description of drawings

Accompanying drawing is only non-limiting for illustrating purpose.

Fig. 1 has shown the exemplary immunization engram analysis of PDS/TTR compound, this compound by contrast epithelial cell, the contrast epithelial cell of processing through acrolein and late period the AD epithelial cell in cell culture medium, express.

Fig. 2 has shown the exemplary survival data through the cortical neuron of contrast epithelial cell or the epithelial medium treatment of AD.

Fig. 3 has shown the positive example results of PHF1 immunity, and this result detects and obtains from the SY5Y cell contacted with the PDS/TTR albumen composition.

Fig. 4 has shown the exemplary immunization trace data that the expression of PDS/TTR compound reduces, and this compound is expressed by the contrast epithelial cell through acrolein, acrolein and T3/T4, acrolein and nifedipine mixture (nitroso nifedipine 55%, oxidized nifedipine 11% and nifedipine 34%) and acrolein, nifedipine mixture and T3/T4 processing.

Fig. 5 has gathered the PDS/TTR-positive cell number, and it carries out immunostaining by the culture to through acrolein, acrolein and T3/T4, acrolein and nifedipine mixture (nitroso nifedipine 55%, oxidized nifedipine 11% and nifedipine 34%) and acrolein, nifedipine mixture and T3/T4 processing and determines.

Fig. 6 shown with the nifedipine of new system and compared the function that the nifedipine mixture does not have calcium channel blocker, and this determines by Laser Scanning Confocal Microscope and calcium fluorescent staining.

The example results prompting nifedipine mixture that Fig. 7 shows suppresses inflammatory cytokine and produces.

The example results prompting NFD-L1 that Fig. 8 shows suppresses inflammatory cytokine and produces.

Fig. 9 has shown the quantitative result of the PHF-1 immunostaining of SY5Y culture, and this SY5Y culture is through acrolein and nifedipine and analog, the combination of mixture and the epithelial cell medium treatment that T3/T4 processes.

Example results prompting nifedipine, oxidized nifedipine, nitroso nifedipine and T3/T4 that Figure 10 shows suppress A β _1-42generate.

Example results prompting nifedipine, nifedipine analog and nifedipine mixture that Figure 11 shows, produce A β in the situation that contain or do not contain T3/T4 to the H4 cell _1-42effect.

The example results that Figure 12 shows is pointed out known calcium channel blocker, as the impact on A β 1-42 generation in H4 glioma culture of Amlodipine, diltiazem, felodipine, Isradipine, nicardipine and Nimodipine.

The impact that the example results prompting NFD-L1 that Figure 13 shows produces A β 1-42 in H4 glioma culture.

The example results prompting nitroso nifedipine that Figure 14 shows significantly suppresses the BACE activity.

The example results prompting NFD-L1 that Figure 15 shows significantly suppresses the BACE activity.

The example results prompting nifedipine mixture that Figure 16 shows, in the situation that contain or do not contain T3/T4, on the impact of PS-1, PEN-2, BACE-1 and Nicastrin.

The example results that Figure 17 shows is pointed out nifedipine, nifedipine mixture and/or the T3/T4 impact on A β 1-40 generation and some A β 1-40 processive enzyme in mouse model.

The example results prompting nitroso nifedipine that Figure 18 shows is processed and is caused A β 1-40 level in mouse model to reduce.

Example results prompting nifedipine, nifedipine mixture and/or T3/T4 that Figure 19 shows reduce H4 culture or the GPCR-3 level in the mouse of medicine acute treatment.Adopt immunoblotting assay to determine the GPCR-3 level.

The example results that Figure 20 shows is pointed out the blood medicine of other kinds, in the situation that contain or do not contain T3/T4, to GPCR-3 level affects in the H4 culture.

After the example results that Figure 21 shows is the nitroso nifedipine processing increased through concentration, H4 cell survival situation.

Figure 22 has gathered the exemplary effects of the enzyme level that the nitroso nifedipine relates to A β process.

Figure 23 has gathered the exemplary effects of the enzyme level that NFD-L1 relates to A β process.

Figure 24 has gathered the exemplary effects of the enzyme level that the nitroso nifedipine relates to A β process in mouse model.

Figure 25 has gathered the exemplary effects of the enzyme level that NFD-L1 relates to A β process in mouse model.

Example results prompting nifedipine, nifedipine mixture and/or T3/T4 that Figure 26 shows, in the mouse brain processing through respective compound, relate to the impact of the enzyme level of Tau phosphorylation.

Figure 27 has shown nifedipine and the nitroso nifedipine exemplary effects to the glutamate transporter level.

Figure 28 has shown that prompting nitroso nifedipine do not induce the example results of hepatic injury in mouse.

Figure 29 has shown the exemplary matching track according to the NLMIXED model, the relation of the MMSE of the correlative study of this model based in human body to the age.

The example results that Figure 30 shows is presented at from detecting in the research that scoring is relevant to neuropathology, and before anatomical object, the A β 1-42 of leaves specimen and A β processive enzyme are as PS-1, Nicas, BACE, APH-1 and PEN-2 level.4 objects are used calcium channel blocker, comprise nifedipine, and 4 objects are not used any calcium channel blocker.Use the ELISA of Invitrogen to measure A β level.Use western blot hybridization and determine protein level for the specific antibody of each albumen.

The example results that Figure 31 shows has shown that in the front leaves specimen of object same as shown in Figure 18, the Tau phosphorylation relates to enzyme level.

The H4 glioma culture that the example results prompting that Figure 32 shows is processed through the nitroso nifedipine flows remarkable increasing in calcium compared with the control.

Figure 33 has shown the synthetic example results of nitroso nifedipine photochemistry.

Figure 34 has shown the example results that NFD-L1 is synthetic.

Definition

Except as otherwise noted, otherwise the implication of scientific and technical terminology used herein is identical with one skilled in the art's of the present invention common understanding.Generally speaking, cell is cultivated the conventional method that program, infection, molecular biology method etc. are all used this area.This type of standard technique can be referring to reference manual, such as Ausubel etc., Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001; With Sambrook etc., Molecular Cloning:ALaboratory Manual, 3 ^rdedition, Cold Spring Harbor Laboratory Press, N.Y., 2001.

For the present invention be should be readily appreciated that more, at first some term is defined.Other definition of following term and other terms are referring to whole specification.

Alzheimer's disease patient: as used herein, the individuality that term " Alzheimer's disease patient ", " AD patient " and " being diagnosed as the individuality of AD " all refer to be diagnosed as AD or consider probably to be diagnosed as Alzheimer's disease (AD).

Animal: as used herein, term " animal " refers to any member of the animal kingdom.In some embodiments, " animal " refers to the people in any developmental stage.In some embodiments, " animal " refers to the non-human animal in any developmental stage.In some embodiments, the non-human animal refers to mammal (for example, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, ox, primate and/or pig).In some embodiments, animal includes, but not limited to mammal, birds, reptile, amphibian, fish, insect and/or worm.In some embodiments, animal can be transgenic animal, gene engineering animal and/or cloned animal.

Approximately: as used herein, term " approximately " or " approximately ", while being applied to one or more desired value, refer to the value approximate with the standard control value.In some embodiments, except context be otherwise noted or situation about clearly meaning (this type of numerical value exceeds except the situation of probable value 100%), term " approximately " or " approximately " refer to the scope of value fall into the both direction up and down of described standard value (higher or lower than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower in.

Biological fluid: as used herein, term " biological fluid " comprises the plurality of liquid sample type that can be used in diagnosis or monitoring detection from individuality.This term comprises other fluid samples of whole blood, serum or blood plasma, cerebrospinal fluid (CSF), urine and biogenetic derivation.This term also is included in it and obtains the sample of processing by any means, as through some component of agent treated, solubilising or enrichment, as albumen or polynucleotides.

Conjoint therapy: term " conjoint therapy ", as used herein, refer to give those situations of two or more different pharmaceutical reagent in overlapping scheme, object is exposed to these reagent simultaneously like this.

Contrast: as used herein, the standard items of term " contrast " implication pointer in the art to institute's comparative result.Typically, use contrast to increase the integrality of experiment by isolated variable, to obtain the conclusion about this class variable.In some embodiments, contrast is with detection reaction or tests reacting or testing to compare of simultaneously carrying out.In an experiment, use " detection " (that is, detection variable).In another experiment, " contrast ", do not used detection variable.In some embodiments, contrast is previously contrast (that is, the detection of carrying out before this or test, or known amount or result before this).In some embodiments, contrast for or comprise the record of printing or otherwise preserving.Contrast can be positive control or negative control.

Give scheme: " giving scheme ", as used herein term, the one group of unit dose that refers to give separately in a period of time (at least one or common more than).Form it for the recommended dose setting of particular therapeutic agent (that is, amount, time, give approach etc.) and given scheme.

Functional: as used herein, " functional " biomolecule is to show character and/or active biomolecule with the form by its feature.

Suppress: as used herein, term " inhibition " refers to reduce or reduce albumen or the activity of gene and/or process or the method for expressing of paying close attention to.Typically, Profilin or gene refer to detect by one or more methods as herein described or well known in the art, the expression of albumen or gene or relative activity reduce at least 10% or more than, for example 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more than, reduce to express or relative activity 1-doubly, 2-doubly, 3-doubly, 4-doubly, 5-doubly, 10-doubly, 50-doubly, 100-doubly or more than.

External: as used herein, term " external " refers to occur in the event in artificial environment, for example at test tube or reaction tube, medium at cell culture medium, but not in multicellular organisms.

In body: as used herein, term " in body " refers to occur in the interior event of multicellular organisms as the non-human animal.

Separate: as used herein, composition that when term " separation " refers to material and/or entity (1) at least some and its initial generation, (natural and/or experimental situation) is relevant separate, and/or (2) manually produce, preparation and/or manufacture.The material separated and/or entity can with being separated at least about 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 98%, approximately 99%, basically 100% or 100% of initial relative other compositions.In some embodiments, the purity of separation agent is higher than approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, basically 100% or 100%.As used herein, if it is substantially free of other compositions, material is " pure ".As used herein, term " cell of separation " refers to not be included in the cell in multicellular organisms.

The individuality of suffering from MCI: as used herein, " individuality of suffering from MCI (mild cognitive impairment) " refers generally to meet the individuality of following forgetful property MCI clinical criteria .Arch Neurol 56:303-308 (1999) such as () Petersen: 1) by question and answer man memory main suit conclusive evidence, 2) the objective memory damage, 3 that causes because of age and education) normal general cognitive function, 4) and ADL not impaired and 5) object do not meet dull-witted standard.

The individuality of suffering from EAD: as used herein, " individuality (early stage or mid-term Alzheimer's disease) of suffering from EAD " is for meeting the individuality of following standard: the cognitive decrease, 2 that higher degree 1) relatively occurs before this) except memory, one or more cognitive decreases, 3) the clinical dementia grade scoring is 0.5 to 1 and 4) dementia that causes of clinical examination eliminating other reasons.

The individuality of suffering from LAD: as used herein, " individuality (moderate or late period Alzheimer's disease) of suffering from LAD refers to meet the individuality (the .Neurology 34:939-48 (1984) such as McKhann) of the standard clinical diagnostic criteria that may suffer from AD.

Lactam: as used herein, " lactam " finger ring acid amides.Typically, the carbon number that its prefix designates exists in ring (except carbonyl group): beta-lactam (has 2 carbon atoms except carbonyl, 4 annular atomses are arranged altogether), gamma-lactam (3 and 5), δ-lactam (4 and 6). in some embodiments, passing type (Ic) or formula (Ic-i) definition are applicable to lactam of the present invention.In some embodiments, being applicable to lactam of the present invention is NFD-L1.

Reference value: as used herein, " reference value " can be absolute value; Relative value; Value with the upper limit and/or lower limit; The scope of value; Mean value; Median, average or contrast or baseline value value relatively with specific.Reference value can be based on the value of individual sample, for example, from the value of the individual sample of suffering from AD, MCI or cognitive disorder, but take from time point more early, or from the value of the AD patient's sample outside detected individuality, perhaps " normally " individuality, be not diagnosed as the individuality of AD.Reference value can be based on the value of a large amount of samples, as from AD patient or normal individual or the biased sample based on comprising or get rid of detected sample.

Sacred disease: as used herein, phrase " sacred disease " refers to disease or the illness of central nervous system.Sacred disease comprises that multiple sclerosis, DPN, nerve degenerative diseases are as AD, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, mild cognitive impairment (MCI) and Frontotemporal dementia.Other exemplary sacred diseases comprise epilepsy, the convulsibility disease, pain, anxiety, depressed, schizophrenia, cognitive decline after anesthesia, opiate tolerance, drug abuse, the ethanol abuse, schizophrenia, neuropathy medicine malin syndrome, appropriate Reye syndrome, Pick's disease, dull-witted, delirium, nerve retrograde affection in Down syndrome, familial Britain type dementia, familial danish type dementia, the Korsakoff disease, olvopontocerebellar atrophy, the dementia that HIV induces and blind, the Multi-infarct dementia, Hereditary motor and sensory neuropathy becomes (HMSN, also referred to as the atrophy of diseased pleural muscle meat or Charcot-Marie-Tooth disease), diabetic peripheral neuropathy with integrated, olvopontocerebellar atrophy, the nerve degeneration of Late-onset, the alcoholic-toxic polyneuropathy, tinnitus, with the pathologic, physiologic symptom.

Normal individual: as used herein, " normally " individual or " health " individuality refers to by or will be estimated and not suffer from AD or MCI by the medical worker, and have simple and easy mental status inspection (MMSE) (referring to Folstein etc., J.Psychiatr.Res 1975; 12:1289-198) scoring maybe will reach the object of MMSE scoring scope 25-30." normally " individuality is age-matched in 5 to 10 years old scope generally, includes but not limited to individuality age-matched, that will estimate.

Albumen: as used herein, term " albumen " refers to polypeptide (that is at least two amino acid chains that, connect by peptide bond between mutually).Albumen can comprise the group (for example, can be glycoprotein, proteoglycans etc.) beyond amino acid and/or can carry out other processing or modification.Those of ordinary skill in the art will understand, " albumen " can be the complete polypeptide chain that produces of cell (with or band signal sequence not), can be maybe its characteristic part.Those of ordinary skill in the art will understand albumen some the time can comprise more than one polypeptide chain, for example by one or more disulfide bond, connect or connect by other means.Polypeptide can contain L-amino acid, D-amino acid or the two has, and can contain any several amino acids well known in the art and modify or analog.Useful modification comprises, such as terminated acetylated, amidatioon etc.In some embodiments, albumen can comprise natural amino acid, alpha-non-natural amino acid, synthesizing amino acid and combination thereof.Be often referred to while using term " peptide " and there is length and be less than approximately 100 amino acid whose polypeptide.

Object: as used herein, term " object " or " patient " refer to passable, for example, for experiment, diagnosis, prevention and/or therapeutic purposes, to it, give any organism according to composition of the present invention.Typical object comprises animal, and (for example, mammal is as mouse, rat, rabbit, non-human primates and people; Insect; Worm; Etc.).

Basically: as used herein, the qualitative condition that term " basically " refers to demonstrate the overall of paid close attention to feature or character or approaches overall scope or degree.It is few that the those of ordinary skill of biological field will be understood the biological and chemical phenomenon,, if having, carry out fully/or reach integrality or reach or avoid absolute results.Therefore, this paper is used term " basically " in order to obtain the integrality of the hidden hunger of a lot of biological and chemical phenomenon own.

Suffer from: the individuality of " suffering from " disease, illness and/or situation has been diagnosed as or has demonstrated one or more diseases, illness and/or situation.

Easily suffer from: individual " easily suffering from " disease, illness and/or situation, and not yet be diagnosed as disease, illness and/or situation.In some embodiments, the individuality of susceptible disease, illness and/or situation may not show the symptom of disease, illness and/or situation.In some embodiments, the individuality of susceptible disease, illness and/or situation will develop into disease, illness and/or situation.In some embodiments, the individuality of susceptible disease, illness and/or situation will can not develop into disease, illness and/or situation.

The treatment effective dose: as used herein, " the treatment effective dose " of term therapeutic agent refers to when the dosage of suffering from or being enough to disease, illness and/or situation are treated, diagnose, prevented and/or postpone its symptom appearance during susceptible disease, illness and/or situation individual.

Therapeutic agent: as used herein, phrase " therapeutic agent " refers to have therapeutic action when giving object and/or excites required biology and/or any agent of pharmacological action.As used herein, term " therapeutic agent " and " reagent " can Alternates.

Treatment: as used herein, term " treatment " refers to that one or more symptoms or the feature for partially or even wholly making specified disease, illness and/or situation alleviates, improves, alleviates, suppresses, stops, postpones its initial, as to reduce its seriousness and/or reduce its incidence any means.In order to reduce the risk of pathologies developed into disease association, can treat to the object that does not show disease symptoms and/or only show the disease early symptom.

Hereinafter the definition of particular functional group and the technical terms of chemistry is described in more detail.For purposes of the present invention, the chemical element of evaluation and Handbook of Chemistry and Physics, the CAS version, the periodic table of elements in the 75th edition in front cover is consistent, to particular functional group's common definition as described therein.In addition, vitochemical rule and particular functional group and reactivity, referring to Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March ' s Advanced Organic Chemistry, 5 ^thedition, John Wiley& Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCHPublishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 ^rdedition, Cambridge University Press, Cambridge, 1987.

Compound of the present invention can exist with specific geometry or stereoisomeric forms in any ratio.All these compounds have been contained in the present invention, comprise cis and transisomer, R-and S-enantiomter, diastereoisomer, (D)-isomer, (L) isomer, its racemic mixture and other mixture thereof, all fall into scope of the present invention.

Aliphatic: as used herein term " aliphatic " or " aliphatic group " refer to hydrocarbyl group, its can be straight chain (, non-branch), side chain or cycloaliphatic (comprise condense, bridge joint and volution fused polycycle), it can be fully saturated maybe can contain one or more unsaturated units, but it is not aromatic.Except as otherwise noted, otherwise aliphatic group contains 1-6 carbon atom.In some embodiments, aliphatic group contains 1-4 carbon atom, and in other embodiments, aliphatic group contains 1-3 carbon atom.Suitable aliphatic group includes, but not limited to linearity or side chain, alkyl, thiazolinyl and alkynyl group, and composition thereof as (cycloalkyl) alkyl, (cycloalkenyl group) alkyl or (cycloalkyl) thiazolinyl.

Thiazolinyl: as used herein term " thiazolinyl ", refer to the univalent perssad derived from the straight or branched aliphatic group, it has at least one carbon-carbon double bond by removing single hydrogen atom.In some embodiments, thiazolinyl contains 2-6 carbon atom.In some embodiments, thiazolinyl contains 2-5 carbon atom.In some embodiments, thiazolinyl contains 2-4 carbon atom.In another embodiment, thiazolinyl contains 2-3 carbon atom.Alkenyl group comprises, for example, and vinyl, acrylic, butylene, 1-methyl-2-butene-1-base etc.

Alkyl: as used herein term " alkyl " refers to remove to the aliphatic group of six carbon atom derived from containing one that the unit price of single hydrogen atom is saturated, the straight or branched alkyl.In some embodiments, alkyl contains 1-5 carbon atom.In another embodiment, alkyl contains 1-4 carbon atom.In another embodiment, alkyl contains 1-3 carbon atom.In another embodiment, alkyl contains 1-2 carbon atom.The example of alkyl comprises, but be not limited to methyl, ethyl, n-propyl group, isopropyl, n-butyl, iso-butyl, the second month in a season-butyl, the second month in a season-amyl group, iso-amyl group, tert-butyl, n-amyl group, neopentyl, n-hexyl, uncle-hexyl, n-heptyl, n-octyl group, n-decyl, n-undecyl, dodecyl etc.

Alkynyl: as used herein term " alkynyl ", refer to the monoradical derived from the straight or branched aliphatic group, it has at least one carbon carbon triple bond by removing single hydrogen atom.In some embodiments, alkynyl contains 2-6 carbon atom.In some embodiments, alkynyl contains 2-5 carbon atom.In some embodiments, alkynyl contains 2-4 carbon atom.In another embodiment, alkynyl contains 2-3 carbon atom.Typical alkynyl group includes, but not limited to acetenyl, 2-propynyl (" propargyl "), 1-propinyl etc.

Amino: as used herein term " amino " refers to formula (NH ₂) shown in group.

Alkoxyl: term " alkoxyl " refers to formula (OR ⁱ) shown in " substituted hydroxy ", R wherein ⁱfor alkyl group defined herein, oxygen groups is connected directly to parent molecule.

Alkylamino: term " alkylamino " refers to formula (NR ^h ₂) shown in " substituted-amino ", R wherein ^hbe hydrogen or alkyl group defined herein independently, nitrogen groups is connected directly to parent molecule.

Aryl-as used herein, term " aryl " refers to have altogether monocycle and the dicyclo ring system of the optional replacement of 5 to 10 rings, wherein at least one ring in system be aromatic and wherein each ring in system comprise three to heptatomic ring.Term " aryl " can with term " aromatic ring " Alternate.In some embodiments of the present invention, " aryl " refers to the aromatic rings system, and it includes, but not limited to benzene, biphenyl, naphthalene, anthracene etc., and it can contain one or more replacements.

Cycloaliphatic: term " cycloaliphatic ", " carbocyclic ring ", " carbocylic radical ", " carbocyclic ring " or " carbocyclic ", independent use or conduct be the part of macoradical more, refer to saturated or the undersaturated cycloaliphatic monocycle of part or dicyclo ring system as described herein, there are 3 to 10 yuan, wherein at random replaced with described aliphatic ring system as hereinbefore defined.Cycloaliphatic includes, but not limited to cyclopropyl, cyclobutyl, cyclopenta, cyclopentenyl, cyclohexyl, cyclohexenyl group, suberyl, cycloheptenyl, ring octyl group, cyclo-octene base and cyclo-octadiene base.In some embodiments, cycloalkyl has 3-6 carbon.Term " cycloaliphatic ", " carbocyclic ring ", " carbocylic radical " or " carbocyclic ring " also comprise the aliphatic ring condensed with one or more aromatic series or non-aromatic ring, as decahydro naphthyl, tetralyl, naphthalane or two ring [2.2.2] octanes, wherein group or tie point are on the aliphatic ring.

Cyano group: term " cyano group ", as used herein, refer to the group shown in formula (CN).

Halogen: term " halogen " and " halogen group " as used herein finger are selected from the atom of fluorine (fluorine-based ,-F), chlorine (chloro ,-Cl), bromine (bromo ,-Br) and iodine (iodo ,-I).

Assorted aliphatic-as used herein, term " assorted aliphatic " or " assorted aliphatic group ", refer to have the hydrocarbyl group of one to five heteroatomic any replacement except carbon atom, its can be straight chain (, non-branch), (" heterocycle ") of side chain or ring-type, and it can be fully saturated maybe can contain one or more unsaturated units, but it is not aromatic.Except as otherwise noted, otherwise assorted aliphatic group contains 1-6 carbon atom, and the hetero atom that wherein 1-3 carbon atom optionally and independently is selected from oxygen, nitrogen and sulphur replaces.In some embodiments, assorted aliphatic group contains 1-4 carbon atom, and the hetero atom that wherein 1-2 carbon atom optionally and independently is selected from oxygen, nitrogen and sulphur replaces.In other embodiments, assorted aliphatic group contains 1-3 carbon atom, and the hetero atom that wherein 1 carbon atom optionally and independently is selected from oxygen, nitrogen and sulphur replaces.Suitable assorted aliphatic group includes, but not limited to linearity or side chain, assorted alkyl, assorted thiazolinyl and assorted alkynyl group.

Heteroaryl-as used herein, term " heteroaryl " is used separately or, as the part of macoradical more, for example " heteroarylalkyl " or " assorted aralkoxy ", refer to have 5 to 10 annular atomses, preferably 5,6 or 9 annular atomses; There are 6,10 or 14 pi-electrons in a ring array; And the group that there is one to five heteroatomic any replacement except carbon atom.Heteroaryl groups comprises, but be not limited to thienyl, furyl, pyrrole radicals, imidazole radicals, pyrazolyl, triazolyl, tetrazole radical, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl group, pyridine radicals, pyridazinyl, pyrimidine radicals, pyrazinyl, indolizine base, purine radicals, naphthyridines base and pteridine radicals.Term " heteroaryl " and " assorted virtue-", as used herein, also comprise assorted aromatic rings and one or more aromatic ring, carbocyclic ring or heterocyclic fused group, wherein group or tie point are on hetero-aromatic ring.Non-limitative example comprises indyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuran group, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolines base, phthalazinyl, quinazolyl, quinoxalinyl, 4H-quinolizine base, carbazyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazine, tetrahydric quinoline group and tetrahydro isoquinolyl.Heteroaryl can also be monocycle or dicyclo.Term " heteroaryl " can with term " hetero-aromatic ring ", " heteroaromatic " Alternate, the ring that any above-mentioned term comprises is to replace arbitrarily.

Hetero atom-as used herein, term " hetero atom " refers to nitrogen, oxygen or sulphur, it comprises nitrogen or the sulphur of any oxidised form, and the basic nitrogen of any tetravalence form.Term " nitrogen " also comprises the nitrogen of replacement.

Heterocycle-as used herein, term " heterocycle ", " heterocyclic radical ", " heterocyclic group " and " assorted cyclic rings " can Alternates, 5 to the 7 yuan of monocycles or 7 to the 10 yuan of bicyclic heterocycles groups that refer to stable any replacement, its be saturated or part undersaturated, except carbon atom, there are one or more hetero atoms as hereinbefore defined.Heteroatom can be connected to produce stable structure with its pendent group in any heteroatom or carbon atom, and annular atoms can be optionally substituted arbitrarily.The example of the heterocyclic group of this type of saturated or fractional saturation comprises, but be not limited to tetrahydrofuran base, tetrahydro-thienyl, pyrrolidinyl, pyrrolidone-base, piperidyl, pyrrolinyl, tetrahydric quinoline group, tetrahydro isoquinolyl, decahydroquinolyl, oxazole alkyl, piperazinyl, alkyl dioxin, dioxolanyl, diazepine base, oxygen azatropylidene base, sulphur azatropylidene base, morpholinyl and the peaceful cyclic group of quinoline.Term " heterocycle ", " heterocyclic radical ", " assorted cyclic rings ", " heterocyclic group ", " heterocyclic moiety " and " heterocycle root " in this article can Alternates, it also comprises heterocycle and one or more aryl, heteroaryl or carbocyclic fused group, as indoline base, 3H-indyl, Chromanyl, Phen base or tetrahydric quinoline group, wherein group or tie point are on heterocycle.Heterocyclic group can be monocycle or two rings.Term " Heterocyclylalkyl " refers to heterocyclically substituted alkyl group, and wherein alkyl and heterocyclic radical part are optionally substituted independently.

Nitro: as used herein term " nitro " refers to formula (NO ₂) shown in group.

Nitroso: as used herein term " nitroso " refers to the group shown in formula (NO).

Part is undersaturated: as used herein, term " part is undersaturated " refers to comprise the cyclic group of at least one two key or triple bond between non-aromatic annular atoms.As herein defined, term " part is undersaturated " is intended to comprise the ring with a plurality of unsaturated positions, rather than comprises fragrance or assorted aromatic group.

Undersaturated: term " undersaturated ", as used herein, refer to have the group of one or more unsaturated units.

Replace arbitrarily: as used herein, compound of the present invention can comprise " replacing arbitrarily " group.Generally speaking, term " replacement ", be no matter term " arbitrarily " before or after, all refer to that one or more hydrogen of special groups are replaced by suitable substituting group.Except so far as otherwise expressly stated, otherwise " replace arbitrarily " but group can have a substituting group at each the position of substitution of group, when an above position of any given structure can be selected from the substituting group replacement of special groups more than one, the substituting group of each position can be identical or different.The substituent combinatorial optimization result that the present invention intends adopting obtains rock-steady structure or chemistry can realize those of compound.Term " stable ", as used herein, refer to when being placed on its production, detection and in some embodiments, its recovery, purifying and for lower time of condition of one or more purposes disclosed herein, compound does not change basically.

Suitable unit price substituting group on the substitutable carbon atom of " replacing arbitrarily " group is halogen independently;-(CH ₂) _0-4r ^o;-(CH ₂) _0-4oR ^o;-O-(CH ₂) _0-4c (O) OR ^o;-(CH ₂) _0-4cH (OR ^o) ₂;-(CH ₂) _0-4sR ^o;-(CH ₂) _0-4ph, it can be by R ^oreplace;-(CH ₂) _0-4o (CH ₂) _0-1ph, it can be by R ^oreplace;-CH=CHPh, it can be by R ^oreplace;-NO ₂;-CN;-N ₃;-(CH ₂) _0-4n(R ^o) ₂;-(CH ₂) _0-4n(R ^o) C (O) R ^o;-N (R ^o) C (S) R ^o;-(CH ₂) _0-4n(R ^o) C (O) NR ^o ₂;-N (R ^o) C (S) NR ^o ₂;-(CH ₂) ₀₄n(R ^o) C (O) OR ^o;-N (R ^o) N (R ^o) C (O) R ^o;-N (R ^o) N (R ^o) C (O) NR ^o ₂;-N (R ^o) N (R ^o) C (O) OR ^o;-(CH ₂) _0-4c (O) R ^o;-C (S) R ^o;-(CH ₂) _0-4c (O) OR ^o;-(CH ₂) _0-4c (O) SR ^o;-(CH ₂) _0-4c (O) OSiR ^o ₃;-(CH ₂) _0-4oC (O) R ^o;-OC (O) (CH ₂) _0-4sR-; SC (S) SR ^o;-(CH ₂) _0-4sC (O) R ^o;-(CH ₂) _0-4c (O) NR ^o ₂;-C (S) NR ^o ₂;-C (S) SR ^o;-SC (S) SR ^o,-(CH ₂) _0-4oC (O) NR ^o ₂;-C (O) N (OR ^o) R ^o;-C (O) C (O) R ^o;-C (O) CH ₂c (O) R ^o;-C (NOR ^o) R ^o;-(CH ₂) _0-4sSR ^o;-(CH ₂) _0-4s (O) ₂r ^o;-(CH ₂) _0-4s (O) ₂oR ^o;-(CH ₂) _0-4oS (O) ₂r ^o;-S (O) ₂nR ^o ₂;-(CH ₂) _0-4s (O) R ^o;-N (R ^o) S (O) ₂nR ^o ₂;-N (R ^o) S (O) ₂r ^o;-N (OR ^o) R ^o;-C (NH) NR ^o ₂;-P (O) ₂r ^o;-P (O) R ^o ₂;-OP (O) R ^o ₂;-OP (O) (OR ^o) ₂; SiR ^o ₃;-(C _1-4the straight or branched thiazolinyl) O-N (R ^o) ₂; Or-(C _1-4the straight or branched thiazolinyl) C (O) O-N (R ^o) ₂, each R wherein ^oaccording to hereinafter described being substituted, it is hydrogen independently, C _1-6aliphatic ,-CH ₂ph ,-O (CH ₂) _0-1ph, or 5-6-unit saturated rings, the unsaturated ring of part or there is 0-4 the heteroatomic aromatic ring independently selected from nitrogen, oxygen or sulphur, or except definition above, two R independently ^oand the middle atom inserted form have 0-4 unit of the heteroatomic 3-12 independently selected from nitrogen, oxygen or sulphur saturated, part is undersaturated, or fragrant monocycle or dicyclo, it can be according to being substituted hereinafter described.

R ^o(or two R independently ^othe ring formed with the atom wherein inserted) the suitable unit price substituting group on be independently halogen ,-(CH ₂) _0-2r ^.,-(halogen R ^.), ,-(CH ₂) _0-2oH ,-(CH ₂) _0-2oR ^.,-(CH ₂) _0-2cH (OR ^.) ₂;-O (haloR ^.) ,-CN ,-N ₃,-(CH ₂) _0-2c (O) R ^.,-(CH ₂) _0-2c (O) OH ,-(CH ₂) _0-2c (O) OR ^.,-(CH ₂) _0-2sR ^.,-(CH ₂) _0-2sH ,-(CH ₂) _0-2nH ₂,-(CH ₂) _0-2nHR ^.,-(CH ₂) _0-2nR ^. ₂,-NO ₂,-SiR ^. ₃,-OSiR ^. ₃,-C (O) SR ^.,-(C _1-4the straight or branched thiazolinyl) C (O) OR ^., or-SSR ^., each R wherein ^.for undersaturated or its front, mean only by one or more halogen atoms, to be replaced during with " halogen ", and it is independently selected from C _1-4aliphatic ,-CH ₂ph ,-O (CH ₂) _0-1ph, or 5-6 unit saturated rings, the unsaturated ring of part or there is 0-4 the heteroatomic aromatic ring independently selected from nitrogen, oxygen or sulphur.At R ^ocomprise=O of suitable divalent substituent on saturated carbon atom and=S.

Suitable divalent substituent on the saturated carbon atom of " replacing arbitrarily " group comprises following :=O ,=S ,=NNR ^* ₂,=NNHC (O) R ^*,=NNHC (O) OR ^*,=NNHS (O) ₂r ^*,=NR ^*,=NOR ^*,-O (C (R ^* ₂)) _2-3o-or-S (C (R ^* ₂)) _2-3s-, wherein each independently goes out terrain R ^*be selected from hydrogen, the C that can replace according to following definitions _1-6aliphatic, or unsubstituted 5-6 unit saturated rings, the unsaturated ring of part or there is 0-4 the heteroatomic aromatic ring independently selected from nitrogen, oxygen or sulphur.The suitable divalent substituent that is connected to the ortho position substitutable carbon atom of " replacing arbitrarily " group comprises :-O (CR ^* ₂) _2-3o-, wherein each independently goes out terrain R ^*be selected from hydrogen, C _1-6aliphatic can be substituted according to following definitions, or unsubstituted 5-6 unit saturated, part is undersaturated or have 0-4 the heteroatomic aromatic ring independently selected from nitrogen, oxygen or sulphur.

At R ^*suitable replacement on aliphatic group comprise halogen ,-R ^.,-(halogen R ^.) ,-OH ,-OR ^.,-O (halogen R ^.) ,-CN ,-C (O) OH ,-C (O) OR ^.,-NH ₂,-NHR ^.,-NR ^. ₂, or-NO ₂, each R wherein ^.for undersaturated or its front, mean only by one or more halogen atoms, to be replaced during with " halogen ", and it is independently selected from C _1-4aliphatic ,-CH ₂ph ,-O (CH ₂) _0-1ph, or have 0-4 saturated independently selected from the heteroatomic 5-6 unit of nitrogen, oxygen or sulphur, part is undersaturated or aromatic ring.

Suitable substituting group on the replaced nitrogen of " replacing arbitrarily " group comprises

or

wherein respectively

be hydrogen independently, the C that can replace according to following definitions _1-6aliphatic, undersaturated-OPh, or have saturated independently selected from the 0-4 of nitrogen, oxygen or sulphur heteroatomic 5-6-unit, part is undersaturated or aromatic ring, or, except definition above, two are independently

and the middle atom inserted form have 0-4 unit of the heteroatomic 3-12 independently selected from nitrogen, oxygen or sulphur saturated, part is undersaturated, or fragrant monocycle or dicyclo.

?

suitable substituting group on aliphatic group be independently halogen ,-R ^.,-(halogen R ^.) ,-OH ,-OR ^.,-O (halogen R ^.) ,-CN ,-C (O) OH ,-C (O) OR ^.,-NH ₂,-NHR ^.,-NR ^. ₂, or-NO ₂, each R wherein ^.for undersaturated or its front, mean only by one or more halogen atoms, to be replaced during with " halogen ", and it is independently selected from C _1-4aliphatic ,-CH ₂ph ,-O (CH ₂) _0-1ph, or 5-6 unit saturated rings, the unsaturated ring of part or there is 0-4 the heteroatomic aromatic ring independently selected from nitrogen, oxygen or sulphur.

Detailed Description Of The Invention

The invention provides, except other aspects, can effectively treat, slow down or stop therapeutic combination and the method for mild cognitive impairment (MCI) or Alzheimer's disease (AD).

As described as the embodiment part, the present invention, partly, based on following unexpected the discovery: (1) known albumen composition PDS/TTR as early diagnosis MCI or Alzheimer's disease biomarker has neurotoxicity and induce Alzheimer's disease characteristic symptom and feature in cell culture; (2) dihydropyridine calcium channel blocker (as nifedipine), and oxidation, nitroso-derivative and mixture (it no longer has the function of calcium channel blocker), and/or T3/T4 effectively reduces or eliminates enzyme and the ability of biochemical pathway and the level that reduces endogenous A β 1-40 peptide in animal model that the PDS/TTR compound induces AD-sample symptom and impact wherein to relate in cell culture; And (3) studies show that of carrying out in human body used dihydropyridine calcium channel blocker obviously to postpone the initial of cognitive decline, this shows to use these compounds can effectively treat Alzheimer's disease.It is shocking, the inventor find oxidation, nitroso nifedipine derivative and mixture no longer have the function of calcium channel blocker.In some embodiments, nitroso nifedipine or derivatives thereof increases stream in calcium.Be not limited to any theory, the ability of these compounds for treating MCI or Alzheimer's disease of estimating may be independent of the ability of its retardance calcium channel.

More surprisingly, the inventor finds that lactam generates, reduces A β processive enzyme and make relevant biochemical route deactivation as NFD-L1 can effectively suppress A β 1-40 in vitro and in vivo, and this and nitroso nifedipine are similar.Be not limited to any theory, once estimate that the nitroso nifedipine is likely a kind of and can be converted into to stoichiometry the prodrug of lactam after giving in vivo.

Like this, the present invention relates to effectively to treat the method and composition of Alzheimer's disease, its nifedipine, oxidation or nitroso nifedipine derivative, lactam based on the treatment effective dose (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1), thyroxine (T4), trilute (T3) and combination thereof.In some embodiments, the invention provides in human subjects and treat, delay, or the method for prevention mild cognitive impairment (MCI) and/or Alzheimer's disease, comprise suffer from or easily suffer from MCI or Alzheimer's disease object treatment effective dose be selected from nifedipine, oxidized nifedipine, the nitroso nifedipine, lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1), thyroxine (T4), the reagent of trilute (T3) and combination thereof, at least one symptom relevant to MCI or Alzheimer's disease or feature are in abundance like this, intensity, the order of severity, or reduce on frequency, or delay to fall ill.In some embodiments, the present invention relates to effectively to treat the method and composition of Alzheimer's disease, compound and the combination thereof shown in its formula (Ia), (Ib), (Ic), (II) based on the treatment effective dose.In some embodiments, be suitable for the function that reagent of the present invention does not have calcium channel blocker.In some embodiments, be suitable for reagent of the present invention and increase stream in calcium.

Further contemplate that method of the present invention can with responsive biomarker and/or cognition detection scoring coupling, with identify the right patient of medical needle comprise in the disease commitment and monitoring therapeuticing effect.Like this, the present invention is specially adapted to treat the patient of commitment, those patients of mild cognitive impairment (MCI) symptom particularly occur and/or stops MCI to develop into Alzheimer's disease.

Describe different aspect of the present invention in detail in following chapters and sections.Use these chapters and sections not to be intended to limit the present invention.Each chapters and sections can be used in any aspect of the present invention.In this application, except separately having clearly, mean, otherwise refer to while using "or" " and/or ".

Therapeutic agent

Be suitable for therapeutic agent of the present invention and comprise that calcium channel blocker (for example, dihydropyridine calcium channel blocker is as nifedipine) and non-calcium channel blocker (for example, the mixture of oxidized nifedipine, nitrosoation-nifedipine, nifedipine and derivative thereof, thyroxine (T4), trilute (T3)).

In some embodiments, be suitable for therapeutic agent of the present invention suc as formula (Ia) or (Ib):

Or its pharmaceutically acceptable salt, wherein:

R ¹and R ²be C independently _1-6aliphatic or cyano group;

R ³and R ⁴c independently _1-6aliphatic;

N is 0,1,2 or 3.

In some embodiments, the compound shown in formula (Ia) is called to " reduction " or " dihydropyridines ".In some embodiments, the compound shown in formula (Ib) is called to " oxidation " or " dehydrogenation ".

In some embodiments, R ¹and R ²be C independently _1-3alkyl.In some embodiments, R ³and R ⁴be C independently _1-4alkyl.In some embodiments, R ¹and R ²for methyl.In some embodiments, R ³and R ⁴for methyl.

In some embodiments, be suitable for therapeutic agent of the present invention and refer to nifedipine, oxidized nifedipine or nitroso nifedipine.As used herein, " nitroso nifedipine " refers to oxidized nifedipine analog as shown below.

In some embodiments, being suitable for therapeutic agent of the present invention comprises, but be not limited to, dihydropyridine compounds is as Amlodipine, Aranidipine, Azelnidipine, Barnidipine, Benidipine, Cilnidipine, clevidipine, Efonidipine, felodipine, isradipine, lacidipine, Manidipine, Lercanidipine, nicardipine, nifedipine, Nilvadipine, Nimodipine, Nisoldipine, nitrendipine and Pranidipine.In some embodiments, being suitable for therapeutic agent of the present invention comprises, but be not limited to oxidation Amlodipine, oxidation Aranidipine, oxidation Azelnidipine, oxidation Barnidipine, oxidation Benidipine, oxidation Cilnidipine, oxidation clevidipine, oxidation Efonidipine, oxidation felodipine, oxidation isradipine, oxidation lacidipine, oxidation Manidipine, oxidation Lercanidipine, oxidation nicardipine, oxidized nifedipine, oxidation Nilvadipine, oxidation Nimodipine, oxidation Nisoldipine, oxidation nitrendipine and oxidation Pranidipine.It is the pyridine variant of described compound that those of ordinary skill in the art will understand " oxidation " dihydropyridine compounds (for example, oxidation Amlodipine, oxidation Nimodipine, oxidation Nilvadipine).

More exemplary treatment agent comprises following:

Wherein Z is H, F, Cl, Br or I;

Wherein Z is H, F, Cl, Br or I.

In some embodiments, be suitable for therapeutic agent of the present invention suc as formula shown in (Ic):

Or its pharmaceutically acceptable salt, wherein:

N is 0,1,2 or 3.

In some embodiments, be suitable for therapeutic agent of the present invention suc as formula shown in (Ic-i):

Or its pharmaceutically acceptable salt, wherein:

R ¹and R ²be C independently _1-6aliphatic or cyano group;

R ³c _1-6aliphatic;

N is 0,1,2 or 3.

As common definition above, R in formula (Ic) ¹for being selected from C _1-6aliphatic, C _1-6the optional substituted radical of assorted aliphatic, aromatic radical, assorted aromatic radical or cyano group.In some embodiments, R ¹replace.In some embodiments, R ¹unsubstituted.In some embodiments, R ¹c _1-6aliphatic.In some embodiments, R ¹c _1-4alkyl.In some embodiments, R ¹methyl, ethyl, propyl group, butyl or isopropyl.In some embodiments, R ¹it is methyl.In some embodiments, R ¹it is isopropyl.In some embodiments, R ¹cyano group.In some embodiments, R ¹c _1-6assorted aliphatic.In some embodiments, R ¹be-OCH ₂cH ₂nH ₂.In some embodiments, R ¹it is aromatic radical.In some embodiments, R ¹it is assorted aromatic radical.

As common definition above, R in formula (Ic) ²for being selected from C _1-6aliphatic, C _1-6the optional substituted radical of assorted aliphatic, aromatic radical, assorted aromatic radical or cyano group.In some embodiments, R ²replace.In some embodiments, R ²unsubstituted.In some embodiments, R ²c _1-6aliphatic.In some embodiments, R ²c _1-4alkyl.In some embodiments, R ²methyl, ethyl, propyl group, butyl or isopropyl.In some embodiments, R ²it is methyl.In some embodiments, R ²it is isopropyl.In some embodiments, R ²cyano group.In some embodiments, R ²c _1-6assorted aliphatic.In some embodiments, R ²be-OCH ₂cH ₂nH ₂.In some embodiments, R ²it is aromatic radical.In some embodiments, R ²it is assorted aromatic radical.

In some embodiments, R ¹and R ²be C independently _1-3alkyl.In some embodiments, R ¹and R ²in at least one be methyl.In some embodiments, R ¹and R ²it is methyl.

As unified definition above, the R in formula (Ic) ³the C that is selected from of optional replacement _1-6aliphatic, C _1-6the group of assorted aliphatic or aryl.In some embodiments, R ¹substituted.In some embodiments, R ¹unsubstituted.In some embodiments, R ¹c _1-6aliphatic.In some embodiments, R ¹c _1-4alkyl.In some embodiments, R ¹methyl, ethyl, propyl group, butyl or isopropyl.In some embodiments, R ¹it is methyl.In some embodiments, R ¹it is isopropyl.In some embodiments, R ¹it is ethyl.In some embodiments, R ¹c _1-6assorted aliphatic.In some embodiments, R ¹be-CH ₂cH ₂oCH ₃.In some embodiments, R ¹it is aromatic radical.

As unified definition above, R in formula (Ic) ⁵be halogen, the optional C replaced _1-6aliphatic, the optional C replaced _1-6assorted aliphatic, hydroxyl, alkoxyl, amino, alkylamino, cyano group, nitro or nitroso.In some embodiments, R ⁵replace.In some embodiments, R ⁵unsubstituted.In some embodiments, R ⁵c _1-6aliphatic.In some embodiments, R ⁵c _1-4alkyl.In some embodiments, R ⁵methyl, ethyl, propyl group, butyl or isopropyl.In some embodiments, R ⁵it is methyl.In some embodiments, R ⁵cyano group.In some embodiments, R ⁵it is halogen.In some embodiments, R ⁵c _1-6assorted aliphatic.In some embodiments, R ⁵it is hydroxyl.In some embodiments, R ⁵it is alkoxyl.In some embodiments, R ⁵amino.In some embodiments, R ⁵it is alkylamino.In some embodiments, R ⁵it is nitro.In some embodiments, R ⁵it is nitroso.

As unified definition above, the n in formula (Ic) is 0,1,2 or 3.In some embodiments, n is 0.In some embodiments, n is 1.In some embodiments, n is 2.In some embodiments, n is 3.

In some embodiments, being suitable for therapeutic agent of the present invention is NFD-L1.

More exemplary treatment agent comprises following:

In some embodiments, be suitable for therapeutic agent of the present invention suc as formula shown in (II):

Or its pharmaceutically acceptable salt, wherein:

X is-CH ₂-,-O-or-NH-; With

Y is-H or-I.

In some embodiments, X is-CH ₂-.In some embodiments, X is-O-.In some embodiments, X is-NH-.

In some embodiments, being suitable for therapeutic agent of the present invention is thyroxine (T4) or trilute (T3):

In some embodiments, be suitable for the mixture that therapeutic agent of the present invention is different compounds as herein described.For example, can be by two or more formulas (Ia) or the compound combination (Ib) to form therapeutic agent.In some embodiments, by two or more combinations in nifedipine, oxidized nifedipine and nitroso nifedipine.In some embodiments, by one or more combinations in T3 and/or T4 and nifedipine, oxidized nifedipine and nitroso nifedipine.In some embodiments, nifedipine, oxidized nifedipine and nitroso nifedipine are combined to form the nifedipine mixture.

Can be with different quality or mol ratio by compound.For example, therapeutic agent of the present invention can be two or more mixtures that prepare with predetermined treatment or mol ratio in nifedipine, oxidized nifedipine, nitroso nifedipine, NFD-L1, thyroxine (T4) and trilute (T3).In some embodiments, be suitable for the mixture that therapeutic agent of the present invention contains nitroso nifedipine and nifedipine.In some embodiments, nitroso nifedipine and nifedipine can with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500 or approximately mix at 1: 1000.In some embodiments, nitroso nifedipine and nifedipine can with quality or molar ratio range approximately 1: 1000 for example, to approximately 1000: 1 (, approximately 1: 500 to approximately 500: 1, approximately 1: 200 to approximately 200: 1, approximately 1: 100 to approximately 100: 1, approximately 1: 10 to approximately 10: 1, approximately 1: 16 to approximately 16: 1, approximately 1: 32 to approximately 32: 1, approximately 1: 64 to approximately 64: 1, approximately 1: 1 to approximately 32: 1, approximately 1: 1 to approximately 10: 1, approximately 100: 1 to approximately 1000: 1, approximately 10: 1 to approximately 100: 1, approximately 1: 1000 to 1: 1, approximately 1: 1 to approximately 1000: 1, or approximately 1: 100 to approximately 1: 10) mix.In some embodiments, be suitable for the mixture that therapeutic agent of the present invention contains oxidized nifedipine and nifedipine.In some embodiments, oxidized nifedipine and nifedipine can with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500 or approximately mix at 1: 1000.In some embodiments, oxidized nifedipine and nifedipine can with quality or molar ratio range approximately 1: 1000 for example, to approximately 1000: 1 (, approximately 1: 500 to approximately 500: 1, approximately 1: 200 to approximately 200: 1, approximately 1: 100 to approximately 100: 1, approximately 1: 10 to approximately 10: 1, approximately 1: 16 to approximately 16: 1, approximately 1: 32 to approximately 32: 1, approximately 1: 64 to approximately 64: 1, approximately 1: 1 to approximately 32: 1, approximately 1: 1 to approximately 10: 1, approximately 100: 1 to approximately 1000: 1, approximately 10: 1 to approximately 100: 1, approximately 1: 1000 to 1: 1, approximately 1: 1 to approximately 1000: 1, or approximately 1: 100 to approximately 1: 10) mix.In some embodiments, be suitable for the mixture that therapeutic agent of the present invention contains nitroso nifedipine and oxidized nifedipine.In some embodiments, nitroso nifedipine and oxidized nifedipine can with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500 or approximately mix at 1: 1000.In some embodiments, nitroso nifedipine and oxidized nifedipine can with quality or molar ratio range approximately 1: 1000 for example, to approximately 1000: 1 (, approximately 1: 500 to approximately 500: 1, approximately 1: 200 to approximately 200: 1, approximately 1: 100 to approximately 100: 1, approximately 1: 10 to approximately 10: 1, approximately 1: 16 to approximately 16: 1, approximately 1: 32 to approximately 32: 1, approximately 1: 64 to approximately 64: 1, approximately 1: 1 to approximately 32: 1, approximately 1: 1 to approximately 10: 1, approximately 100: 1 to approximately 1000: 1, approximately 10: 1 to approximately 100: 1, approximately 1: 1000 to 1: 1, approximately 1: 1 to approximately 1000: 1, or approximately 1: 100 to approximately 1: 10) mix.In some embodiment, be suitable for the mixture that therapeutic agent of the present invention contains nitroso nifedipine, oxidized nifedipine and nifedipine.In some embodiments, nitroso nifedipine, oxidized nifedipine and nifedipine mix with quality or mol ratio in approximately 5: 1: 3,5: 2: 2,6: 3: 1,10: 4: 1,3: 1: 5,2: 5: 5 or 1: 1: 1.In some embodiments, the mixture that therapeutic agent contains T3 and T4.In some embodiments, T3 and T4 can with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500 or approximately mix at 1: 1000.In some embodiments, T3 and T4 can be mass or molar ratio in the range from about 1:1000 to about 1000:1 (e.g., from about 500:1 to about 1:500, about 1:200 to about 200:1, from about 100:1 to about 1:100, from about 1:10 to about 10:1, about 1:16 to about 16:1, about 1:32 to about 32:1, about 1:64 to about 64:1, about 1:1 to about 32:1, about 1:1 to about 10:1, about 100:1 to about 1000:1, from about 10:1 to about 100:1, from about 1:1000 to about 1:1, from about 1 : 1 to about 1000, or about 1:100 to about 1:10) mixing.In some embodiments, can carry out further combination to produce therapeutic agent required for the present invention to different compounds as herein described and mixture.For example, the T3/T4 mixture can with nifedipine as herein described, nifedipine derivative (for example, oxidation or nitroso nifedipine) or nifedipine mixture in any one combination.

In some embodiments, be suitable for therapeutic agent of the present invention comprise nitroso nifedipine and lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) mixture.In some embodiments, nitroso nifedipine and lactam are (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) can be with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500, or approximately mix at 1: 1000.In some embodiments, nitroso nifedipine and lactam are (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) can with quality or molar ratio range approximately 1: 1000 for example, to approximately 1000: 1 (, approximately 1: 500 to approximately 500: 1, approximately 1: 200 to approximately 200: 1, approximately 1: 100 to approximately 100: 1, approximately 1: 10 to approximately 10: 1, approximately 1: 16 to approximately 16: 1, approximately 1: 32 to approximately 32: 1, approximately 1: 64 to approximately 64: 1, approximately 1: 1 to approximately 32: 1, approximately 1: 1 to approximately 10: 1, approximately 100: 1 to approximately 1000: 1, approximately 10: 1 to approximately 100: 1, approximately 1: 1000 to 1: 1, approximately 1: 1 to approximately 1000: 1, or approximately 1: 100 to approximately 1: 10) mix.In some embodiments, be suitable for therapeutic agent of the present invention contain lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) and the mixture of oxidized nifedipine.In certain embodiments, a lactam (or compounds represented by the formula (Ic) (Ic-i), as NFD-L1) and nifedipine oxide mass or molar ratio may be from about 1000:1 to about 500 : 1, about 200:1, about 100:1, about 64:1, about 32:1, about 16:1, about 10:1, about 8:1, about 5:1, about 4:1, about 3 : 1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:8, about 1:10, about 1:16, about 1 : 32, about 1:64 to about 1:100, about 1:200, about 1:500, or about 1:1000 mixing.In some embodiments, lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1) and oxidized nifedipine can with quality or molar ratio range approximately 1: 1000 for example, to approximately 1000: 1 (, approximately 1: 500 to approximately 500: 1, approximately 1: 200 to approximately 200: 1, approximately 1: 100 to approximately 100: 1, approximately 1: 10 to approximately 10: 1, approximately 1: 16 to approximately 16: 1, approximately 1: 32 to approximately 32: 1, approximately 1: 64 to approximately 64: 1, approximately 1: 1 to approximately 32: 1, approximately 1: 1 to approximately 10: 1, approximately 100: 1 to approximately 1000: 1, approximately 10: 1 to approximately 100: 1, approximately 1: 1000 to 1: 1, approximately 1: 1 to approximately 1000: 1, or approximately 1: 100 to approximately 1: 10) mix.In some embodiments, be suitable for therapeutic agent of the present invention contain lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1), the mixture of oxidized nifedipine and nifedipine.In some embodiments, lactam (for example, formula (Ic) or (Ic-i) shown in compound, as NFD-L1), oxidized nifedipine and nifedipine mix with quality or mol ratio in approximately 5: 1: 3,5: 2: 2,6: 3: 1,10: 4: 1,3: 1: 5,2: 5: 5 or 1: 1: 1.In some embodiments, the mixture that therapeutic agent contains T3 and T4.In some embodiments, T3 and T4 can with quality or mol ratio approximately 1000: 1, approximately 500: 1, approximately 200: 1, approximately 100: 1, approximately 64: 1, approximately 32: 1, approximately 16: 1, approximately 10: 1, approximately 8: 1, approximately 5: 1, approximately 4: 1, approximately 3: 1, approximately 2: 1, approximately 1: 1, approximately 1: 2, approximately 1: 3, approximately 1: 4, approximately 1: 5, approximately 1: 8, approximately 1: 10, approximately 1: 16, approximately 1: 32, approximately 1: 64, approximately 1: 100, approximately 1: 200, approximately 1: 500 or approximately mix at 1: 1000.In some embodiments, T3 and T4 can be mass or molar ratio in the range from about 1:1000 to about 1000:1 (e.g., from about 500:1 to about 1:500, about 1:200 to about 200:1, from about 100:1 to about 1:100, from about 1:10 to about 10:1, about 1:16 to about 16:1, about 1:32 to about 32:1, about 1:64 to about 64:1, about 1:1 to about 32:1, about 1:1 to about 10:1, about 100:1 to about 1000:1, from about 10:1 to about 100:1, from about 1:1000 to about 1:1, from about 1 : 1 to about 1000, or about 1:100 to about 1:10) mixing.In some embodiments, different compounds described herein and mixture can further combine, to produce therapeutic agent required for the present invention.For example, the T3/T4 mixture can with for example, compound shown in nifedipine as herein described, nifedipine derivative (, oxidation or nitroso nifedipine), formula (I-c) (for example, NFD-L1) or the combination of nifedipine mixture.

Biomarker for the identification of patient or monitor therapy

Can use different biomarkers to differentiate and suffer from, easily suffer from MCI or Alzheimer's disease or have object or the patient of ill risk.As used herein, biomarker is a kind of distinctive biomolecule, and there is the situation difference in it in for example, for example, sample from a kind of phenotype state (, ill) and another kind of phenotype state (, anosis) object.If as calculated not on the same group in the mean value of biomarker expression or median have the significant difference with statistical significance, think that biomarker there are differences between different phenotype states.Biomarker, be used alone or in combination, and the relative risk that provides object to belong to one or another kind of phenotype state detects.Thereby it is the useful label of judgement disease (diagnosis), curative effect of medication (diagnosis and treatment) and drug toxicity.

For example, the inventor delivered recently contain prostaglandin-D2-synzyme and thyroxine transport protein～level of 55kD protein complex (PDS/TTR compound) can be used as susceptibility and the specific diagnosis biomarker of MCI and AD, refer to U.S. Patent Publication No. 2008/0026405, its disclosure is incorporated in this reference.

Typically, the PDS/TTR compound is present in cerebrospinal fluid, and it is a kind of susceptibility and specific biological label of disease.The formation of PDS/TTR compound is positioned choroid plexus, and epithelial assembling is positioned near telocoele.The function of choroid plexus is blood-CSF barrier.Choroid plexus makes water, salt and specific little molecule enter CSF from blood, but the protein in effective tissue blood enters CSF.The required albumen of CSF is synthesized by choroid plexus.Like this, choroid plexus also has the function as the CSF source.The epithelial cell that will separate from the fresh choroid plexus that AD patient's autopsy examination obtains to late period is cultivated and increases medium.The epithelial cell culture medium of AD is checked to the PDS/TTR compound level that rear discovery is compared with compared with control cells wherein raises.Thereby, can for discriminating, suffer from compare the level of the PDS/TTR compound with rising with normal control, easily suffer from MCI or Alzheimer's disease or have object or the patient of ill onset risk.

In some embodiments, be suitable for biomarker of the present invention and comprise at least one in thyroxine transport protein and/or prostaglandin-H2D isomerase albumen, and be selected from thyroxine transport protein, prostaglandin-H2D isomerase, beta-2-microglobulin, bladder chalone C, superoxide dismutase [Cu--Zn], blood plasma retinol-in conjunction with albumen, phosphatidyl-ethanolamine-in conjunction with at least one in albumen, carbonic anhydrase 2 and/or transferrins.By the relation between acquisition and standard items testing result, determine the situation of mild cognitive impairment or Alzheimer's disease.

In some embodiments, can use NF-M, tau (total protein; T-tau and different phosphorylation form; P-tau) and/or the derivative of amyloid precusor protein (APP), comprise A β ₄₀with A β ₄₂as the biomarker of differentiating the patient crowd who uses the present composition and method treatment.In some embodiments, need the object for the treatment of to have compared with the control abnormal protein biology marker complex level, wherein the protein biology marker complex comprises (i) beta-amyloyd 40 (A β 40), (ii) beta-amyloyd 42 (A β 42), (iii) A β 40 and the ratio of A β 42 and (iv) one or more in the ratio of the tau of phosphorylation and total tau.

The other biological label of reporting in the literature can be used for differentiate that the patient that the present invention treats includes, but not limited to Fahnestock etc., J.Neural.Transm.Suppl.2002 (62): 241-52 (2002); Masliah etc., Neurobiol.Aging 16 (4): 549-56 (1995); Power etc., Dement.Geriatr.Cong.Disord.12 (2): 167-170 (2001); Burbach etc., J.Neurosci.24 (10): 2421-30 (2004), Li et al, Neuroscience 113 (3): 607-15 (2002) and Sanna etc., those that describe in J.Clin.Invest.111 (2): 241-50 (2003), its disclosure is incorporated in this reference.

In some embodiments, utilize the fluid sample obtained from object to measure biomarker.In some embodiments, fluid sample is selected from CSF, serum, whole blood, blood plasma, urine, ascites, saliva, organizes diffusate, irrigating solution and combination thereof.

Can use several different methods to carry out the quantitative and qualitative analysis detection to biomarker.For example, when detecting albumen composition (as PDS/TTR), can utilize the first component in sandwich enzyme-linked immunity test (ELISA) capture complexes (for example, PDS), to utilize probe to catch the second component (for example, TTR).Other illustrative methods is referring to US patent publication No. 2008/0026405, and it is incorporated to this paper by reference.Other method is well known in the art, can be for the present invention.

Typically, the detection level of biomarker is contrasted with one or more or reference level compares.Those of ordinary skill in the art will understand, and according to the different aspect of invention, can change the suitable reference level for comparing with AD biomarker detection level.Suffer from or easily suffer from the object of AD or MCI susceptible for discriminating, suitable " reference level " is generally the level shown in healthy individual, especially, is the healthy individual of age-matched.Reference level can with patient's sample Parallel testing.Reference level can also be predetermined level or the level based on the past data.For example, suitable reference level can be never to suffer from the average level obtained in the crowd of AD or MCI.Typically, the level that suitable reference level obtains in the crowd of age-matched (for example, mean value or median).

Typically, with healthy individual or crowd contrast or reference level is compared, the individual biomarker for the treatment of that needs as herein described have level higher or that raise.

For the purpose for the treatment of monitoring, suitable reference level is generally healthy individual or suffers from for example, level as shown in the individuality of Alzheimer's disease (, having pre-determined the stage, as MCI, EAD or LAD).Reference level can with patient's sample Parallel testing.Reference level can also be predetermined level or the level based on the past data.For example, suitable level can be from the crowd who does not suffer from AD or MCI, or has been diagnosed as the crowd's of MCI or AD (for example, EAD or LAD) average level.Perhaps, suitable reference level can be the past reference level of particular patient, for example, and from the level of same individual sample, but time point more early (for example, before the treatment or in treatment time point) more early.Typically, the level that suitable reference level obtains in the crowd of age-matched (for example, mean value or median).

Fen Lei be (not for AD patient is pressed to layer, by AD patient be divided into slightly, the layered approach of moderate and severe stage A D), suitable reference level is general for example, since (being diagnosed as moment AD, EAD or LAD) or the crowd of MCI in the level (for example, mean value or median) that obtains.

In some embodiments, can use suitable biomarker (as～55kDa PDS/TTR compound) level monitoring curative effect.Typically, desirable therapeutic purpose is the PDS/TTR compound level in reduction or reduced objects, does not contain so detectable compound in the fluid sample obtained from object.More conservative, auxiliary therapeutic purpose is for stoping the increase of any～55kDa PDS/TTR compound level.Therefore, the those of ordinary skill of medical field can determine, based on suitable biomarker level, whether given therapeutic scheme has reached selected therapeutic purpose.In this way, the those of ordinary skill of medical field can also contrast or reference level compares with suitable by the mensuration level to suitable biomarker, to determine the effective dose of therapeutic agent as herein described.

Typically, use the crowd of age-matched to determine different reference level.Ideally, the crowd of age-matched has the identical age with individuality to be measured, but the crowd of age approximate match is also to accept.The crowd of age approximate match and the age gap of individuality to be measured can 1,2,3,4 or 5 years old in, or it can be the age groups that comprises Individual Age to be measured.Age approximate match crowd's increment can 2,3,4,5,6,7,8,9 or 10 years old for example, with interior (, " the 5 years old increment " group as 62 years old older individuals reference point can comprise 58-62 year older individuals, 59-63 year older individuals, 60-64 year older individuals, 61-65 year older individuals or 62-66 year older individuals).

The process that detected value and reference point are compared can be by being suitable for determining that any conventional method of AD biomarker detected value and reference point type realizes.For example, can adopt quantitatively or the qualitative detection technology is carried out " detections ", can be according to adopted detection technique difference, the mode that change compares detected value and reference point.For example, the detected value used in method of the present invention is the most common is quantitative values (for example, the quantitative detection of concentration, as the nanogram number of AD label in every ml sample, or absolute quantity).When adopting qualitative detection, can pass through the check number Value Data, the expression of check data (that is, check diagram is as block diagram or line chart) compares.As a non-limitative example, if measured value be at least about reference point 95% the time, it is generally acknowledged that measured value is substantially equal to or is greater than reference point (for example, can think that measured value 1.71 is substantially equal to reference point 1.80).If measured value be less than reference point 95% the time, think that measured value is less than or for example, lower than reference point (, can think that measured value 1.7 is less than reference point 1.80).

Cognitive function detects

Can also adopt multiple cognition detection method to differentiate object or the patient who suffers from, easily suffers from or exist MCI or Alzheimer's disease risk.Two kinds of exemplary cognitive detection methods are simple and easy mental status inspection (MMSE) and clinical dementia grade (CDR) scoring.

In some embodiments, adopt the MMSE scoring to differentiate the object that needs the compositions and methods of the invention treatment.The comprehensive grading that the MMSE scoring detects for relating to multiple cognitive function.The peak of the MMSE total points that may exist is 30 minutes.Can adopt MMSE to be classified to the seriousness of dementia or other diseases situation Patients ' Cognitive obstacle.Table 1 has shown the MMSE scoring of ordinary representation cognitive function degree.

Table 1

The MMSE scoring	Cognitive function
		27-30	Normal cognitive function
21-26	Mild cognitive impairment
		11-20	The moderate cognitive disorder
0-10	The severe cognitive disorder

In some embodiments, needing the MMSE scoring for the treatment of target is 21-26 (mild cognitive impairment), 11-20 (moderate cognitive disorder) or 0-10 (severe cognitive disorder).

In some embodiments, adopt the CDR scoring to differentiate the object that needs the compositions and methods of the invention treatment.CDR scoring consists of the parts of six independent scorings: memory, directed, judge and deal with problems, community's affairs, family and hobby and personal nursing.Table 2 has shown the CDR scoring of ordinary representation cognitive function degree.

Table 2

The MMSE scoring	Cognitive function
		0	Without cerebral damage
0.5	Utmost point mild dementia
		1	Mild dementia
2	Moderate dementia
		3	Severe dementia

In some embodiments, CDR scoring may be suffered from, easily suffer from higher than 0 indicated object or be had MCI or an Alzheimer's disease risk.In some embodiments, needing the CDR scoring for the treatment of target may be 0.5 (utmost point mild dementia), 1 (mild dementia), 2 (moderate dementia) or 3 (severe dementia).

In some embodiments, can adopt cognition detection scoring (as MMSE scoring or CDR scoring) monitoring curative effect.Typically, effectively treatment should improve the cognition detection scoring.Thereby, by the relatively cognition detection scoring for the treatment of front and back or therapeutic scheme different time points, the those of ordinary skill of medical field can determine that whether given therapeutic scheme is effective.For example, relative cognition detection front based on treatment or that the therapeutic scheme different time points is determined is marked, and the effective dose of therapeutic agent described herein can be determined or adjust to the those of ordinary skill of medical field.

Pharmaceutical composition and giving

The present invention comprises the pharmaceutical composition that comprises those therapeutic agents as herein described.In some embodiments, pharmaceutical composition of the present invention comprises therapeutic agent and the pharmaceutically acceptable carrier for the treatment of effective dose.

As used herein, that term " pharmaceutically acceptable carrier " refers to is avirulent, inert solid, semi-solid liquid filling agent, thinner, and encapsulating material, the preparation assistant agent of any type, or be only sterile aqueous matrix, as salt solution.Can as some example of the material of pharmaceutically acceptable carrier, be sugar, as lactose, dextrose plus saccharose, starch be as corn starch and potato starch, and cellulose and derivative thereof are as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; The powdery tragcanth; Fructus Hordei Germinatus, gelatin, talcum powder; Excipient is as cocoa butter and suppository wax; Oil is as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycol is as propane diols, and polyalcohol is as glycerine, sorbierite, mannitol and polyethylene glycol; The ester class is as ethyl oleate and ethyl laurate, agar; Buffer is as magnesium hydroxide and aluminium hydroxide; Alginic acid; Apirogen water; Isotonic saline solution, Lin Geshi liquid; Ethanol and phosphate buffer, and other nontoxic compatible materials that use in pharmaceutical preparation.

According to the Formulation principle, in composition, can also exist wetting agent, emulsifier and lubricant as lauryl sodium sulfate and dolomol and colouring agent, releasing agent, seed coating medicine, sweetener, flavouring and flavouring agent, preservative and antioxidant.The example of pharmaceutically acceptable antioxidant includes, but not limited to water soluble antioxidant as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfite, sodium metabisulfite, sodium sulphite etc.; Oil-soluble inhibitor, as ascorbyl palmitate, BHA (BHA), BHT (BHT), fourth of the twelve Earthly Branches phosphatide, n-propyl gallate, alpha-tocopherol etc.; And metal-chelator is as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid etc.

" the treatment effective dose " of term therapeutic agent or referred to as " effective dose ", as used herein, finger needs the therapeutic agent for the treatment of target to be enough to alleviate, to improve, to stablize and/or postponement and MCI according to suitable scheme, or the progress (for example, postponing the progress of abundance, intensity, seriousness or frequency) of initial and postponement MCI or the Alzheimer's disease of at least one symptom relevant to Alzheimer's disease or feature.Yet, be appreciated that and use accumulated dose the every day that can determine therapeutic agent of the present invention and composition in the scope of medical worker's medical diagnosis.Any specific patient's specific treatment effective dose level will change according to many factors, comprise treated illness and the order of severity of illness; The activity of specific compound used; Particular composition used; Patient's age, body weight, health status, sex and diet; The time that gives of specific compound used, give approach and excretion rate; The treatment duration; Medicine with specific compound coupling used or overlapping use; And the known similar factor of medical field.

In some embodiments, the treatment effective dosage ranges of therapeutic agent of the present invention can be, for example, and 0.01 to 100mg/kg body weight or higher.In some embodiments, the treatment effective dosage ranges of therapeutic agent of the present invention is approximately 0.1 for example, to about 50mg/kg body weight (, approximately 0.1 to about 35mg/kg, approximately 0.1 to about 15mg/kg, approximately 6.25 to about 35mg/kg, approximately 12.5 to about 35mg/kg, approximately 6.25 to about 25mg/kg, about 35mg/kg).In some embodiments, the treatment effective dose scope of therapeutic agent is for example the about 0.01mg of every dosage, to about 2.5g (, about 0.01mg is to about 2.0g, about 0.01mg to about 1.5g, about 0.01mg to about 1.0g, every dosage).In some embodiments, the treatment effective dose scope of therapeutic agent is every dosage approximately 0.01 to about 1000mg (for example, approximately 0.01 to about 500mg, approximately 0.01 to about 250mg, approximately 0.01 to about 200mg, approximately 0.01 to about 150mg, approximately 0.01 to about 100mg, approximately 0.01 to about 50mg, approximately 0.01 to about 10mg, approximately 0.01 to about 5mg, approximately 0.01 to about 2.5mg, approximately 0.01 to about 2.0mg, approximately 0.01 to about 1.5mg, approximately 0.01 to about 1.0mg, approximately 0.01 to about 0.5mg, approximately 0.01 to about 0.1mg).In some embodiments, therapeutic agent (especially, the nitroso nifedipine) effective dose scope is that the about 100mg of every dosage for example, to about 5g (, about 100mg is to about 3g, about 100mg to about 2.5g, about 100mg to about 2g, about 100mg to about 1.5g, about 100mg to about 1000mg, about 100mg to about 500mg, about 100mg to about 250mg).In some embodiments, the treatment effective dose of therapeutic agent can be the about 0.01mg of every dosage, about 0.05mg, about 0.1mg, about 0.5mg, about 1mg, about 5mg, about 10mg, about 25mg, about 50mg, about 100mg, about 500mg, about 1000mg, about 1.5g, about 2g, about 2.5g, about 3g or about 5g.Typically, the total amount that amount as herein described is all reactive compounds in composition.For example, if contain the mixture of nifedipine, nitroso nifedipine and oxidized nifedipine in composition, treating effective dose is nifedipine, nitroso nifedipine and oxidized nifedipine amount sum.

In some embodiments, the treatment effective dose of therapeutic agent described herein for example, for being not enough to induce the amount of bad reaction (, hepatotoxicity wind agitation) in human subjects.

In some embodiments, therapeutic agent as herein described gives once every day.In some embodiments, therapeutic agent as herein described gives repeatedly every day, for example every day twice, three times or four times.In some embodiments, total dose range every day of therapeutic agent be every day multiple dose or single dose for example give about 0.01mg, to about 5g (, every day, multiple dose or single dose gave about 0.01mg to about 4.0g, about 0.01mg to about 3.0g, about 0.01mg to about 2.5g, about 0.01mg to about 2.0g, about 0.01mg to about 1.5g, about 0.01mg to about 1.0g).In some embodiments, total dose range every day of therapeutic agent be every day single dose or multiple dose give approximately 0.01 to about 1000mg (for example,, approximately 0.01 to about 500mg, approximately 0.01 to about 250mg, approximately 0.01 to about 200mg, approximately 0.01 to about 150mg, approximately 0.01 to about 100mg, approximately 0.01 to about 50mg, approximately 0.01 to about 10mg, approximately 0.01 to about 5mg, approximately 0.01 to about 2.5mg, approximately 0.01 to about 2.0mg, approximately 0.01 to about 1.5mg, approximately 0.01 to about 1.0mg, approximately 0.01 to about 0.5mg, approximately 0.01 to about 0.1mg.In some embodiments, therapeutic agent (especially, the nitroso nifedipine) total dose range every day be every day multiple dose or single dose for example give about 50mg, to about 5g (, about 50mg is to about 4g, about 100mg to about 3g, about 100mg to about 2.5g, about 100mg to about 2g, about 100mg to about 1.5g, about 100mg to about 1000mg, about 100mg to about 500mg, about 100mg to about 250mg).In some embodiments, every TDD of therapeutic agent can be about 0.01mg, about 0.05mg, about 0.1mg, about 0.5mg, about 1mg, about 5mg, about 10mg, about 25mg, about 50mg, about 100mg, about 500mg, about 1000mg, about 1.5g, about 2g, about 2.5g, about 3g, about 3.5g, about 4g, about 4.5g or about 5g.Typically, the total amount that amount as herein described is all reactive compounds in composition.For example, if contain the mixture of nifedipine, nitroso nifedipine and oxidized nifedipine in composition, treating effective dose is nifedipine, nitroso nifedipine and oxidized nifedipine amount sum.

In some embodiments, therapeutic agent as herein described per month, every two weeks, weekly, twice or time give on every Wendesdays weekly.In these examples, giving dosage every day mentioned above is the mean value that gives dosage every day.

In some situation, maintaining higher activating agent dosage in patient's blood may be very important, particularly early stage what treat.Therefore, at least in the starting stage, preservation dose is relatively high/or in the preset time section level of substantial constant, for example at least about six hours or above, for example at least about 12 hours or above, for example at least about twenty four hours or more than, may be very important.

Compound of the present invention can give separately or with the other drug that affects central or peripheral nervous system, particularly for the specific brain regions district, associating or simultaneously give.

Pharmaceutical composition of the present invention can give with any approach, comprises oral, subcutaneous, vein, transdermal, abdominal cavity, intramuscular, in the ventricles of the brain, brain essence, in sheath, encephalic, oral cavity, mucous membrane, in nose, rectum, in ear, conjunctiva, epidermis, electric osmose, in uterine neck, in nasal sinus, in tracheae, in intestines, outside dura mater, outside amnion, external, haemodialysis, infiltration, interstitial, in abdomen, in amnion, in artery, in joint, in courage, in bronchi, in capsule, intracardiac, in cartilage, in chamber, in brain, in the brain pond, in cornea, in hat, in coronary artery, in cavernous body, intracutaneous, in dish, in pipe, in duodenum, in dura mater, in epidermis, in esophagus, in stomach, in gums, in ileum, in focus, in lymphatic vessel, in marrow, in meninges, intramuscular, intraocular, in ovary, in pericardium, in pleura, in prostate, in lung, in hole, in synovial membrane, in tendon, in sheath, in thorax, in tubule, in knurl, in tympanum, in uterus, in blood vessel, intravenous injection, intravenous drip, in ventricle, in bladder, in vitreum, iontophoresis, lavation, throat, nasal feeding, the occlusive dressing technology, eye is used, oropharynx, outside stomach and intestine, through skin, outside dura mater, enclose nerve, periodontal, respiratory tract, after ball, soft tissue, under arachnoid, under conjunctiva, hypogloeeis, under mucous membrane, local, transdermal, saturating mucous membrane, through placenta, through tracheae, through tympanum, ureter, urethra, with vagina, give.In some embodiments, in oral via being selected from, subcutaneous, the vein of pharmaceutical composition of the present invention, transdermal, abdominal cavity, intramuscular, the ventricles of the brain, in brain essence, sheath, in encephalic, oral cavity, mucous membrane, nose and the approach that gives of rectum give.In some embodiments, drug composition oral of the present invention gives.

Comprise pharmaceutically acceptable emulsion, micro emulsion, solution, suspension, syrup and elixir for the oral liquid dosage form given, wherein contain this area inert diluent commonly used, as water, isotonic solution or salt solution.Such composition can also comprise, as assistant agent, as wetting agent; Emulsifier and supensoid agent; Sweetener, flavouring and flavouring agent.

Can use suitable dispersion or wetting agent and supensoid agent to prepare injectable formulation according to the common practise of this area, for example sterile water for injection or oil-based suspension.Sterile injectable preparation can also be sterile injectable solution, suspension or emulsion in nontoxic injection thinner or solvent, for example, and the solution in 1,3-BDO.Operable carrier and solvent are water, ringer's solution, U.S.P. and isotonic sodium chlorrde solution.In addition, usually use aseptic, fixed oil as solvent or suspendible matrix.That for this purpose, can use any gentleness can not comprise synthetic monoglyceride or diglyceride by the property sent out oil.In addition, in injectable formulation, can use fatty acid as oleic acid.

The injectable formulation sterilizing for example can be filtered by the filter of holding back bacterium, or, by adding bactericidal agent to form aseptic solid composite, can before use it be dissolved in or be dispersed in sterile water or other sterile injectable matrix.

For the effect of prolong drug, the absorption of the medicine while giving by subcutaneous or intramuscular injection of usually wishing to slow down.Reach the suspension that the most conventional method of this purpose is exactly the poor crystal of injection water dissolubility or indefiniteness material.The absorption rate of medicine depends on the dissolution rate of medicine, and the dissolution rate of medicine depends on the physical state of medicine conversely, for example crystalline size and crystal structure.The another kind of method that drug absorption is postponed gives for the form with solution or suspension in oil.Can also form matrix and biodegradable polymer by medicament microcapsule and prepare the Injectable depot form as polyactide-poly-glycosides.By controlling the ratio of medicine and polymer and composition and polymer, can control the rate of release of medicine.Other examples of biodegradable polymer comprise poe and poly-acid anhydrides.Can also prepare Injectable depot by drug encapsulation being entered to the liposome or the micro emulsion that there is compatibility with body tissue.

By medicine and suitable nonirritant excipient are mixed with medicine is carried out to the suppository that rectum gives, as cocoa butter and polyethylene glycol, it is solid at normal temperatures but is liquid under rectal temperature, thereby it will melt and discharge medicine in rectum.

Include, but not limited to capsule, tablet, pill, pulvis, caplets and particle for the oral solid dosage forms given.In this type of solid dosage forms, therapeutic agent can be mixed as sucrose, lactose or starch with at least one inert diluent.This type of preparation can also comprise other materials beyond inert diluent, and for example tablet lubricants and other sheet agent aids are as dolomol and microcrystalline cellulose.In capsule, tablet and pill, preparation can also comprise buffer.Can also use other controlled release coat of enteric coatings core while preparing tablet and pill.

Can also add the solid constituent of similar type as filler in the soft filling and hard-filled gelatin capsule of using this type of excipient, as lactose and high molecular weight polyethylene glycol etc.

Can also use one or more excipient mentioned above that reactive compound is made to the microencapsulation form.Can prepare tablet, dragee, capsule, pill and granule with the solid dosage forms of dressing and shell, as enteric coatings and known other dressings of field of pharmaceutical preparations.It can optionally contain opacifier, and it can also be the composition of release of active ingredients only, or preferably, the specific part in enteron aisle discharges, and optionally, with delayed mode, discharges.The example of the embedding component that can use comprises polymer and wax.

The formulation that supplies the compounds of this invention part or transdermal to give further comprises ointment, paste, emulsifiable paste, lotion, gel, pulvis, solution, spray, inhalant or paster.Under aseptic condition by active component and pharmaceutically acceptable carrier and mix with required arbitrarily preservative or buffer as requested.Also comprise within the scope of the invention eye-drops preparations, auristilla, spongaion, pulvis and solution.

Except reactive compound of the present invention, can also contain excipient in ointment, paste, emulsifiable paste and gel as animal and plant fat, oil, wax, paraffin, starch, tragcanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talcum powder and zinc oxide or its mixture.

Go out beyond reactive compound of the present invention, pulvis and spray can also contain the mixture of excipient as lactose, talcum powder, silicic acid, aluminium hydroxide, calcium silicates and polyamide powder and these materials.Usually spray can also contain propellant, as Chlorofluorocarbons (CFCs).

Percutaneous plaster can be by the reactive compound controlled-release delivery to body.Can prepare this type of preparation by compound being dissolved or being dispersed in suitable matrix.Can also use sorbefacient to increase compound transdermal amount.Can be by using rate controlling membranes or compound being scattered in to speed control in polymer substrate or gel.

Pharmaceutical composition as herein described can be made to quick-release or controlled release (discharging also referred to as slowly-releasing, sustained release or prolongation) preparation.It is known that multiple slowly-releasing or prolongation delivery formulations or equipment are those of ordinary skill in the art.Its example includes, but are not limited to U.S. Patent number 5,674, and 533,5,059,595,5,120,548,5,073,543,5,639,476,5,354,556 and 5,733, described in 566, its disclosure is incorporated in this reference.Can use this type of formulation that slowly-releasing or the controlled release of one or more active components are provided, use for example Hydroxypropyl methylcellulose, other polymer substrates, gel, permeable membrane, osmosis system, multiple coatings, particulate, liposome, microballoon or its combination of variation ratio that required release characteristics is provided.It is easy selecting suitable controlled release preparation known to a person of ordinary skill in the art and using it for therapeutic agent of the present invention.For example, the present invention includes solid oral dosage form, such as but not limited to, tablet, capsule, caplets and the capsule tablets of controlled release preparation (that is, slowly-releasing, prolongation discharge or sustained release) made.

The benefit of controlled release preparation comprises that prolong drug is active, reduces the compliance that gives frequency and increase the patient.For example, controlled release or prolongation delivery formulations can remain in patient body and obtain constant dosage level, to strengthen sending through blood-brain barrier.

Most of controlled release preparation is designed to initial release and produces rapidly the therapeutic agent (active component) of a dosage of required curative effect, and other dosage of sustained release in order to maintain therapeutic or the prophylactic effects of this level in a long period section.In order to maintain medicine this constant level in vivo, the speed that medicine discharges from preparation must be able to substitute the dose of organism metabolism and excretion.Can discharge by the control of multiple conditional stimulus active component, include but not limited to DH, temperature, enzyme, water or other physiological conditions or compound.

In some embodiments, two or more therapeutic agents can be combined and given.Two or more therapeutic agents can be used as every kind of the part that multiple dose gives scheme and give separately.Perhaps, a part that can be using those reagent as unitary agent, in single composition together with compound of the present invention.If give the part of scheme as multiple dose, give, two kinds of therapeutic agents can be simultaneously, in succession or generally in space, in the time period of five hours, give.

As used herein, term " associating ", " associating " and relational language refer to therapeutic agent of the present invention simultaneously or sequential giving.For example, compound of the present invention can with the other treatment agent respectively in unit formulation or in the lump in same unit formulation simultaneously or sequential giving.Thereby, the invention provides a kind of single unit formulation, it comprises described compound, additional therapeutic agent and pharmaceutically acceptable carrier, assistant agent or excipient.

In a plurality of embodiments, particularly, in a plurality of embodiment, provide the present invention.Following embodiment provides explanatory and nonrestrictive description to scope of the present invention and applicability.

Embodiment

the neurotoxicity of embodiment 1:PDS/TTR compound

The result that the epithelial cell culture medium of Alzheimer's disease patient is detected shows, with compared with control cells, compare, wherein the level of PDS/TTR compound raises, and this shows effective biomarker that can be using the PDS/TTR compound as the Alzheimer's disease early diagnosis.Referring to U.S. Patent Application Publication No. 20080026405, its disclosure is incorporated in this reference.The present embodiment has shown that the PDS/TTR compound also has neurotoxicity except the biomarker that can be used as disease.

At first, find acrolein (a kind of accessory substance of peroxidatic reaction of lipid, the unsaturated three carbon aldehyde of α, β) cause the normal control epithelial cell to express the PDS/TTR compound in medium, its generation level is with suitable from Alzheimer's disease patient's epithelial cell.In these experiments, use the method for having set up to set up rapidly the epithelial primary culture of choroid plexus after autopsy.In the MEM growth medium, make AD and normal control culture grow to fusion, contain 2% hyclone and 1% epidermal growth factor (EGF) in this medium.The normal control culture is used instead to the selectivity MEM medium that contains the N2 fill-in, and used solvent (contrast) or 5 μ M acrolein to process 72 hours.To use the medium that contains the N2 fill-in instead from the culture of AD object continues to cultivate 72 hours.After processing, collect the medium in each blake bottle, use the desalination of PD-10 post.Then protein frozen drying wash-out obtained, resuspended in 25 μ l water, adopt Western blot and analyzed for the specific antibody of PDS and TTR.The example results of the immunoblotting assay that Fig. 1 is the PDS/TTR compound, this compound is from expressing in contrast epithelial cell, the contrast epithelial cell of processing through acrolein and the epithelial cell culture medium from Alzheimer's disease patient in late period.As shown in Figure 1, the expression that acrolein makes to contrast PDS/TTR compound in epithelial cell increases to suitable with the epithelial cell of AD in late period (LAD).

Then use the LAD epithelial cell medium treatment cortical neuron that contains the PDS/TTR compound.For determining by LAD choroid plexus epithelium culture or the PDS/TTR compound that produces through the normal control culture that solvent or 5 μ M acrolein are processed whether primary cortical neuron is produced to harmful effect, normal control epithelium culture is used instead containing the medium of N2 fill-in and only used solvent (contrast) or 5 μ M acrolein processing 16 hours.Using the LAD culture instead the N2 medium continues to cultivate 16 hours.After processing, collect all types of medium (LAD; The normal control of only processing with solvent or with the normal control of 5 μ M acrolein processing) and add in primary neurons of rats culture and (cultivate 7 days).Use treated primary culture instead conditioned medium, continue to cultivate 16 hours, use MTT reduction detection method to measure cell viability.Exemplary the results are shown in Figure 2.As shown in Figure 2, compared with the control, through the survival rate of the cortical neuron of LAD epithelium medium treatment, significantly reduce (Fig. 2).This experiment shows that the PDS/TTR compound itself has neurotoxicity.

For the further neurotoxic effect of research PDS/TTR compound, SY5Y neuroglia blastoma cell is exposed to from LAD or the epithelial conditioned medium of normal control 16 hours.After being exposed to conditioned medium, using 70% methyl alcohol/30% acetone fixed cell and use anti-PHF-1 antibody to carry out immunohistochemical staining to it.Seen at AD NFT (neurofibrillary tangles), PHF-1 identifies abnormal phosphorylation Tau.As shown in Figure 3, with approximately 3% comparing of the SY5Y cell of processing through control medium, through the PHF-1 positive of the SY5Y cell of LAD epithelial cell medium treatment, approach 25%.The double helix silk is the precursor of neurofibrillary tangles.Thereby the PHF1 immunity positive is to form the typical characteristics that late period, neuron tangled in Alzheimer's disease.Like this, this experiment has shown that the PDS/TTR compound promotes the double helix silk to form in SY5Y neuroglia blastoma, and prompting PDS/TTR compound can promote amyloid beta (A β) to produce in H4 neuroglia blastoma cell.

For determining whether the compound produced in the epithelium culture affects the inflammatory cytokine path, by Human astrocyte knurl culture with 2.5X 10 ⁵the density of individual cells/well is seeded in culture plate, is exposed in conditioned medium 24 hours.After being exposed to medium, collecting cell from each hole, the ELISA of commodity in use detects the level of inflammatory cytokine (IL-6, TNF-α, TGF-β and IL-6).Testing result shows that the PDS/TTR compound activates two kinds of inflammatory cytokine paths (that is, IL-6, TNF-α) (data are unlisted) in the astrocytoma culture, and surperficial PDS/TTR plays a role in neural inflammation.

Generally speaking, in the present embodiment experimental results show that the PDS/TTR compound cause can be directly as the change of the multiple biochemical indicator of Alzheimer's disease mark.

embodiment 2: the inhibition that nifedipine, nifedipine analog mixture and/or T3/T4 express PDS/TTR

The detection of describing in embodiment 1 provides a kind of discriminating can be for the instrument of the potential therapeutic agent of PDS/TTR compound neuroprotective unit cell.The inventor observes compound as nifedipine (1,4-dihydro-2,6-dimethyl-4-(2-nitrobenzophenone)-3,5-pyridinedicarboxylic acid dimethyl ester, CAS#21829-25-4 (Sigma Aldrich)), be used for the treatment of hypertensive calcium channel blocker; Or the nifedipine analog is as the oxidized derivatives (2 of nifedipine, 6-dimethyl-4-(2-nitrobenzophenone)-3,5-pyridinedicarboxylic acid dimethyl ester, CAS#67035-22-7 (Sigma Aldrich)) or the nitroso-derivative (2 of nifedipine, 6-dimethyl-4-(2-nitroso phenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, CAS#50428-14-3 (Sigma Aldrich)), be used alone or in combination and all can in cell culture, effectively suppress the expression of PDS/TTR compound.In addition, also T3 and T4 are estimated, find that it can effectively suppress the expression of PDS/TTR compound.

Especially, as described in Example 1, use 5 μ M acrolein, 5 μ M acrolein to add that 0.5 μ M T3/0.5 μ MT4,5 μ M acrolein add 1 μ M nifedipine mixture (nitroso nifedipine 55%, oxidized nifedipine 11% and nifedipine 34%) or 5 μ M acrolein add 1 μ M nifedipine mixture and 0.5 μ M T3/0.5 μ M T4 processes epithelial cell.Determine that by immunoblotting assay as described above each culture secretes to the amount of the PDS/TTR in medium.Example data is shown in Fig. 4.Result shows, through acrolein add T3/T4, acrolein adds the nifedipine mixture or acrolein adds the nifedipine mixture and T3/T4 processes the expression of cell significantly lower than only through acrolein, processing the expression of cell.

In addition, adopt immunostaining to determine the PDS/TTR-positive cell, and count and relatively pass through the quantity of PDS/TTR-positive cell in processing and undressed cell culture.The exemplary Fig. 5 that the results are summarized in.As shown in Figure 5, only in the sample that acrolein is processed the quantity of PDS/TTR-positive cell be in unprocessed contrast the PDS/TTR-positive cell quantity approximately 600%.And on the contrary, with unprocessed the contrast, compare, add T3/T4, acrolein through acrolein and add the quantity that nifedipine mixture or acrolein add PDS/TTR-positive cell in the sample that nifedipine mixture and T3/T4 process and significantly reduce.

For determining that whether the nifedipine mixture is by stoping calcium channel performance function, we have estimated the impact of nifedipine mixture on calcium channel.Use the neural rubber master batch cell plastid of new system nifedipine or nifedipine mixture pretreatment SY5Y knurl culture 16 hours, then transfer them to without in the calcium medium and add 5 μ M Fura-2 fluorescent dyes.Then use without calcium medium washing culture and be exposed in the calcic medium, using laser confocal microscope to measure the fluorescence that Ca is combined with Fura-2 at excitation wavelength 340nm place.Every ware, to 50 to 100 cell imagings, detects 3 wares altogether.Example results is shown in Fig. 6.What is interesting is, as shown in Figure 6, compare with the new system nifedipine, the nifedipine mixture has extremely low calcium channel blocker active (～30%), and it shows that these compounds may play a role by substituting new mechanism.

Thereby the experiment of describing in the present embodiment shows that the mixture of nifedipine analog and T3/T4 are used alone or in combination and all can in epithelial cell, effectively suppress the expression of PDS/TTR, and this effect may be irrelevant with calcium channel.And T3/T4 can strengthen the effect of nifedipine analog.

embodiment 3: the nifedipine analog suppresses inflammatory cytokine and produces

There is report to show that inflammatory response element (cell factor) increases in the Alzheimer's disease patient.The inventor detects nifedipine mixture and each analog thereof in the astrocytoma culture.By the Human astrocyte oncocyte with 2.5X 10 ⁵the density of individual cells/well is inoculated in 6 well culture plates, cultivates 24 hours.Then culture is used instead to the selectivity MEM medium of serum-free and used the nifedipine mixture and each analog is processed 24 hours.The each processing used 3 6-orifice plates.After processing, the ELISA that collects medium in each hole commodity in use detects the level of IL-1 β, IL-6, TNF-α and TGF-β.Example results is shown in Fig. 7.As shown in Figure 7, after nifedipine mixture or oxidized nifedipine processing, the level of the IL-1 secreted in medium, IL-6 and TNF-α significantly reduces, and shows that these compounds have direct positive role to neural inflammation.

embodiment 4:NFD-L1 suppresses inflammatory cytokine and produces

Use and the similar approach shown in embodiment 3, in the astrocytoma culture, NFD-L1 is detected.As shown in Figure 8, after NFD-L1 processes, the level of the IL-1 secreted in medium, IL-6 and TNF-α significantly reduces, and shows that NFD-L1 has direct positive role to neural inflammation.Result in the present embodiment shows that lactam can effectively suppress inflammatory disease as NFD-L1 in central nervous system.

embodiment 5: nifedipine, nifedipine mixture and/or T3/T4 reduce the level of PHF-1

As described in Example 1, after SY5Y neuroglia blastoma cell is exposed to the remarkable LAD epithelial cell medium raise of PDS/TTR compound level, with those of the medium that is exposed to undressed control cultures, compare, its PHF-1 immunostaining significantly increases.In this experiment, adopt the method described in embodiment 1 that the SY5Y cell is exposed in the epithelial cell medium of the combined treatment of the mixture of acrolein and nifedipine, nifedipine analog, nifedipine analog and T3/T4.As shown in Figure 9, nifedipine mixture and nifedipine/nifedipine analog adds the level that T3/T4 significantly reduces PHF-1.

suppress the generation of A β 1-42 in embodiment 6:H4 neuroglia blastoma cell

In the present embodiment, the H4 neuroglia blastoma cell that the inventor uses stable transfection to cross expression amyloid precusor protein (APP) is further studied the generation that whether nifedipine, nifedipine analog (for example, oxidized nifedipine or nitroso nifedipine) and/or T3/T4 can suppress A β 1-42.The stable transfection composition is crossed the neural rubber master batch cell plastid of the H4 knurl of expression amyloid (APP) and secrete A β in medium _1-42.Use 1 μ M new system nifedipine, 1 μ M oxidized nifedipine, 1 μ M nitroso nifedipine or 0.5 μ M T3/0.5 μ M T4 to process these H4 cells 16 hours.Use ELISA (Invitrogen) to measure the A β level in medium.As shown in figure 10, after new system nifedipine, oxidized nifedipine, nitroso nifedipine or T3/T4 process, significantly reduce the generation of A β 1-42.

And, further detect and containing or do not containing under the condition of T3/T4, the impact on A β 1-42 production in the H4 cell of nifedipine, nifedipine analog and nifedipine mixture.Example results is summarized in Figure 11.As shown in FIG., T3/T4 promotes the inhibitory action that nifedipine, nifedipine analog and nifedipine mixture produce A β 1-42.

Whether the calcium channel blocker that then we have detected other can suppress A β 1-42 in H4 neuroglia blastoma culture produces.Used in this experiment known calcium channel blocker, as Amlodipine, diltiazem, felodipine, Isradipine, nicardipine and Nimodipine.Especially, containing or, containing using 1 each drug treating of μ M H4 cell under the condition of T3/T4, cell is being cultivated 16 hours among selectivity MEM (serum-free), use Invitrogen ELISA detection is secreted to the A β 1-42 in medium.Example results is shown in Figure 12.As shown in figure 12, nicardipine makes the secretion of A β demonstrate significantly reduced trend (p<0.10), and Nimodipine makes the formation of A β demonstrate significantly reduced trend (p<0.05).Other drug can significantly not change the formation level of A β.When T3/T4 and Nimodipine and diltiazem coupling, the combination of calcium channel blocker and T3/T4 significantly reduces the formation of A β.T3/T4 and other drug coupling can significantly not reduce the formation of A β.

embodiment 7: in H4 neuroglia blastoma cell, NFD-L1 suppresses the generation of A β 1-42

Use and the similar approach shown in embodiment 6, detect the inhibition that NFD-L1 produces A β 1-42 in H4 neuroglia blastoma culture.As shown in figure 13, NFD-L1 suppresses the generation of A β 1-42.The results suggest lactam of the present embodiment can effectively suppress the generation of A β 1-42 as NFD-L1.

embodiment 8: to the inhibition of beta-secretase (BACE) and gamma secretase activity

Nifedipine, nifedipine analog and nifedipine mixture can effectively suppress A β _1-42peptide produces this surprising discovery and impels the β to A _1-42the mechanism that peptide reduces makes further research.Estimate A β _1-42generation depend on the activity of beta-secretase (BACE), this enzyme is the enzyme at beta-secretase cracking site cracking amyloid precusor protein, and the activity that depends on the gamma secretase compound formed by presenilin-1 (PS-1), nicastrin, APH-1 and PEN-2, it is in the cracking of gamma secretase cracking site.The inventor has detected A β in its culture model system _1-42whether the inhibition generated is by due to the inhibition of BACE and/or gamma secretase activation.

The Invitrogen commercial kit of the restructuring BACE that use comprises fluorogenic substrate and purifying detects the activity of BACE.As shown in figure 14, nifedipine is used separately or can make the activity of BACE slightly suppress with the thyroxine coupling; Yet the nitroso nifedipine is used separately or can significantly suppress the activity (Figure 14) of BACE with the thyroxine coupling.

To through the nifedipine mixture separately or with the H4 culture of T3/T4 Combined Treatment in the protein level of each component is detected in BACE and gamma secretase compound result show, the level of the alone remarkable reduction PS-1 of nifedipine mixture and PEN-2.The level of BACE-1 reduces, but it does not have significant.Yet the nifedipine compound adds T3/T4 can significantly reduce PS-1, PEN-2, BACE-1 and Nicastrin.Any processing is all on not impact of APH-1.Example results is shown in Figure 16.Use SABC and laser confocal microscope to determine protein level (50-100 the cell/ware in each part of cell; 3 wares/experiment), use immunoblotting assay to be verified total cell homogenates.For the specific antibody of each albumen all purchased from commercial supplier.

Experimental results show that nifedipine mixture and oxidation and nitroso-derivative in the present embodiment, and/or T3/T4 directly acts on the enzyme of being responsible for producing A β.

the inhibition of embodiment 9:NFD-L1 to beta-secretase (BACE)

Use and the similar approach shown in embodiment 8, detected the inhibitory action of NFD-1 to the BACE activity.As shown in figure 15, NFD-L1 suppresses BACE.The present embodiment shows that lactam can effectively suppress the activity of beta-secretase (BACE) as NFD-1.

embodiment 10: the inhibition that in body, A β 1-40 produces

Vitro data shown in giving above, the inventor has started acute exposure research in 3 monthly age C57-black-6 (C57BL/6) mouse.In this research, continuous three days, lumbar injection (IP) gave six groups of C57BL/6 mouse (six every group) solvents (2%DMSO/98% polyethylene glycol-3000 (PEG-3000)), 25mg/kg nifedipine or nifedipine mixture, T3/T4 (10mg/kg T3 and 10mg/kg T4) respectively, the nifedipine mixture adds T3/T4 and nifedipine adds T3/T4.Put to death animal after injecting for the third time 1 hour.The serum of getting brain and finally obtaining is freezing in liquid nitrogen immediately, for before analyzing, it being kept to-80 ℃.

The homogenate of a brain hemisphere is detected to (Invitrogen ELISA) for A β 1-40, another homogenate is used for measuring protein level.In addition, adopt immunoblotting assay to detect Notch, the Nicast of the necessary substrate cracking of PS-1, BACE, PS-1, the level of APH-1.As shown in figure 17, with the animal that gives solvent, compare, give the mouse that T3/T4 and nifedipine mixture add T3/T4 and demonstrate moderate but significant A β _1-40level reduces (25%).Giving during nifedipine, nifedipine mixture add the mouse of T3/T4, the level of PS-1, Nicast, APH-1 significantly reduces.In giving nifedipine, nifedipine and adding the mouse of T3/T4 and independent T3/T4, the level of BACE albumen significantly reduces.And on the contrary, in the reason group, the level of the Notch of cracking does not have significant difference (Figure 17) throughout.

Again brain is extracted, utilize GC/MS to analyze the level of nifedipine wherein and analog thereof.All found oxidized nifedipine in the sample of all analyses, this shows that the ingredients of a mixture has passed through blood-brain barrier, can use it for neuro-protective like this.This experiment shows that these derivatives have the required brain permeability for the treatment of Alzheimer's disease.

In the present embodiment experimental results show that nifedipine recklessly Nehe without and oxidation and nitroso-derivative and/or T3/T4 reduce in vivo the level of endogenous A β 1-40 peptide.

embodiment 11: the inhibition that the nitroso nifedipine produces A β 1-40 in vivo

Carry out further experiment and can effectively suppress the generation of A β 1-40 in vivo to show the nitroso nifedipine.Especially, IP injection gives C57/B16 mouse (6 every group) and take the nitroso nifedipine that concentration that 2%DMSO/98%PEG-300 is solvent increases for three days on end.The last injection was put to death animal after 15 minutes.Get rapidly brain, be split into two hemisphere, and by its quick-frozen in liquid nitrogen.Brain is placed in to dry ice and transports, be placed under-80 ℃ before analyzing.Use the Dounce homogenizer by the homogenate in the diethylamine that contains the adequate proteins enzyme inhibitor (200mg weight in wet base/mL) of a hemisphere of each brain.By homogenate 16, centrifugal 30 minutes of 000X g, get 50 μ L soluble proteins and A β 1-40 carried out quantitatively according to the explanation of production firm with Covance ELISA.The % that result is processed animal with solvent means.The analysis result demonstration is used 35mg/kg nitroso nifedipine to process the level (Figure 18) that can significantly reduce A β 1-40.

Like this, the present embodiment has proved that the nitroso nifedipine effectively suppresses the generation of A β 1-40 in vivo.

embodiment 12: the in vitro and in vivo of orphan G-protein-coupled receptor 3 (GPCR-3) suppresses

Carry out the present embodiment and can suppress orphan G-protein-coupled receptor 3 (GPCR-3) to detect whether nifedipine, nifedipine mixture and/or T3/T4, the enzyme of GPCR-3 for playing an important role in the stability maintaining gamma secretase compound (according to discussion above, it plays an important role in the process of APP cracking formation A β) as described above.

Use 1 μ M mixing nifedipine, 1 μ M mixing nifedipine to add 0.5 μ M T3/0.5 μ M T4,0.5 μ M T3/0.5 μ M T4,1 μ M new system nifedipine, 1 μ M new system nifedipine adds 0.5 μ M T3/0.5 μ M T4 and processes H4 neuroglia blastoma cell 16 hours.By adopting the GPCR-3 specific antibody, utilize the analysis of the Western marking to measure the level of GPCR-3.As shown in figure 19, nifedipine mixture, new system nifedipine and/or T3/T4 significantly reduce the expression of GPCR-3 in the H4 cell.

In addition, adopt the GPCR-3 level in the definite above described C57BL/6 mouse of embodiment 10 of immunoblotting assay.Example results also is shown in Figure 19.Nifedipine mixture and T3/T4, new system nifedipine and T3/T4 also can reduce the GPCR-3 expression in mouse.

Thereby the present embodiment has proved that nifedipine mixture and oxidation thereof and nitroso-derivative and/or T3/T4 reduce the level of GPCR-3 in vitro and in vivo.

For determining whether nifedipine mixture and oxidation thereof and nitroso-derivative and/or T3/T4 regulate by blood pressure the path related to and reduce the GPCR-3 level, and we detect as atenolol, captopen and enantopirl known blood-pressure drug in the H4 cell.Especially, use 1 each medicine of μ M containing or do not containing processing H4 cell under the condition of T3/T4, cell is cultivated 16 hours in selectivity MEM (serum-free) medium, used laser confocal microscope and the anti-GPCR-3 antibody test of specificity GPCR-3 level.Example results is shown in Figure 20.As shown in figure 20, only captopen+T3/T4 causes the level of GPCR-3 significantly to change (reduction).This experiment shows that nifedipine mixture and oxidation thereof and nitroso-derivative and/or T3/T4 make the minimizing of GPCR-3 not rely on the blood pressure path.

embodiment 13: the effect of the enzyme level that the nitroso nifedipine relates to during A β is processed

Carry out this experiment and determine the effect of nitroso nifedipine to the enzyme level that relates in A β processing.The survival rate of the H4 cell that at first the nitroso nifedipine based on through cumulative concentration is processed is determined the suitable concentration of nitroso nifedipine.Especially, with 2.5X 10 ⁵the density of individual cells/well is seeded to culture plate by H4 neuroglia blastoma cell, makes its adherent spending the night.For culture is changed selectivity MEM, and use the nitroso nifedipine of cumulative concentration to process 16 hours.After processing, add MTT, making its final concentration is 0.5mg/mL, and culture is hatched 30 minutes.After MTT processes, discard medium, use DMSO to dissolve by mitochondria MTT is transformed and obtains formazan crystal and measure its absorbance at 650nm.Data mean (Figure 21) with the mean value ± SEM% that accounts for contrast MTT reduction.The H4 culture is compared in the result of study prompting with nifedipine stronger to the tolerance of nitroso nifedipine.Based on survival data, select 2.5 μ M nitroso nifedipines for follow-up research.

For determining the effect of nitroso nifedipine to the enzyme level that relates in A β process, with 2.5X 10 ⁵the density of individual cell/ware was inoculated the H4 neuroglia blastoma culture of expressing APP and was made its adherent spending the night.Use culture instead selectivity MEM and use 2.5 μ M or 0.5 μ M nitroso nifedipine separately or 2.5 μ M nitroso nifedipine+nifedipines (0.1 and 0.01 μ M) or 1 μ M T3/T4 process.After processing, in PBS, the washing culture is also fixed 30 minutes with 70% methyl alcohol/30% acetone three times under-20 ℃ of conditions.Then use BACE, PS-1, GPCR-3, nicastrin and ADAM (being responsible for the enzyme of α secretase cracking) to carry out immunohistochemical staining to culture.30 to 50 cell imagings (each processed group 3 ware) in 4-5 visual field of every ware selection (Figure 22).Observe similar result through nifedipine, nitroso nifedipine after processing, it all makes BACE albumen significantly reduce.With nifedipine by contrast, the nitroso nifedipine can not cause the level of PS-1 or nicastrin to occur significantly to change.Also with nifedipine by contrast, the nitroso nifedipine causes GPCR-3 significantly to increase.The nitroso nifedipine also causes the level of ADAM-10 significantly to raise, and it is responsible for the β at α secretase site cracking A.The cracking of α secretase causes A β 1-42 to reduce.

the effect of embodiment 14:NFD-L1 to the enzyme level that relates in A β process

Use and the similar approach shown in embodiment 13, adopt NFD-L1 to process H4 neuroglia blastoma culture, to determine the effect of NFD-L1 to the enzyme level that relates in A β process.As shown in figure 23, similar with the nitroso nifedipine, NFD-L1 causes BACE albumen significantly to reduce, and causes ADAM-10 significantly to increase.NFD-L1 also causes PS-1 and NCT significantly to reduce.The present embodiment has proved that lactam has remarkable effect as NFD-L1 processes to the enzyme level related in A β process.

embodiment 15: the effect of nitroso nifedipine to the enzyme level that relates in A β process in body

For determining the effect of nitroso nifedipine to the enzyme level that relates in A β process in body, use gives the mouse of solvent or 35mg/kg nitroso nifedipine, utilize ELISA to carry out quantitatively A β level, utilize immunoblotting assay to carry out quantitatively (Figure 24) to the protein level related in A β process.The IP injection causes A β 1-40 and presenilin-1 significantly to reduce in 3 days.Data also show that the PS-1 of nitroso nifedipine mediation suppresses can not cause the Notch-1 level of cracking to reduce, and can cause the Notch-1 level of cracking significantly to raise on the contrary.In addition, use the nitroso nifedipine to process and cause the level of ADAM-10 significantly to raise, it has the function of α secretase.The rising of estimating the ADAM-10 level causes APP to increase in the cracking of α secretase site, makes A β generate and reduces.

the effect of embodiment 16:NFD-L1 to the enzyme level that relates in A β process in body

Use and the similar approach shown in embodiment 15, determine the effect of NFD-L1 to the enzyme level that relates in A β process in body.As shown in figure 25, similar with the nitroso nifedipine, lumbar injection NFD-L1 causes A β 1-40 and presenilin-1 significantly to reduce.Data also show that the PS-1 of NFD-L1 mediation suppresses to cause the Notch-1 level of cracking significantly to raise.In addition, using NFD-L1 to process causes the ADAM-10 level significantly to raise.Thereby the present embodiment has proved that lactam has remarkable effect as NFD-L1 processes to the enzyme level related in A β process in body.

embodiment 17: to the inhibition of Tau phosphorylation

In the present embodiment, we have detected the phosphorylation whether nifedipine, nifedipine mixture, its oxidation and nitroso-derivative and/or T3/T4 can reduce Tau albumen.Tau protein phosphorylation result causes double helix silk and the self assembly of pencil silk, and its pathogenesis with Alzheimer's disease is relevant.

Although relate to multiple kinases in the Tau phosphorylation, it is main relevant with GSK-3 β (GSK-3 β) that the Hyperphosphorylationof in AD is considered to.In be present in～95% the double helix silk of GSK-3 β that used specificity-phosphoric acid-Tau Identification of the antibodies.GSK-3 β is a kind of composition active kinase, can its Ser 9 phosphorylations be made to its deactivation by using protein kinase B (Akt).Akt is a kind of serine/threonine kinase, its signal by phosphatidyl kinases (PI3K) mediation regulates and controls, and it activates by threonine residues (Thr-308) phosphorylation by phosphatidyl kinases dependant kinase 1 (PDK1) regulation and control with by Ser 473 phosphorylations of PDK α/TORC2 kinase regulatory.In addition, can be coupled to G by GCPR _{α 12/13}heterotrimeric G protein is regulated the activation of Akt/GSK-3 β path.Found G α ₁₂activation stimulate RhoA and effector Rho kinases (ROCK) thereof.ROCK is at the further trans-activation receptor tyrosine kinase of ser160 phosphorylation (RTK), its activation PI3K signal path, cause the phosphorylation of Akt/GSK-3 β/activation (referring to summary New etc., " Gprotein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis; " FEBS J, 2007; 274:6025-36).The Akt of phosphorylation increases phosphorylation and the deactivation of GSK-3 β, and then reduces the phosphorylation of Tau.

As described in Example 10 in the mouse brain that nifedipine, nifedipine mixture and/or T3/T4 process, we have detected level and the phosphorylation state of the albumen that tau phosphorylation path relates to.As shown in figure 26, giving during nifedipine or nifedipine mixture add the mouse of T3/T4, the level of phosphorylation ROCK (p-ROCK) and GSK-3 β (p-GSK-3 β) significantly raises.Also can significantly the raise level of p-GSK-3 β of the independent use of T3/T4.Various processing on the total protein level of ROCK all less than impact.In treated mouse, the level of p-25 slightly reduces.These results suggest nifedipines, nifedipine mixture and/or T3/T4 can make the Tau phosphorylation reduce in vivo.

embodiment 18: the effect to glutamate transport of nifedipine and nitroso nifedipine

For detecting the effect of nitroso nifedipine to glutamate transport, with 2.5X 10 ⁵the density of individual cells/well is inoculated in the neuroastrocytoma culture in culture plate, makes its adherent spending the night.Use culture instead selectivity MEM and use 2.5 μ M nitroso nifedipines to process 16 hours.Use PBS by culture washing three times and fix 30 minutes with 70% methyl alcohol/30% acetone under-20 ℃ of conditions.After fixing, use, for main glutamate transporter EAAT1 or EAAT2 (Glut-1) specific antibody, culture is carried out to immunostaining.Then, use laser confocal microscope by the culture imaging, use Leica software to carry out quantitatively staining power.In each visual field, 30 to 50 cells are carried out to imaging, each culture dish is selected 5 visuals field.Analysis result shows that the nitroso nifedipine causes EAAT2 significantly to increase, but EAAT1 does not change (Figure 27).EAAT2 in the AD brain significantly reduces.The acute C57/Bl6 mouse 25mg/kg nifedipine that gives, used the tissue specimen from these mouse to carry out immunoblotting assay (20 μ g sample protein) detection EAAT2 for three days on end.Analysis result shows that nifedipine significantly increases EAAT2.In a word, these Notes of Key Data nifedipines and nitroso nifedipine raise the level of the crucial glutamate transporter that changes in the AD brain.

embodiment 19: hepatotoxicity wind agitation research

For determining whether to use the nitroso nifedipine to cause hepatotoxicity wind agitation, we select the commercialization alkaline phosphatase enzyme reagent kit (Diagnostic Chemicals Limited) used clinically to carry out quantitative detection to the alkaline phosphatase enzyme level in the final blood serum sample of the nitroso nifedipine mouse from giving the dosage increase.Testing result shows that the nitroso nifedipine of each dosage all can not cause the level of serum alkaline phosphatase significantly to raise, and when dosage is 35mg/kg, in fact it cause this level significantly to reduce (Figure 28).These Notes of Key Data nitroso nifedipines can not induced hepatic injury.

embodiment 20: the research that the people is correlated with

Carry out research that the people is correlated with to determine the impact of calcium channel blocker on human patients.In first research, by object be divided into contrast, there is APOE4 (with the incidence of Alzheimer's disease, increasing relevant gene) contrast, use the object of dihydropyridine calcium channel blocker and there is APOE4 and use the object of dihydropyridine calcium channel blocker.Adopt MMSE (simple and easy mental status inspection) to detect the cognitive function of determining each object.Can determine that model of fit is to the age curve.Figure 29 has shown the example results of the NLMIXED model of MMSE.This model shows to compare with the object that does not use calcium channel blocker, uses the cognitive decrease of calcium channel blocker object to postpone 4 years.

This human body correlative study has proved the morbidity of using dihydropyridine calcium channel blocker to delay cognitive decrease, and the prompting dihydropyridine calcium channel blocker can be used in the treatment nerve degenerative diseases, as Alzheimer's disease.

In second research, we have carried out autopsy to 8 objects that amount to from the Neuropsychology correlative study.4 objects are used calcium channel blocker to comprise nifedipine, and 4 objects are not used any calcium channel blocker.The Application standard method has been measured A β 1-42 in object frontal lobe sample and the A β processive enzyme level as PS-1, Nicas, BACE, APH-1 and PEN-2.Especially, use Invitrogen ELISA to measure A β 1-42 level, adopt immunoblotting assay and determine protein level for the specific antibody of each albumen.As shown in figure 30, with the object of not medication, compare, the A β 1-42 level of medication object significantly reduces.With the object of not medication, compare, some A β processive enzyme in those objects of medication, comprise that PS-1, Nicas significantly reduce.What is interesting is, compare with the object of not medication, the BACE of those objects of medication, APH-1 and PEN-2 level raise.

In addition, also detected the enzyme level relevant with the Tau phosphorylation in object frontal lobe sample.As shown in figure 31, with those not the object of medication compare, the phosphorylation p-Akt of medication object, p-GSK-3 β and p-ROCK level all raise.Medication is not with Akt, GSK-3 β and the ROCK total protein of medication object are on close level.As discussed above, the p-Akt of activation makes GSK-3 β phosphorylation, and then it is inactivated and makes the Tau phosphorylation reduce.These results can reduce the Tau phosphorylation with using calcium channel blocker, and its conclusion that can be used for the treatment of Alzheimer's disease is consistent.

The calcium channel blocker that studies have shown that these human bodies are relevant can reduce the level of A β 1-42 and reduce some A β 1-42 processive enzyme in human patients, and make the enzyme-deactivating relevant to the Tau phosphorylation, show that calcium channel blocker may be effective to the treatment Alzheimer's disease.

embodiment 21: the nitroso nifedipine increases stream in calcium

Carry out this and test to determine whether the nitroso nifedipine has the function of calcium channel blocker.Add 5 μ M Fluo-4AM effect 30 minutes in H4 neuroglia mother cell culture, use the Locke's Fluid containing glucose to wash three times and use solvent, 1 μ M nifedipine or 2.5 μ M nitroso nifedipines to process 1 hour.Add 40 μ L 100mM KCl to make cell depolarization, after depolarising, use laser confocal microscope quantitatively to detect 30 seconds (n=75-100 cell/ware, 3 wares/group) to calcium level.Consistent with expected results, after depolarising, nifedipine causes stream in Ca significantly to reduce.And on the contrary, the sample of processing through the nitroso nifedipine is significantly increase (Figure 32) of stream in Ca after depolarising.

Thereby the present embodiment proved, different from nifedipine, the nitroso nifedipine does not have the effect of calcium channel blocker.It is shocking, the nitroso nifedipine increases stream in calcium.Be not limited to any theory, estimate that nitroso nifedipine and derivative thereof are not treat MCI or Alzheimer's disease by relying on the new mechanism that blocks calcium channel.

embodiment 22: nitroso nifedipine synthetic

Used in the present embodiment the photochemistry synthetic method to synthesize the nitroso nifedipine.Especially, in the Pyrex culture tube, use the 10mL acetonitrile to dissolve nifedipine (20mg), add a cover and under the 250W Halogen lamp LED (3M EVW) irradiate 30 minutes.Utilize Rotary Evaporators to remove desolventizing with products of separated, obtain blue-green oil (18.1mg, yield 94%).The GC/MS analysis result shows, to the conversion ratio of nitroso nifedipine higher than 98.5%.Example results is shown in Figure 33.

embodiment 23:NFD-L1's is synthetic

The nitroso nifedipine (10mg, 30.5 μ mol) that will be dissolved in 5mL ethanol mixes with the glutathione (93mg, 305 μ mol) be dissolved in 5mL water, and under 37 ℃, reaction is 2 hours.After 2 hours, add water and use ethyl acetate to extract product.Except desolventizing, obtain white solid NFD-L1 in Rotary Evaporators, its yield is 85% (purity>95%).The exemplary in nature spectrogram is shown in Figure 34.

embodiment 24: to the treatment of human patients

Give to determine through the MMSE scoring mankind patient's nitroso nifedipine suffering from MCI, dosage is 1000mg every day.Every day three times, oral patient's nitroso nifedipine tablets that gives.

Give to determine through the CDR scoring another mankind patient's nitroso nifedipine of suffering from early stage Alzheimer's disease (EAD), dosage is 800mg every day.Every day three times, oral patient's nitroso nifedipine tablets that gives.

Give to determine via the PDS/TTR compound level in patient's fluid sample mankind patient's nitroso nifedipine of patient MCI, dosage is 1000mg every day.Four times a day, oral patient's nitroso nifedipine tablets that gives.

Identity-definition

Above some non-limiting embodiment of the present invention is described.Those skilled in the art adopt conventional experimental technique, can obtain specific implementations of the present invention described herein multiple be equal to alternative.Those of ordinary skill in the art will understand, and according to the definition of following claim, under the prerequisite that does not break away from purport of the present invention and scope, can carry out multiple change and revision to the present invention.

Unless with the obvious contradiction of context or be otherwise noted, otherwise in the claims, article can refer to one or more as " one " and " one " and " being somebody's turn to do ".Unless with the obvious contradiction of context or be otherwise noted, if in a group membership one, more than one or all be present in, in or when relevant to given product or process, between the one or more members in claim or specification in a group, think " or " relation.The present invention includes such embodiment, in a group membership wherein be present in definitely, in or relevant to given product or process.The present invention also comprises such embodiment, in a group membership wherein more than one or all be present in, in or relevant to given product or process.And, be appreciated that the present invention includes change, combination and change that the one or more restrictions to one or more claim or specification relevant portion, key element, clause, descriptive term etc. are carried out introduces other claims.For example, can be revised any claim that is independent of other claims, be present in the one or more restrictions in any other claims that are independent of same claim basis with introducing.And, when right requires to comprise composition, clearly mean or have the apparent contradiction of those of ordinary skills or an irrationality except context separately has, otherwise be appreciated that it comprised by composition for disclosed herein by composition for any method of purpose, comprised the method for preparing composition according to any preparation method disclosed herein or additive method well known in the art.In addition, present invention includes the composition prepared according to any means for preparing composition disclosed herein.

When key element exists with tabular form, as Ma Kushi group form, can be understood as each subgroup that also discloses key element, any key element can be deleted from this group.Note also that, term " comprises " and means openly, and it allow to introduce other key elements or step.Should be understood that generally speaking, the aspect of invention or invention refers to comprise specific factor, feature, step etc., some embodiment of the aspect of invention or invention by or basically by, for example key element, feature, step etc. form.Its purpose does not have specifically described those embodiments herein for being reduced at.It is applicable to each embodiment that the present invention comprises one or more key elements, feature, step etc., the embodiment that the present invention also provides by or basically has been comprised of those key elements, feature, step etc.

When given certain scope, it comprises end points.And, except the understanding according to context and/or those of ordinary skills separately have the prompting or clearly the indication, be appreciated that the value with Range Representation can be any specific value in described scope in different embodiments of the present invention, except context separately has situation about clearly meaning, it is accurate to 1/10th of scope lower limit unit.Except the understanding according to context and/or those of ordinary skills separately has prompting or clearly means, it is also understood that the value with Range Representation can be the arbitrarily small scope in given range, wherein endpoint table among a small circle is shown and 1/10th of scope lower limit unit identical accuracy.

In addition, be appreciated that and any specific embodiment of the present invention can be got rid of clearly from any one or more claim.Can from any one or more claim, get rid of any embodiment, key element, feature, application or the aspect of the present composition and/or method.For concise and to the point purpose, do not particularly point out in this article all embodiments that one or more key elements, feature, purpose or aspect are excluded.

Being incorporated to of list of references

Whole publications that the application quotes and patent document are all whole with reference to being incorporated at this, its with the content of each publication or patent document be incorporated to this paper be equal to.

Claims

1. be suitable for the pharmaceutical composition for the treatment of sacred disease in human subjects, described pharmaceutical composition comprises the reagent for the treatment of effective dose, and pharmaceutically acceptable carrier, described reagent is selected from lower group: nifedipine, oxidized nifedipine, nitroso nifedipine, lactam, thyroxine (T4), trilute (T3) and combination thereof.

2. pharmaceutical composition according to claim 1, wherein said sacred disease is nerve degenerative diseases.

3. pharmaceutical composition according to claim 2, wherein treat nerve degenerative diseases and comprise treatment, delay or stop mild cognitive impairment (MCI) and/or Alzheimer's disease.

4. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent is not calcium channel blocker.

5. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent increases stream in calcium.

6. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent comprises nifedipine.

7. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent comprises oxidized nifedipine.

8. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent comprises the nitroso nifedipine.

9. according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent comprises lactam.

10. according to the described pharmaceutical composition of any one in claim 1-3, wherein said lactam is the compound shown in formula (Ic).

11., according to the described pharmaceutical composition of any one in claim 1-3, wherein said lactam is the compound shown in formula (Ic-i).

12., according to the described pharmaceutical composition of any one in claim 1-3, wherein said lactam is NFD-L1.

13., according to the described pharmaceutical composition of any one in claim 1-3, wherein said pack is containing the mixture of nitroso nifedipine, oxidized nifedipine and nifedipine.

14., according to the described pharmaceutical composition of any one in claim 13, wherein said mixture comprises 55% nitroso nifedipine, 11% oxidized nifedipine and 34% nifedipine.

15., according to the described pharmaceutical composition of any one in claim 1-3, wherein said pack is containing the mixture of nitroso nifedipine and lactam.

16. pharmaceutical composition according to claim 15, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

17. pharmaceutical composition according to claim 15, wherein said lactam is NFD-L1.

18., according to the described pharmaceutical composition of any one in claim 1-3, wherein said pack is containing the mixture of lactam, oxidized nifedipine and nifedipine.

19. pharmaceutical composition according to claim 18, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

20. pharmaceutical composition according to claim 18, wherein said lactam is NFD-L1.

21., according to the described pharmaceutical composition of any one in aforementioned claim, wherein said reagent further comprises thyroxine (T4) and/or trilute (T3).

22., according to the described pharmaceutical composition of any one in aforementioned claim, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1000mg.

23. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 200mg.

24. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 100mg.

25. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 50mg.

26. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 10mg.

27. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 5mg.

28. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 2.5mg.

29. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 2.0mg.

30. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1.5mg.

31. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1.0mg.

32. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 0.5mg.

33. pharmaceutical composition according to claim 22, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 0.1mg.

34., according to the described pharmaceutical composition of any one in aforementioned claim, wherein said treatment effective dose is not enough to induce bad reaction in human subjects.

35. pharmaceutical composition according to claim 34, wherein said bad reaction is hepatotoxicity wind agitation.

36., according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent is that the scope of nitroso nifedipine and treatment effective dose is that the about 10mg of every dosage is to about 2.5g.

37., according to the described pharmaceutical composition of any one in claim 1-3, wherein said reagent is that nitroso nifedipine and treatment effective dose are not enough to induce hepatotoxicity wind agitation.

38. according to the described pharmaceutical composition of any one in aforementioned claim, wherein said pharmaceutical composition is made preparation and is given by certain approach, that described approach is selected from is oral, subcutaneous, in vein, transdermal, abdominal cavity, intramuscular, the ventricles of the brain, in brain essence, sheath, in encephalic, oral cavity, mucous membrane, nose or rectum give.

39., according to the described pharmaceutical composition of any one in aforementioned claim, wherein said pharmaceutical composition is made into preparation and gives for oral.

40., according to the described pharmaceutical composition of any one in aforementioned claim, wherein said pharmaceutical composition is made into quick releasing formulation.

41., according to the described pharmaceutical composition of any one in claim 1-39, wherein said pharmaceutical composition is made into sustained release preparation.

42. treat, delay or stop the method for sacred disease in human subjects, described method comprises

Treat the reagent of effective dose to the object of suffering from or easily suffer from sacred disease, described reagent is selected from lower group: nifedipine, oxidized nifedipine, nitroso nifedipine, lactam, thyroxine (T4), trilute (T3) and combination thereof, at least one symptom relevant to described sacred disease or feature reduce on abundance, intensity, the order of severity or frequency like this, or delay morbidity.

43., according to the described method of claim 42, wherein said sacred disease is nerve degenerative diseases.

44., according to the described method of claim 43, wherein said nerve degenerative diseases is mild cognitive impairment (MCI) or Alzheimer's disease.

45., according to the described method of any one in claim 42-44, wherein said reagent is not calcium channel blocker.

46., according to the described method of any one in claim 42-44, wherein said reagent increases stream in calcium.

47., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are cognitive decline.

48., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are to produce amyloid beta.

49., according to the described method of claim 48, the generation of wherein said amyloid beta comprises the generation of A β 1-40.

50., according to the described method of claim 48, the generation of wherein said amyloid beta comprises the generation of A β 1-42.

51., according to the described method of any one in claim 48-50, wherein by the activity that increases the alpha-secretase enzyme, reduce the generation of amyloid beta.

52., according to the described method of claim 51, wherein said alpha-secretase activity is the ADAM-10 activity.

53., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are the beta-secretase activity.

54., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are the gamma-secretase activity.

55., according to the described method of claim 54, wherein by the activity that suppresses presenilin-1 (PS-1), nicastrin, APH-1 and/or PEN-2, make the activity decreased of gamma-secretase.

56., according to the described method of claim 54, wherein by the activity that suppresses orphan's G albumen-coupled receptor 3 (GPCR-3), make the activity decreased of gamma-secretase.

57., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are to form the double helix silk.

58., according to the described method of claim 57, wherein said at least one symptom or feature are Protein tau phosphorylations in brain.

59., according to the described method of any one in claim 42-44, wherein said at least one symptom or feature are immunity or the inflammatory situations of central nervous system.

60., according to the described method of claim 59, wherein by the level that reduces one or more cell factors in central nervous system, make described immunity or inflammatory situation alleviate.

61., according to the described method of claim 60, wherein said one or more cell factors comprise IL-1.

62., according to the described method of claim 60, wherein said one or more cell factors comprise IL-6.

63., according to the described method of claim 59, wherein said one or more cell factors comprise TNF-α.

64., according to the described method of any one in claim 42-63, wherein said reagent comprises nifedipine.

65., according to the described method of any one in claim 42-63, wherein said reagent comprises oxidized nifedipine.

66., according to the described method of any one in claim 42-63, wherein said reagent comprises the nitroso nifedipine.

67., according to the described method of any one in claim 42-63, wherein said reagent comprises lactam.

68. according to the described method of claim 67, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

69., according to the described method of claim 67, wherein said lactam is NFD-L1.

70., according to the described method of any one in claim 42-63, wherein said reagent comprises the mixture of nitroso nifedipine, oxidized nifedipine and nifedipine.

71., according to the described method of claim 70, wherein said mixture comprises 55% nitroso nifedipine, 11% oxidized nifedipine and 34% nifedipine.

72., according to the described method of any one in claim 42-63, wherein said reagent comprises the mixture of nitroso nifedipine and lactam.

73. according to the described method of claim 72, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

74., according to the described method of claim 72, wherein said lactam is NFD-L1.

75., according to the described method of any one in claim 42-63, wherein said reagent comprises the mixture of lactam, oxidized nifedipine and nifedipine.

76. according to the described method of claim 75, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

77., according to the described method of claim 75, wherein said lactam is NFD-L1.

78., according to the described method of any one in claim 64-77, wherein said reagent further comprises thyroxine (T4) and/or trilute (T3).

79., according to the described method of any one in claim 42-78, the treatment effective dose of wherein said reagent is enough to increase the glutamate transporter level in the human subjects brain.

80. according to the described method of claim 79, the level that the level of wherein said glutamate transporter is neuroglia glutamate transporter EAAT2.

81., according to the described method of any one in claim 42-80, wherein said treatment effective dose is not enough to induce bad reaction in human subjects.

82. 1 described method according to Claim 8, wherein said bad reaction in human subjects is hepatotoxicity wind agitation.

83., according to the described method of any one in claim 42-82, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1000mg.

84. 3 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 200mg.

85. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 100mg.

86. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 50mg.

87. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 10mg.

88. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 5mg.

89. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 2.5mg.

90. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 2.0mg.

91. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1.5mg.

92. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 1.0mg.

93. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 0.5mg.

94. 4 described methods according to Claim 8, the scope of wherein said treatment effective dose is that every dosage is approximately 0.01 to about 0.1mg.

95., according to the described method of any one in claim 42-44, wherein said reagent is that the scope of nitroso nifedipine and treatment effective dose is that the about 10mg of every dosage is to about 2.5g.

96., according to the described method of any one in claim 42-44, wherein said reagent is that the scope of lactam and treatment effective dose is that the about 10mg of every dosage is to about 2.5g.

97. according to the described method of claim 96, wherein said lactam be formula (Ic) or (Ic-i) shown in compound.

98., according to the described method of claim 96, wherein said lactam is NFD-L1.

99. according to the described method of any one in claim 42-98, wherein said reagent gives by certain approach, that described approach is selected from is oral, subcutaneous, in vein, transdermal, abdominal cavity, intramuscular, the ventricles of the brain, in brain essence, sheath, in encephalic, oral cavity, mucous membrane, nose and rectum give.

100., according to the described method of any one in claim 42-100, wherein said reagent gives by oral.

101., according to the described method of any one in claim 42-100, wherein said reagent gives once per month.

102., according to the described method of any one in claim 42-100, wherein said reagent gives once in every two weeks.

103., according to the described method of any one in claim 42-100, wherein said reagent gives once weekly.

104., according to the described method of any one in claim 42-100, wherein said reagent gives once every day.

105., according to the described method of any one in claim 42-100, wherein said reagent gives twice every day.

106., according to the described method of any one in claim 42-100, wherein said reagent gives three times every day.

107., according to the described method of any one in claim 42-100, wherein said reagent gives four times every day.

108. according to the described method of any one in claim 42-107, the biomarker horizontal abnormality of described object compared with the control wherein, wherein said biomarker comprises:

At least one in thyroxine transport protein and/or prostaglandin-H2D isomerase albumen, and

The albumen that at least one the second is different, the different albumen of described the second is selected from thyroxine transport protein, prostaglandin-H2D isomerase, beta-2-microglobulin, bladder chalone C, superoxide dismutase [Cu--Zn], blood plasma retinol-in conjunction with albumen, phosphatidyl-ethanolamine-in conjunction with albumen, carbonic anhydrase 2 and/or transferrins.

109., according to the described method of claim 108, wherein said biomarker comprises prostaglandin-D2-synzyme and thyroxine transport protein (PDS/TTR compound).

110. according to the described method of any one in claim 42-107, the biomarker horizontal abnormality of described object compared with the control wherein, wherein said biomarker comprises (i) amyloid beta 40 (A β 40), (ii) amyloid beta 42 (A β 42), (iii) A β 40 and the ratio of A β 42 and (iv) one or more in the ratio of the tau of phosphorylation and total tau.

111., according to the described method of any one in claim 108-110, wherein in the fluid sample from described object, determine described biomarker.

112. according to the described method of claim 111, wherein said fluid sample is selected from CSF, serum, whole blood, blood plasma, urine, ascites, saliva, organizes diffusate, irrigating solution and combination thereof.

113. according to the described method of any one in claim 108-112, biomarker level in wherein said contrast prompting object, described object is selected from healthy individual, suffers from patient, the object before treatment and the combination thereof of determining the Alzheimer's disease in stage.

114., according to the described method of any one in claim 108-113, wherein reduce with the described biomarker level of comparing described object that contrasts.

115., according to the described method of any one in claim 108-113, wherein raise with the described biomarker level of comparing described object that contrasts.

116., according to the described method of any one in claim 108-115, wherein said method further comprises that the abnormal level based on described biomarker at first determines the step of the treatment effective dose of described reagent.

117., according to the described method of any one in claim 108-116, there is cognitive disorder in the detection of wherein said object scoring prompting.

118., according to the described method of claim 117, wherein said detection scoring is MMSE (simple and easy mental status inspection (Mini Mental Status Examination)) scoring.

119., according to the described method of claim 118, wherein said MMSE scoring is lower than 27.

120., according to the described method of claim 119, wherein said MMSE scoring scope is 21-26.

121., according to the described method of claim 117, wherein said detection scoring is clinical dementia grade (CDR) scoring.

122., according to the described method of claim 121, wherein said CDR scoring is higher than 0.

123., according to the described method of claim 122, wherein said CDR scoring is 0.5 or 1.

124. the solid orally ingestible that comprises nitroso nifedipine and nifedipine, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 1: 1.

125., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 2: 1.

126., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 4: 1.

127., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 8: 1.

128., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 16: 1.

129., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 32: 1.

130., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 64: 1.

131., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 100: 1.

132., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 200: 1.

133., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 500: 1.

134., according to the described solid orally ingestible of claim 124, the mass ratio of wherein said nitroso nifedipine and nifedipine is at least about 1000: 1.

135. the solid orally ingestible that comprises lactam and nifedipine, the mass ratio of wherein said lactam and nifedipine is at least about 1: 1.

136., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 2: 1.

137., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 4: 1.

138., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 8: 1.

139., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 16: 1.

140., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 32: 1.

141., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 64: 1.

142., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 100: 1.

143., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 200: 1.

144., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 500: 1.

145., according to the described solid orally ingestible of claim 136, the mass ratio of wherein said lactam and nifedipine is at least about 1000: 1.

146., according to the described solid orally ingestible of any one in claim 136-145, wherein said lactam is NFD-L1.

147., according to the described solid orally ingestible of any one in claim 136-146, it further comprises one or more pharmaceutically acceptable excipient.

148., according to the described solid orally ingestible of claim 147, wherein said one or more pharmaceutically acceptable excipient comprise at least one in adhesive, buffer, thinner, dispersant, softening agent, film forming agent, glidant, opacifier, preservative, solvent, stabilizing agent, surfactant, supensoid agent and/or tension regulator.

149., according to the described solid orally ingestible of any one in claim 136-148, wherein said solid orally ingestible is made into preparation for slowly-releasing.

150., according to the described solid orally ingestible of any one in claim 136-148, wherein said solid orally ingestible is made into preparation for quick-release.

151. the compound shown in a formula (Ic):

Or its pharmaceutically acceptable salt, wherein:

R ⁵halogen, the optional C replaced _1-6aliphatic, the optional C replaced _1-6assorted aliphatic, hydroxyl, alkoxyl, amino, alkylamino, cyano group, nitro or nitroso; And

N is 0,1,2 or 3.

152., according to the described compound of claim 151, wherein said compound is the compound shown in formula (Ic-i):

Or its pharmaceutically acceptable salt, wherein:

R ¹and R ²be C independently _1-6aliphatic or cyano group;

R ³c _1-6aliphatic;

N is 0,1,2 or 3.

153. according to the described compound of claim 151, wherein R ¹it is methyl.

154. according to the described compound of claim 151, wherein R ²it is methyl.

155. according to the described compound of claim 151, wherein R ¹and R ²it is methyl.

156. according to the described compound of claim 151, wherein R ³it is methyl.

157., according to the described compound of claim 151, wherein n is 0.

158., according to the described compound of claim 151, wherein said compound is NFD-L1.

159. pharmaceutical composition, described pharmaceutical composition comprises the described compound of any one and pharmaceutically acceptable carrier in claim 151-158.

160. treatment in human subjects, delay or stop the method for sacred disease, described method to comprise to the object of suffering from or easily suffer from sacred disease to give the described compound of any one in claim 151-158.

161., according to the described method of claim 160, wherein said sacred disease is nerve degenerative diseases.

162., according to the described method of claim 161, wherein said nerve degenerative diseases is mild cognitive impairment (MCI) or Alzheimer's disease.

163. suppress the method for beta-secretase (β ACE) in human subjects, described method comprises to human subjects and gives the described compound of any one in claim 151-158.

164. regulate the method for inflammatory situation in the central nervous system of human subjects, described method comprises to human subjects and gives the described compound of any one in claim 151-158.