CN101098708A

CN101098708A - Treatment of an intestinal adenoma and/or adenocarcinoma by inhibition of Notch pathway activation

Info

Publication number: CN101098708A
Application number: CN 200580046283
Authority: CN
Inventors: J·C·克利夫尔斯; M·E·范吉恩; J·H·范埃斯
Original assignee: Hubrecht Laboratorium
Current assignee: Hubrecht Institute
Priority date: 2004-11-10
Filing date: 2005-11-10
Publication date: 2008-01-02
Also published as: ZA200704794B

Abstract

The invention relates to the field of biochemistry and medicine. More specifically the invention relates to methods and pharmaceuticals for the treatment of intestinal neoplasia. The invention discloses the surprising finding that inhibitors of Notch pathway activation (for example gamma-secretase inhibitors) are extremely useful in the treatment of intestinal adenoma and/or adenocarcinoma.

Description

By suppressing Notch pathway activation treatment enteric adenoma and/or adenocarcinoma

The present invention relates to biochemistry and medical domain.More specifically, the present invention relates to be used for the treatment of the method and the medicine of enteric adenoma (being also referred to as polyp) and/or adenocarcinoma.

Small intestinal is the main position of gastral largest portion and digestion and absorption.Except that the chyme that receives from stomach, the The initial segment duodenum of small intestinal also receives from the bile of gallbladder and from the digestive enzyme of pancreas.Pancreatin produces with inactive form, only just becomes activated in duodenal lumen.Small intestinal is divided into three parts: duodenum, jejunum and ileum.

The enteric cavity surface is covered by many finger-shaped or cytofilas that are called the long 0.5-1.5mm of fine hair fully.The center of fine hair is mucodermal extension, and its surface is covered by the monolayer columnar epithelium.The fine hair substrate is the monolayer tubular structure that is called enteraden or Lieberkuhn's glands nest to the opening on enteric cavity surface.These crypts extend to muscular layer of mucosa downwards.The monolayer cylindrical cell that is serving as a contrast them is the cylindrocellular extendible portion of monolayer that covers fine hair.The main cell type of epithelium is enterocyte or absorptive cell.Each enterocyte has about 3000 microvilluss on its enteric cavity surface, it appears as the lip-deep fuzzy striated border of fine hair under optical microscope.Fine hair and microvillus are called as circular folds with submucosal fold, make the sorbent surface of small intestinal amass about 600 times of increase.

Small intestine epithelium is made up of following cell type: enterocyte, goblet cell, paneth's cell, enteroendocrine cell, little gauffer cell and undifferentiated cell.With discuss in more detail in these cells some.

Enterocyte (being also referred to as absorptive cell) is the high cylindrical cell with microvillus and basal nuclei, is specifically designed to substance transportation.They are connected with each other by the connection complex and link to each other with other cell type.Aminoacid and monosaccharide absorb through active transport, and monoglyceride and fatty acid pass through microvillose membrane passively.Absorbed material enters the fenestrated capillary in the proper mucous membrane below epithelium just in time or enters lymph lacteal vessel (most lipids and hdl particle).The life-span of enterocyte is 5-6 days.

Goblet cell is a mucous secreting cell, is the second the abundantest epithelial cell.They are scattered between other cell type.The mucus of goblet cell is very large glycoprotein, and savings is on the cell top.The elongated shape base portion of cell accommodates nucleus and organelle.Abundance from duodenum to the terminal ileum goblet cell increases.Their life-span also is 5-6 days.

Undifferentiated cell is a stem cell, only is present in the crypts base portion, produces the cell of all other types.The cell that is destined to become goblet cell or enterocyte experiences about twice extra division after leaving stem cell bank, migrate to fine hair from crypts, and will come off in top of villi.

Large intestine is made up of colon, caecum, vermiform appendix, rectum and anal canal.The major function of colon is heavy absorbed electrolyte and water and discharges indigested food and refuse.Mucosa looks like level and smooth from the cardinal principle level, because it does not have fine hair.There is countless straight tubular glands.They extend to muscular layer of mucosa always.Body of gland and surface are lined with simple columnar epithelium, its cell type with small intestinal is described identical.But paneth's cell is not present among the adult usually, and enteroendocrine cell is rare.Column absorptive cell and goblet cell enrich.Than more extensive along the surface, its number is many more towards the rectum far-end more in crypts for goblet cell.Mucus helps to increase passing through of solid colonic contents, and coated bacteria and particulate matter.Absorptive cell has irregular short microvillus, though their secretion glycocalyxs, also proof does not contain digestive enzyme.Absorptive cell active transport electrolyte.Water is passively along with electrolyte also is absorbed.With identical at small intestinal, there is undifferentiated cell in the crypts base portion.

Although relevant for multiple cancer form (from be in appearance solid tumor and in the associated transitions of health different parts to hemocyte leukemia in systemic circulation, from fully optimum pernicious to aggressive) extensive knowledge, but effectively treat cancer be still the difficulty, general therapeutic is limited to three types: radiotherapy, chemotherapy and operative treatment.

In fact also there is not at present the probability of carrying out more special treatment at the basic reason of specific cancer or one group of cancer.At attempting providing these certain drug to carry out extensive efforts by drug discovery, described drug discovery attempts to identify the medicine that is used for certain cancer treatment.

The generation of cancer is usually since a cellular change, and this variation causes this first cell unrestrictedly to be grown and division becomes and do not stop splitted cell mass.These change the sudden change of the normally chronic generation of key gene or the accumulation of other change, and the cell mass of sudden change loses that it is original, usually is special feature in this variation, and obtains increasing carcinous character.In the cell that changes, the normal growth regulating of cell is handled and is lost its function.Usually it is no longer controlled in cancerous cell only to express a spot of gene transcription in described cell type.

To the transcription factor of dormancy can for example cause cancer to the activation of genetic transcription typical non-limiting growth and tumor character in particular cell types.

Example is the sudden change of suppressor gene, and suppressor gene plays a role by producing the protein that suppresses the approach of transcribing usually, and does not re-use this approach in the cell of becoming privileged.The suppressor gene of sudden change no longer helps to keep the cell stunt to end.At or intervene the growth of control cell or the medicine of transcribing specific protein-protein in the approach or protein-DNA interaction of growing can be considered to be used for the typical drug candidate of certain cancer treatment, particularly when these approach make a mistake and cause unconfined cell enlargement.

The approach of transcribing makes a mistake and causes the representative instance that cancer takes place to be found in adenomatous polyposis coli (APC).The sudden change of this gene is modal one of the disease incident that causes of philtrum, and the colorectal polyp that caused by APC sudden change will take place during ordinary life about 50% crowd.Thousands of colorectum tumor takes place in the individuality of heredity APC sudden change.APC albumen with at least six kinds other may relate to the protein interaction that exchanges APC relative growth control: beta-catenin is white, γ-catenin, tubulin, EB1, hDLG and ZW3/GSK beta kinase.Having the colon cancer cell of sudden change APC, to contain a large amount of monomeric kytoplasm beta-catenins white.Introduce wild type APC again and reduce the white total amount of beta-catenin.

Particularly in the past during the decade, the molecular genetic analysis of colorectal carcinoma is found, adenomatous polyposis coli (APC) tumor suppressor gene that is accredited as the gene that causes familial adenomatous polyposis (FAP) is at first being brought into play the speed limit effect in the colorectum tumor forms: it suddenlys change in the colorectum tumor that majority distributes, in mice and people at two APC allele of tumorigenic initial stage inactivation all.

In addition, although the colorectum tumor is represented the sign of FAP, planting is the wide spectrum infringement that the APC sudden change causes ectoderm, mesoderm, entoderm source usually.In fact, FAP patient has the high risk that develops into fibroma durum (fibroma), duodenum and gastric tumor, the congenital hypertrophy of retinal epithelium (CHRPE), epidermoid cyst, osteoma, cns tumor etc.Back one observed result clearly illustrates that in the tissue homeostasis of many different parts of apc gene in keeping whole organism and plays a significant role.

The Notch signal transduction is controlled the destiny decision-making (Artavanis-Tsakonas etc., 1999) of spatial distribution and cell in whole regnum animale.The single-transmembrane receptor that the Notch gene code is big.Interaction between Notch receptor and the part causes the Proteolytic enzyme cracking of receptor.Free Notch born of the same parents' internal area (NICD) of gained is indexed into nucleus, combine with transcription factor RBP-J κ (CSL or CBF1) this its, so activation target gene is transcribed, and (Baron 2003, Mumm﹠amp; Kopan2000).The Notch target gene that is preferably characterized is to send out shape division enhancer (hairy/enhancerof split, HES) transcription repressor.HES albumen is prevented the expression (Heitzler etc., 1996, Oellers etc., 1994) of downstream gene again.

Although the Notch pathway component is all expressed (Schroder﹠amp in the mice intestinal; But also do not obtain at present hereditism's evidence that these components participate in control epithelial cell destiny Gossler2002).The known HES-1 deficient animals of representing the Notch target gene in other tissue shows that mucous secreting cell is relative with enteroendocrine cell to be increased, and it is reduced to cost (Jensen etc., 2000) relatively with absorptive cell.Disclose as gene knockout, the downstream target Math-1 (Jensen etc., 2000) (Zheng etc., 2000) that the HES-1 that infers in intestinal prevents guarantees towards secretion pedigree necessary (Yang etc., 2001).These results' explanation is shown that the Notch signal transduction has changed leave the destiny that transition amplification compartment turns to the differentiation pit cell of enterocyte phenotype.In this route, Notch signal transduction activating transcription factor gene is HES1 for example, and HES1 prevents again such as genes such as Math1, drives noble cells and leaves the secretion pedigree.

Notch comes initial freely exploitation to be used for the use of the inhibitors of gamma-secretase of Alzheimer to the indirect support of the control of enterocyte destiny.Notch is one of known substrate of several gamma-secretases.Processing is essential step behind this pathway activation to gamma-secretase to the Proteolytic enzyme of Notch.As a result, one of effect of inhibitors of gamma-secretase is to remove Notch pathway activation (DeStrooper etc., 1999, Kopan﹠amp; Goate 2000).Size and number that the rodent toxicologic study of carrying out with these inhibitor discloses muciparous goblet cell all increase (Searfoss etc., 2003; Wong etc., 2004; Milano etc., 2004).Because the research of these kinds, a plurality of inhibitors of gamma-secretase likely stop in they are used for the treatment of the further clinical development of Alzheimer, because it is unusual to suspect that seriously they bring out intestinal.

The invention discloses wonderful discovery, the inhibitor of Notch pathway activation (for example inhibitors of gamma-secretase) is extremely useful for treatment enteric adenoma and/or adenocarcinoma.Inhibitor for treating enteric adenoma and/or adenocarcinoma with the Notch pathway activation cause conversion/malignant cell inhibition of proliferation, make them be divided into for example goblet cell of mitosis after date cell (promptly no longer carrying out (as seen) division).The cell of these differentiation has the life-span of lacking (5-6 days) relatively, is removed by body in its dead back, and enteric adenoma and/or adenocarcinoma reduce to small part on size/volume.

In the first embodiment, the invention provides a kind of method that is used to change adenoma and/or adenocarcinoma cell destiny, this method comprises influences the Notch pathway activation.More specifically, the invention provides a kind of (external and/or body in) and be used to induce adenoma and/or adenocarcinoma cell to be formed with the method for a postmitotic cell, this method comprises the Notch pathway activation that suppresses at least in part in described adenoma and/or the adenocarcinoma cell.The example of mitosis after date cell is a goblet cell.

In another embodiment, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal.

The Notch pathway activation takes place with following incident usually.Notch strides the film surface receptor, and it can be activated by the water-disintegrable cracking of multiple protein, and a kind of cracking wherein is by a kind of albumen composition cracking with proteinase activity that is called gamma-secretase.(γ)-and secretase is a kind of protease, it exercises its cracking activity in film.(γ)-and secretase is a kind of multicomponent enzyme, by at least four kinds of different albumen, promptly presenilin (presenilin 1 or 2), nicastrin, PEN-2 and APH-1 form.Presenilin is the catalytic center of gamma-secretase.When combining with part, Notch receptor occurred conformation changes, and extracellular domain is come off by the effect of metalloproteases ADAM protease.Play a role followed by the gamma-secretase complex then, cause discharging Notch born of the same parents' internal area (NICD).NICD is indexed into nucleus, this itself and CSL (the conjugated protein J κ of C-promotion-binding factor/recombination signal sequence/nothing hair inhibitive factor/Lagl) interact.The combination of NICD is converted into activator with CSL from transcription repressor, causes the Notch target gene expression.

The present inventor has studied the expression in the adenoma of Notch approach various ingredients and target gene spontaneous generation in the min mice of APC sudden change, and the min mice of APC sudden change is the reliable animal model of familial adenomatous polyposis and intestinal cancer.The present inventor has set up that a plurality of components comprise Notch2 and the Delta-like-1 expression in adenoma in the Notch approach.In addition, the present inventor has determined that also Notch target gene Hesl expresses in adenoma, and this shows that active Notch signal transduction takes place in these adenomas.In the step then, the present inventor has determined the effect of the inhibitor of Notch pathway activation to adenoma.

By the inhibitor of Notch pathway activation is provided to the pernicious/transformant that participates in enteric adenoma and/or adenocarcinoma, the destiny of described cell is changed to turning to postmitotic destiny, for example turns to the goblet cell type.This cell has the life-span of lacking (5-6 days) relatively, provides inhibitor soon with death, causes the amount of enteric adenoma and/or adenocarcinoma cell to reduce, and promptly at least a volume reduces in enteric adenoma and/or the adenocarcinoma.

By the inhibitor of Notch pathway activation is provided, the multiplication capacity of enteric adenoma and/or adenocarcinoma is reduced at least in part.Preferably, this reduce by scanning or exploratory operation as seen.More preferably, reducing of enteric adenoma and/or adenocarcinoma is completely, promptly do not have (visible) remaining pernicious/transformant.

The animal of this paper is defined as non-human animal or people.

The Notch pathway activation (to the small part inhibition, but fully preferred) suppress and can realize by different approaches, this paper following (non-limiting) summarizes these approach.Preferred part suppresses, and promptly carries out in adenoma and/or adenocarcinoma cell and does not disturb Notch approach signal transduction in non-adenoma and/or the non-adenocarcinoma cell.

In addition, the Notch pathway activation is defined as the pathway activation of the different allele variants that comprise Notch sample molecule and/or Notch (sample) molecule.

Enteric adenoma is normally defined benign tumor such as polyp.Adenocarcinoma is normally defined malignant tumor, is also referred to as colorectal carcinoma.Term " enteric adenoma and/or adenocarcinoma " is defined as the metastasis that also comprises from described adenocarcinoma in this article.Preferably, described metastasis is from the enteraden cancer.This metastasis can be positioned at (will) any position of health of the individuality of treatment, for example the enteraden cancer metastasis is positioned at lung or brain or the kidney or the liver of described individuality.

In addition, method of the present invention is fit to treatment enteric adenoma and/or adenocarcinoma and/or from their metastasis of any size.At present a shortcoming of the oncotherapy that uses is that the existence of the described treatment blood vessel (blood vessel generation) that need depend on new formation usually is delivered to described tumor or its metastasis with the chemical compound that will use in the described treatment.The present invention depends on conversion/malignant cell inhibition of proliferation and they differentiation to mitosis after date cell.Therefore, compare with some traditional treatments, little tumor or little metastasis (any neovascularization is not arranged as yet) can early many time points are treated.

In preferred embodiments, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal, and wherein said Notch pathway activation is suppressed at least in part by offering described animal inhibitors of gamma-secretase.This inhibitors of gamma-secretase is to be peptide molecule or non-peptide molecule or half peptide molecule, preferably micromolecule in nature for example.The initial definition of gamma-secretase is used for Alzheimer.Critical step is the APP proteolysis in the pathogenesis of Alzheimer, causes beta-amyloyd peptide (A β) to form, and beta-amyloyd peptide is the major protein component of this sick characteristic brain speckle.APP (as Notch) is at first in extracellular domain (passing through beta-secretase in this case) cracking, and the remainder of APP is generated A β peptide by the gamma-secretase cracking in film.The inhibition that A β peptide is generated by blocking-up gamma-secretase activity is one of the most promising therapeutic strategy of Alzheimer pathology progress that slows down at present.Several companies have developed inhibitors of gamma-secretase simultaneously, for example DAPT (N-[N-(3,5-difluoro phenylacetyl group)-L-alanyl]-S-phenylglycine tertiary butyl ester).In addition, tested (the gamma-secretase enzyme inhibition activity of hexichol nitrogen  (DBZ), BZ (benzene phenodiazine ), LY-411,575 etc. chemical compound from chemical classes AS (aryl sulfonic acid amides), DBZ., summarize in 2004 for example at Harrison etc. about the summary of inhibitors of gamma-secretase, wherein inhibitors of gamma-secretase is divided into sulfonamide/sulfone and benzene phenodiazine /benzo lactams.Several in these inhibitors of gamma-secretase have entered I phase and II clinical trial phase stage.Those skilled in the art know that, (described method comprises the Notch pathway activation that suppresses at least in part in the described animal to be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, wherein said Notch pathway activation is suppressed at least in part by offering described animal inhibitors of gamma-secretase) can promptly be undertaken by providing at least a or two or more inhibitors of gamma-secretase at least by the combination that different inhibitors of gamma-secretase are provided.Know that gamma-secretase can work with (at least) two kinds of approach usually: APP approach and Notch approach.Chemical company has developed simultaneously to one or another kind of special gamma-secretase, and is for example special but do not disturb the gamma-secretase of Notch approach to the APP approach.Know that not disturbing the gamma-secretase of Notch approach is useless in the method for the invention.Therefore, preferably can either disturb the Notch approach again interfering AP P approach gamma-secretase or preferably can disturb the gamma-secretase of Notch approach specifically.This inhibitor can be for example at Harrison etc., finds in 2004, and the document is incorporated herein by reference.

In preferred embodiments, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal, wherein said Notch pathway activation is suppressed at least in part by offering described animal inhibitors of gamma-secretase, and wherein said inhibitors of gamma-secretase is DAPT or hexichol nitrogen  (DBZ) or benzene phenodiazine  (BZ).

DAPT, DBZ or BZ induce adenoma and/or adenocarcinoma cell to be formed with a postmitotic cell effectively, so these chemical compounds have similar effect in the method for the invention.But the IC50 of DBZ is 1.7 nM, and the IC50 of BZ is 2.2 nM, and the IC50 of DAPT is 10 nM, if therefore compare with DAPT, uses the DBZ or the BZ of low amount can obtain similar result.

As mentioned above, gamma-secretase is an albumen composition.Suppress the another kind of mode of Notch pathway activation at least in part and finish, because think that to have only complex to be only activated by the formation that suppresses described albumen composition at least in part.This is for example by providing one of described component to realize as the negative molecule of dominance or by a part/molecule that described complex is provided, and wherein said part/molecule comprises and prevents the sudden change that further forms the sudden change of complex or cause instability (nonactive) albumen composition.The another kind of mode that suppresses the Notch pathway activation at least in part is the catalysed partial that specificity suppresses described complex, and promptly specificity suppresses presenilin.

In another preferred embodiment, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal, and wherein said Notch pathway activation is suppressed at least in part by reducing ligand-mediated Notch to activate at least in part.As mentioned above, the Notch pathway activation in conjunction with Notch receptor experience conformation change after, this incident, makes extracellular domain come off by the effect of ADAM protease from part.It will be apparent to those skilled in the art that can be at least in part with the bonded part of Notch, but preferably suppressed by multiple strategy fully.Preferably reducing described ligand-mediated Notch at least in part by the negative part of the dominance that offers described animal Notch activates.The example of natural Notch part is Delta, Jagged and Serrate albumen.The negative part of dominance promptly can combine with Notch but further not activate the part of Notch approach (blocking-up Notch pathway activation) basically, can for example assign to prepare in conjunction with micromolecule based on the joint portion of described native ligand from described native ligand.When the negative part of dominance contacted with Notch, described dominance feminine gender part combined with Notch and does not further activate the Notch approach.Preferably, being connected of the negative part of described dominance and Notch/in conjunction with long period and native ligand in conjunction with by partly, preferably blocking-up/inhibition fully, Notch pathway activation quilt (at least in part) inhibition as a result.The example of the negative part of dominance is for example to comprise the Delta of disappearance in the cell and the mutant (Sun and Artavanis-Tsakonas, 1996) of Serrate.

In another embodiment preferred, described ligand-mediated Notch activates by offering the negative part of described animal dominance and is reduced at least in part.In principle, every type Notch molecule, promptly Notch1,2,3,4 or its function fragment and/or functional deriv all can be used for this purpose.Function fragment be from one of these molecules (or its equivalent) can with the bonded any fragment of Notch part (N-terminal fragment, C-terminal fragment or interior segments or their any combination).This function fragment can for example exist as the film binding compounds or as non-film binding compounds.By the negative Notch of dominance with can utilize combining of part, described part can not with natural/originally/function on available Notch combine, so the Notch pathway activation is suppressed at least in part.Functional deriv be for example suddenly change (point mutation, insertion) back make with part combine still may but Notch molecule that the bonded signal of this sudden change prevention part is transmitted.Functional deriv also can be from other species.In another preferred embodiment, described ligand-mediated Notch activates by offering described animal and can block the antibody of Notch part and Notch interphase interaction at least in part and reduced at least in part.This antibody for example be at the ligand binding moiety of Notch or in the part with the interactional part of Notch.The preparation of antibody is in the routine work of this area, and the further details of therefore relevant this respect does not provide.Do not rely on employed ligand-mediated Notch and activate the inhibition type, the result is the same: the formation of (to small part) NICD is suppressed, and this finally causes conversion/malignant cell to be formed with a postmitotic cell (for example goblet cell).

As mentioned above, the Notch pathway activation (to small part, but fully preferred) suppress and can realize by different way.In another embodiment preferred, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal, and wherein said Notch pathway activation is suppressed at least in part by offering described animal ADAM protease inhibitor.The Notch part is with after Notch combines, and Notch receptor experience conformation change makes extracellular domain come off by the effect of ADAM protease.ADAM represents disintegrin (disintegrin) and metalloproteases.By the material that can suppress to finish the protease that extracellular domain comes off is provided, the Notch pathway activation is by at least in part, but preferably fully suppresses, and promptly do not have NICD and forms and take place.Under the situation of enteric adenoma and/or adenocarcinoma, this causes increasing adenoma and/or adenocarcinoma cell, and to change mitosis after date cell into be goblet cell.In addition, the part of Notch pathway activation suppresses preferably to avoid as much as possible any possible side effect of not expecting.

It will be apparent to those skilled in the art that suppressing the activated different modes of Notch at least in part also can make up with for example reinforced effects.

Sudden change activation by the Wnt approach causes enteric adenoma, the sudden change of described Wnt approach to activate the disappearance that modal reason is intestinal neoplasms suppressor gene APC (Kinzler and Vogelstein, 1996).We have determined the Wnt target gene program in the colorectal cancer cell recently, have therefore found adenoma cell and have increased significant symmetry (van de Wetering etc., 2002) between the crypts CFU-GM.Whether be extended down to the Notch approach for studying this symmetry, we have studied the expression in the adenoma of multiple Notch pathway component and target gene spontaneous generation in the min mice of APC sudden change.Generally speaking, as before (Schroder and Gossler, 2002) report that the expression of receptor and part is followed by the expression of crypts in the adenoma.For example, Fig. 1 has provided the expression of Notch2 and Delta-like-1 in the adenoma of min mice of APC sudden change.More importantly, not only in crypts, take place as the expression of the HES1 of active Notch signal transduction indication, also in APC ^MinAs one man observe in the adenoma of all size in the mice intestinal (Fig. 1).This observed result shows that as in crypts, Notch and Wnt approach also have activity in increasing adenoma cell.Embodiment disclosed herein proves that unexpectedly the enteric epithelium ancestral cells had both needed the Wnt approach also to need the Notch approach could keep not breaking up.This paper is open, and the conversion/malignant cell that has constitutive activity for Wnt cascade wherein is true equally: need the Notch activity to keep transformation/malignant state.

In preferred embodiments, the invention provides a kind of be used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, this method comprises the Notch pathway activation that suppresses at least in part in the described animal, also comprises the Wnt pathway activation that suppresses at least in part in the described animal.

By offering the inhibitor of the cell Wnt pathway activation of suffering from enteric adenoma and/or adenocarcinoma, pernicious/non-transformed cell differentiation is also dead soon after formation, therefore reduces the size/volume of enteric adenoma and/or adenocarcinoma.By the compound action of Notch pathway activation inhibitor and Wnt pathway activation inhibitor, enteric adenoma and/or adenocarcinoma cell differentiation cause reducing at least in part the intestinal malignant tumor.It will be apparent to those skilled in the art that by the independent effect of Wnt approach restrainer and also can treat enteric adenoma and/or adenocarcinoma.

In another embodiment, the invention provides the Notch approach restrainer and be used for the treatment of purposes in the medicine of enteric adenoma and/or adenocarcinoma in preparation.To as described in the method for the present invention, can use multiple inhibitor as above, therefore stop the Notch pathway activation at least in part to reduce the formation of NICD at least in part.The example of preferred Notch inhibitor is: inhibitors of gamma-secretase such as DAPT or hexichol nitrogen  (DBZ) or benzene phenodiazine  (BZ), can reduce activated inhibitor of ligand-mediated Notch (for example by the negative part of the dominance of Notch or by the negative Notch of dominance or by blocking the antibody of Notch part and Notch interphase interaction at least in part) or ADAM protease inhibitor.In addition, can be supplemented with Wnt approach restrainer or be used in combination in this inhibitor with existing treatment such as chemotherapy, operation or radiotherapy.

In preferred embodiments, the invention provides Notch pathway activation inhibitor and be used for the treatment of purposes in the medicine of enteric adenoma and/or adenocarcinoma in preparation, wherein said enteric adenoma and/or adenocarcinoma betide among the patient who suffers from genetic syndrome familial adenomatous polyposis (FAP).FAP is caused by the genetic mutation of adenomatous polyposis coli (APC) gene.Polyposis mainly is meant " a lot of polyp ".Polyp is the little tissue growth on the intestinal wall.Modal polyp and also be that unique polyp that can become intestinal cancer is adenomatous polyp.In FAP, in whole colon, become hundred to thousands of polyps.These polyps begin to form about 16 years old usually.If do not extract, these polyps almost always develop into cancer.The individuality that is had APC sudden change by diagnosis not only has the risk of suffering from colorectal carcinoma, and with the risk of common people's faciation high other types of cancer of trouble than having.Weak FAP (attenuated FAP) is the lower FAP variant of a kind of order of severity (AFAP), and wherein less polyp takes place the people.AFAP takes place so early unlike FAP usually, but it also has similar trouble cancer excessive risk.In case be diagnosed as FAP, the patient finally stands preventative (Asia) total colectomy usually.Carry out method of the present invention or purposes if suffer from the patient of FAP, then can partly avoid using this colectomy now.APC used herein ^MinMice is the homologue of people FAP substantially.

In another embodiment, the invention provides a kind of pharmaceutical composition, it comprises at least two kinds of Notch pathway activation inhibitor or comprises at least a Notch pathway activation inhibitor and at least a Wnt approach restrainer.

Pharmaceutical composition of the present invention can provide with the form of powder, solution in (non-) is liquid, aqueous or suspensoid or as Emulsion.Described pharmaceutical composition can further contain pharmaceutically acceptable carrier and/or diluent.Described pharmaceutical composition can oral administration, parenteral, intramuscular, intravenous, non-through intestinal (parentally), intraperitoneal or colorectum (for example using suppository) administration.Preferred oral and colorectum are sent, and want area for treatment because can easily arrive by these route of delivery.In addition, preferred local delivery is to avoid any side effect of not expecting.

About dosage, notice that those skilled in the art can determine the effective dose of DAPT for example and/or DBZ and/or BZ according to for example known pharmacokinetics.In addition, determine experiment by using one or more dosage of knowing, those skilled in the art also can determine effective dose.

Except the purposes of medicine in above-mentioned any method, medicine of the present invention also is very useful as the interpolation (remaining (residual)) to other treatment.For example, if decision surgical removal adenoma or adenocarcinoma, medicine of the present invention before the operation and/or during and/or provide afterwards to induce adenoma and/or adenocarcinoma cell to be formed with a postmitotic cell or further to reduce adenoma and/or adenocarcinoma at least in part or treat remaining (may be sightless) residual adenoma and/or adenocarcinoma cell.This is for example by carrying out with liquefaction medicine of the present invention washing abdominal cavity or by directly medicine of the present invention being injected to doubtful zone.

Below describe and to explain the present invention in more detail, but it does not limit the present invention.

Experimental section

Experiment I

Material and method

SABC antibody

Use following antibody.Mouse anti-Ki67 (1: 100; Novocastra), mouse anti-beta-catenin is white (1: 50; Transduction Labs).

Tissue sample preparation, SABC and in situ hybridization

With the whole excision of intestinal, wash gently to remove any fecal content thing with cold PBS, use formolage then.Small intestinal is rolled into ring closely, at room temperature in formalin, fixes 16 hours.With tissue slice (2-6 μ m).After dewaxing and the aquation, seal buffer (120mM Na with peroxidase ₂HPO ₄, 43mM citric acid, 30mM NaN ₃, 0.2%H ₂O ₂PH5.8) pretreatment 15 minutes of at room temperature will cutting into slices.(10mM boils in pH6.0) to repair antigen at sodium citrate buffer solution with sample.After 20 minutes, will boil dish and slowly cool to room temperature.The incubation of antibody is to spend the night among the BSA among the PBS under 4 ℃, at room temperature carries out 1 hour for lysozyme, Ki67.All use Envision in all cases ⁺Test kit (DAKO) is as second class grade chemical.The incubation time is 30 minutes.Use the DAB colour developing.Slide is redyed and sealing with hematoxylin then.The probe that in situ hybridization uses is (Schroder etc., 2002) as described.

People's pathology paraffin embedding sample to fine sign carries out similar in situ hybridization, but obvious end user RNA sequence is as probe.

Like alizarin blue dyeing

After dewaxing and the aquation, will be organized in and like in alizarin blue (1%, in 0.5% acetic acid) incubation 5 minutes.Wash with water then, incubation is 1 minute in dimethyl diaminophenazine chloride.Dehydration is washed in dimethylbenzene, and is used the Pertex sealing rapidly.

APC ^MinThe pharmacology of Notch signal transduction suppresses in the mice

Handle the APC in 8 ages in week with the inhibitors of gamma-secretase (DAPT:100mg/kg is in Semen Maydis oil) that two kinds of different oral administration are sent ^MinMice 2.5,6.5 or 15 days separates intestinal then, and carries out histological examination.

The result

By the sudden change of Wnt approach activate cause enteric adenoma, the sudden change of described Wnt approach to activate the disappearance that modal reason is intestinal neoplasms suppressor gene APC (summary is seen Kinzler and Vogelstein, 1996; Bienz and Clevers, 2000).We have reported at colorectal cancer cell aspect the expression of Wnt expression of target gene program and have increased to have significant symmetry between the crypts CFU-GM (van de Wetering etc., 2002) recently.For whether the symmetry between research crypts and the intestinal neoplasms is extended down to the Notch approach, we have studied multiple Notch pathway component and target gene at APC ^MinExpression in the mice in the adenoma of spontaneous generation.Generally speaking, as before (Schroder and Gossler, 2002) (Table I) report that the expression of receptor and part is expressed followed by crypts in the adenoma.For example, Fig. 1 shows the expression of Notch2 and Delta-like-1 in the adenoma.More importantly, as the expression of the Hesl of active Notch signal transduction indication (Schroder and Gossler, 2002) take place in crypts not only, also at APC ^MinAs one man observe in the adenoma of all size in the mice intestinal on (Fig. 1, right side).This observed result shows that as in crypts, Notch and Wnt approach also have activity simultaneously in increasing adenoma cell.

Then we whether propose the Notch pathway activities be essential problem for the undifferentiated proliferation phenotype that keeps adenoma cell.We select to suppress by using inhibitors of gamma-secretase DAPT that the Notch approach is carried out the pharmacology.We begin the APC to 15 ages in week then ^MinMice is treated, and these mices in this age have 30-60 in small intestinal can have 1-3 adenoma through detected adenoma of naked eyes or polyp in colon.The mice treatment is reached 15 days, then intestinal is carried out histological examination.According to trial test and the therapeutic scheme delivered, DAPT that will be in Semen Maydis oil is with the dosage of 100mg/kg orally give APC once a day ^MinMice.The effect of not observing unanimity in the 2.5th day or 6.5 days in treatment.But, change widely in adenoma, having taken place with the 15th day of this compounds for treating.Shown in Fig. 2 and 3, the number that increases the Ki67 positive cell significantly reduces.In Fig. 2, by the beta-catenin visible adenoma of colon of vision (left side) that dyes in vain.It should be noted that the propagation in adjacent normal crypts is uninfluenced.The big kitchen range of concentrating of alizarin blue positive goblet cell has all appearred in the whole adenoma liking.The white positive of karyon beta-catenin of stagnation and noble cells shows that these cells are from the negative adenoma of Apc-.In Fig. 3, provided the example of small intestinal adenoma.Equally, the left side shows that the adenoma tissue staining is a dark-brown.The cell proliferation that Ki67 dyeing (right side) proves stops after with Compound D APT treatment.

A large amount of evidences show that the Wnt cascade is the crypts CFU-GM of transition amplification in mice and the people's intestinal and the main drive of adenoma and adenocarcinoma proliferation potential back.Present data show that active Notch signal transduction bringing into play the effect of no less important in the undifferentiated state that keeps crypts CFU-GM and Apc sudden change tumor cell.Though the Wnt cascade is activated through sudden change when conversion process begins, the Notch approach may keep not suddenling change in tumour progression very much.But can predict late and have strong selection pressure to existing activation Notch approach to suddenly change in the tumor.This activated mutant does not appear in the newspapers in colorectal carcinoma.

Usually be considered to provide quite disadvantageous target as the Wnt cascade that in colorectal carcinoma, is activated, because the proteic cascade of Apc tumor suppressor gene downstream sections is driven by the protein-protein interphase interaction fully to drug development.Prove directly that as this paper the representative of Notch approach is used for the treatment of another targeted drug strategy of intestinal neoplasms such as familial adenomatous polyposis or sporadic colorectal carcinoma.The multiple inhibitors of gamma-secretase of having developed the number of chemical source is used for the treatment of Alzheimer.Increase at animal toxicity research midgut goblet cell number is considered to the topmost side effect of not expecting of these chemical compounds.But we think that this Notch dependent interaction becomes these inhibitors of gamma-secretase to be used for the attractive treatment pattern of colorectal carcinoma.

Experiment II

Material and method

SABC antibody

Use following antibody.Mouse anti-Ki67 (1: 100; Novocastra), mouse anti-beta-catenin is white (1: 50; Transduction Labs), the anti-Math1 of rabbit is (1: 50; Dr Jane Johnson gives).

Tissue sample preparation, SABC and in situ hybridization

With the whole excision of intestinal, wash gently to remove any fecal content thing with cold PBS, use formolage then.Small intestinal is rolled into ring closely, at room temperature in formalin, fixes 16 hours.With tissue slice (2-6 μ m).After dewaxing and the aquation, seal buffer (120mM Na with peroxidase ₂HPO ₄, 43mM citric acid, 30mM NaN ₃, 0.2%H ₂O ₂PH5.8) pretreatment 15 minutes of at room temperature will cutting into slices.(10mM boils in pH6.0) to repair antigen at sodium citrate buffer solution with sample.After 20 minutes, will boil dish and slowly cool to room temperature.Under 4 ℃, the incubation of antibody is to spend the night among the BSA among the PBS at the antibody of Math1, at the white antibody of Ki67 and beta-catenin incubation 1 hour at room temperature.All use Envision in all cases ⁺Test kit (DAKO) is as second class grade chemical.Use the DAB colour developing.Then slide is redyed and sealing with hematoxylin.

Inhibitors of gamma-secretase DBZ

By Syncom, Groningen, the DBZ (Milano etc., 2004) of the synthetic 3g of the Netherlands customization, purity＞99.9%.DBZ is suspended in 0.5% (weight/volume) hydroxypropyl emthylcellulose (Methocel E4M) and 0.1% (weight/volume) Tween 80 in water imperceptibly.

With inhibitors of gamma-secretase DBZ treatment animal

Every day was with 0,3,10 and 30 μ mol/Kg inhibitors of gamma-secretase DBZ peritoneal injection C57B16 mices totally 5 days.

The APC in 8 ages in week of our begin treatment ^MinMice, each two mice are all used 0,3,10 or 30 μ mol/Kg DBZ injection for curing.Every 48 hours intraperitoneal injection DBZ, carried out altogether 10 days, by the series section intestinal is carried out histological examination then.

The result

As another instrument of blocking the Notch signal transduction in vivo, we have synthesized inhibitors of gamma-secretase DBZ 18, purity＞99.9%.DBZ has blocked the Notch cracking in the test based on cell, its IC50＜2 nM (not shown)s.In the experiment of determining dosage, with this chemical compound with 0,3,10 and 30 μ mol/Kg peritoneal injections every day in the C57B16 mice, totally 5 days.When higher dosage (10 and 30 μ mol/Kg), PAS dyeing shows peritoneal injection goblet cell conversion complete (being respectively Fig. 4 C and D) after 5 days.In addition, cell proliferation stops fully, observed variation as broad as long (result does not show) when histology's label (Ki67 and Mathl) discloses the tissue variation with the CSL disappearance.When 3 μ mol/Kg, PAS dyeing shows that the goblet cell number increases (Fig. 4 B) slightly.Hereditism and pharmacological result unite and show that the Notch signal transduction is correctly to keep crypts CFU-GM undifferentiated state necessary in the crypts compartment.

Whether essential for the undifferentiated proliferation phenotype that keeps adenoma cell problem, we select the approach from pharmacology's inhibition Notch about the Notch pathway activities.In experiment I, this is undertaken by using inhibitors of gamma-secretase DAPT.In experiment II, we select to suppress (Wong etc., 2004) by using inhibitors of gamma-secretase DBZ that the Notch approach is carried out pharmacology.We begin the APC to 8 ages in week ^MinMice is treated, and these mices in this age have 30-60 in small intestinal can have 0-3 adenoma through the detected adenoma of naked eyes (polyp) in the colon.Each two mice carries out histological examination by the series section to intestinal then with 0,3,10 or 30 μ mol/Kg DBZ treatment 10 days.To beta-catenin white dyeing show adenoma, adenoma be embedded in usually accumulation (accumulation) hypertrophy but in the unconverted normal crypts (Fig. 5 A, C).The DBZ of 10 or 30 μ mol/Kg has easily induced Math1+/PAS+/Ki67-goblet cell (Fig. 5 D, M-O) in adenoma, and the effect minimum of 3 μ mol/Kg, together to the same (not shown) of the effect of normal pit cell.Even in same animal, in each adenoma, also observe different conversion ratios.Be the Quantitative yield rate, 100 adenomas of the mice of the 10 μ mol/Kg DBZ treatment of using by oneself analyzed by measuring the nuclear percentage ratio of Math1+.In 8% adenoma, greater than all epithelial 50% being converted into the Math1+ cell.In 20% adenoma, 10-50% takes place transform.28% shows the conversion of 1-10%, and 46% does not show that then goblet cell transforms.At untreated APC ^MinNever observing goblet cell in the mice transforms.In in 100 adenomas analyzing each, observe＜1% Math1+ goblet cell.These observed results show that adenoma cell may be forced to differentiation when the Notch approach suppresses.

Experiment III

Material and method

SABC antibody

Tissue sample preparation and SABC

With the whole excision of intestinal, wash gently to remove any fecal content thing with cold PBS, use formolage then.Small intestinal is rolled into ring closely, at room temperature in formalin, fixes 16 hours.With tissue slice (2-6 μ m).After dewaxing and the aquation, seal buffer (120mM Na with peroxidase ₂HPO ₄, 43mM citric acid, 30mM NaN ₃, 0.2%H ₂O ₂PH5.8) pretreatment 15 minutes of at room temperature will cutting into slices.(10mM boils in pH6.0) to repair antigen at sodium citrate buffer solution with sample.After 20 minutes, will boil dish and slowly cool to room temperature.The incubation of antibody is to spend the night among the BSA among the PBS, at room temperature carries out 1 hour at Ki67 and the white antibody of beta-catenin.All use Envision+ test kit (DAKO) as second class grade chemical in all cases.Use the DAB colour developing.Then slide is carried out counterstain and sealing with hematoxylin.

Gamma-secretase inhibitors DBZ and BZ

By Syncom, Groningen, DBZ and the BZ (Milano etc., 2004) of the synthetic 3g of the Netherlands customization, purity＞99.9%.DBZ is suspended in 0.5% (weight/volume) hydroxypropyl emthylcellulose (Methocel E4M) and 0.1% (weight/volume) Tween 80 in water imperceptibly, BZ is suspended among 6% (volume/volume) ethanol/94% (volume/volume) Lahrafil M, 1944 CS imperceptibly.

With gamma-secretase inhibitors DBZ and BZ treatment animal

Every day was with 0,3,10 and 30 μ mol/Kg gamma-secretase inhibitors BZ peritoneal injection C57B16 mices totally 5 days.

For oral research, medicine (DBZ and BZ) is given the APC in 8 ages in week ^MinMice (10,20 or 30 μ mol/Kg) carries out histological examination by the series section to intestinal then.

The result

As another instrument of blocking the Notch signal transduction in vivo, we have also synthesized inhibitors of gamma-secretase BZ, purity＞99.9%.BZ and DBZ have blocked the Notch cracking in based on the test of cell, its IC50 is respectively 2.2 and the 1.7nM (not shown).In the experiment of determining dosage, with the BZ chemical compound with 0,3,10 and 30 μ mol/Kg peritoneal injections every day in the C57B16 mice, totally 5 days.When higher dosage (10 and 30 μ mol/Kg), PAS dyeing show peritoneal injection after 5 days goblet cell transform fully.Observed variation is as broad as long after organizing variation and treat (see and test II) when CSL lacking (result does not show) and with DBZ.These results reconfirm that the Notch signal transduction is correctly to keep crypts CFU-GM undifferentiated state necessary in the crypts layer.

Whether essential for the undifferentiated proliferation phenotype that keeps adenoma cell problem, we select the approach from pharmacology's inhibition Notch about the Notch pathway activities.In experiment I, this is undertaken by using inhibitors of gamma-secretase DAPT.In experiment II, we select by using inhibitors of gamma-secretase DBZ after the IP administration Notch approach to be suppressed (Wong etc., 2004) from the pharmacology.In experiment III, we select by after inhibitors of gamma-secretase DBZ that uses variable concentrations and the administration of BZ oral administration the Notch approach being suppressed from the pharmacology.Use variable concentrations principle behind to be that these chemical compounds of high concentration may not only influence intestinal neoplasms, and influence normal structure.

We begin the APC to 8 ages in week ^MinMice is treated, and these mices in this age have 30-60 in small intestinal can have 0-3 adenoma through the detected adenoma of naked eyes (polyp) in the colon.Each three mice was treated maximum 12 days with 10,15 or 30 μ mol/Kg BZ or DBZ, by the series section intestinal was carried out histological examination then.To beta-catenin white dyeing show adenoma, adenoma be embedded in usually accumulation hypertrophy but in the still unconverted normal crypts.Variation has taken place in the 12nd day adenoma with compd B Z and the treatment of DBZ oral administration.As if when concentration was 15 μ mol/Kg, the number of propagation Ki67 positive cell significantly reduced in the adenoma, and that most of normal bowel is organized is uninfluenced.

In a word, such as the Ki67/ beta-catenin dye in vain proof, with compd B Z and DBZ to APC ^MinAs if cell proliferation in the adenoma of its mouse oral clothes treatments back stops and normal structure is uninfluenced.

Table I

Gene	The crypts epithelium	Adenoma
Gene	The crypts epithelium	Adenoma	Notch1	+/-	+/-
Notch2	+	+	Notch1	+/-	+/-
Notch2	+	+	Notch3	+	+
Notch4	-	+/-	Notch3	+	+
Notch4	-	+/-	Delta1	++	+
Delta3	+	+	Delta1	++	+
Delta3	+	+	Delta4	+	+

Jagged1	+/-	+/-
Jagged1	+/-	+/-	Jagged2	-	-
			Jagged2	-	-
			Hesl	+	+

The accompanying drawing summary

Fig. 1

(example of the target gene (Hesl (right side)) of Delta-like 1 (left side), Notch2 (centre) and activation Notch approach is at APC for the Notch pathway component of in situ hybridization proof ^MinExpression in the mice adenoma.

Fig. 2

(upper diagram) or untreated (bottom graph) APC with the DAPT treatment as described herein ^MinThe analysis of mice adenoma of colon.Engrain in left upper portion/bottom graph shows to have the zone of representing the adenoma tissue in the white epithelium of high-level beta-catenin.Middle upper portion/bottom graph has provided the dyeing of cell cycle/propagation label Ki67.Notice the remarkable decline that causes proliferation activity in the adenoma with the DAPT treatment.Right upper portion/the increase of goblet cell when bottom graph demonstration is treated with DAPT, dark cell is indicated when being dyeed by the alizarin indigo plant of love.

Fig. 3

(upper diagram) or untreated (bottom graph) APC with the DAPT treatment as described herein ^MinThe analysis of mouse small intestine adenoma.Engrain in left upper portion/bottom graph shows to have the zone of representing the adenoma tissue in the white epithelium of high-level beta-catenin.Right upper portion/bottom graph has provided the dyeing of cell cycle/propagation label Ki67.Notice the remarkable decline that causes proliferation activity in the adenoma with the DAPT treatment.

Fig. 4

Gamma-secretase inhibitors DBZ makes the propagation pit cell be converted into mitosis after date goblet cell.To the C57B16 mice with every day 0,3,10 and 30 μ mol/Kg peritoneal injection gamma-secretase inhibitors DBZ, totally 5 days.When concentration was 3 μ mol/Kg, PAS dyeing showed that the goblet cell number slightly reduces (B), and during 10 and 30 μ mol/Kg, and the propagation pit cell is to the conversion of mitosis after date goblet cell (being respectively C and D) fully.

Fig. 5

By gamma-secretase inhibitors DBZ APC ^MinProliferative cell is to the conversion of mitosis after date goblet cell in the tumor.

APC ^MinMice was treated 10 days with 0 μ mol/Kg DBZ (A, B, E, F, G, K) or 10 μ mol/KgDBZ (C, D, L, M, N, O), by the series section intestinal was carried out histological examination then.White dyeing shows adenoma (figure A, C, E, L) to beta-catenin.The expression (O is than K) of Math1 (D than B, than M under the high-amplification-factor than F) and Pas has been induced in DBZ treatment, and has reduced the expression (N is than G) of Ki67.

Fig. 6

As described herein with BZ treatment or untreated APC ^MinThe intestinal of mice and the analysis of enteric adenoma.

To the C57B16 mice with every day 0,3,10 and 30 μ mol/Kg peritoneal injection gamma-secretase inhibitors BZ, totally 5 days.When being 3 μ mol/Kg, PAS dyeing shows that the goblet cell number slightly reduces (not shown), and when being 10 and 30 μ mol/Kg, and the propagation pit cell is to the conversion of mitosis after date goblet cell (A) fully.

Oral administration as described herein gives (upper diagram) or untreated (bottom graph) APC of BZ or DBZ treatment ^MinThe analysis of the adenoma of mice.Engrain shows to have the zone of representing the adenoma tissue in the white epithelium of high-level beta-catenin among the figure (B, C).Figure (D, E) has provided the dyeing of cell cycle/propagation label Ki67.Notice with DBZ treatment (not shown) or with BZ and treat the remarkable decline that (E is than D) causes proliferation activity in the adenoma, and as if surrounding tissue uninfluenced.

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Claims

1. method that is used to change adenoma and/or adenocarcinoma cell destiny, it comprises influences the Notch pathway activation.

2. one kind is used to induce adenoma and/or adenocarcinoma cell to be formed with the method for a postmitotic cell, and it comprises the Notch pathway activation that suppresses at least in part in described adenoma and/or the adenocarcinoma cell.

3. one kind is used for reducing at least in part the enteric adenoma that animal exists and/or the method for adenocarcinoma, and it comprises the Notch pathway activation that suppresses at least in part in the described animal.

4. according to each the method for claim 1-3, wherein activate and suppress described Notch pathway activation at least in part by weakening ligand-mediated Notch at least in part.

5. according to each the method for claim 1-3, wherein by providing inhibitors of gamma-secretase to suppress described Notch pathway activation at least in part.

6. according to the method for claim 5, wherein said inhibitors of gamma-secretase is DAPT or hexichol nitrogen  (DBZ) or benzene phenodiazine  (DB).

7. according to the method for claim 4, wherein weaken described ligand-mediated Notch at least in part and activate by the negative part of the dominance that Notch is provided.

8. according to the method for claim 4, wherein activate by providing the negative Notch of dominance to weaken described ligand-mediated Notch at least in part.

9. according to the method for claim 4, wherein activate by providing the antibody that to block Notch part and Notch interphase interaction at least in part to weaken described ligand-mediated Notch at least in part.

10. according to each the method for claim 1-3, wherein by providing the ADAM protease inhibitor to suppress described Notch pathway activation at least in part.

11. according to each the method for claim 1-10, it further comprises and suppresses the Wnt pathway activation at least in part.

12.Notch approach restrainer is used for the treatment of purposes in the medicine of enteric adenoma and/or adenocarcinoma in preparation.

13. according to the purposes of claim 12, wherein said Notch approach restrainer is an inhibitors of gamma-secretase.

14. according to the purposes of claim 14, wherein said inhibitors of gamma-secretase is DAPT or hexichol nitrogen  (DBZ) or benzene phenodiazine  (DB).

15. according to the purposes of claim 13, wherein said Notch approach restrainer is to weaken the activated inhibitor of ligand-mediated Notch.

16. according to the purposes of claim 15, wherein said inhibitor is the negative part of the dominance of Notch.

17. according to the purposes of claim 15, wherein said inhibitor is the negative Notch of dominance.

18. according to the purposes of claim 13, wherein said inhibitor is the antibody that can block Notch part and Notch interphase interaction at least in part.

19. according to the purposes of claim 13, wherein said inhibitor is the ADAM protease inhibitor.

20. according to each the purposes of claim 13-19, it further comprises the Wnt approach restrainer.

21. according to each the purposes of claim 13-20, wherein enteric adenoma and/or adenocarcinoma betide among the patient who suffers from genetic syndrome familial adenomatous polyposis (FAP).

22. a pharmaceutical composition, it comprises at least two kinds of Notch pathway activation inhibitor or comprises at least a Notch pathway activation inhibitor and at least a Wnt approach restrainer.