CN1738533A

CN1738533A - Methods for inhibiting cancer and scar formation

Info

Publication number: CN1738533A
Application number: CNA200380108713XA
Authority: CN
Inventors: 哈姆迪·K·哈姆迪; 拉克尔·卡斯特利翁
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-12-09
Filing date: 2003-12-04
Publication date: 2006-02-22
Also published as: NZ540607A

Abstract

Methods for inhibiting cancer, scar formation, disrupting the cellular cytoskeleton, and conferring resistance from infection are disclosed. Such methods comprise the administration of oleuropein and/or the products of its hydrolysis in therapeutically effective amounts. To that end, a variety of pharmaceutical formulations and routes or administration are disclosed and may be utilized to treat a wide variety of diseases.

Description

Suppress cancer and synulotic method

The cross reference of related application

The application is a part continuation application, require U.S. Patent application No.10/657,414 priority, be entitled as the method that suppresses angiogenesis, submit on September 8th, 2003, it is U.S. Patent application No.10/153, and 003 continuation application is entitled as the method that suppresses angiogenesis, submit on May 22nd, 2002, now issued and be U.S. Patent No. 6,632,798, also require U.S. Provisional Patent Application No.60/431,780 priority is entitled as the inhibition method for cancer, submits on December 9th, 2002.

Remarks: the research/development of federal funding: unavailable

Background of invention

The present invention relates to the method that effectively suppresses cancer, cicatrization, destruction cytoskeleton and give anti-infection property.More properly, the present invention relates to treat with the method for cancer, cicatrization and infection diseases associated with at the living animal of suffering from this class disease, comprise the method for sending this activity among the mankind.

Cancer is cell and the tumor growth that loses adjusting.Although the survival of cell, differentiation, motion and invasion and attack in wound healing, fetus and embryonic development and the process in the formation of corpus luteum, endometrium and placenta be natural and regulated.These processes are critical to healthy and growth, are subjected to the very well adjusting of oncogene, tumor suppressor gene and countless growth factors, but they can cause that when imbalance tumour forms.But, the gene alteration that is caused by the nature-nurture factor can cause the growth and the tumour of pathogenicity and imbalance.The pathogenicity cell insufficiency of accommodation that causes cancer and other diseases is undesirable, is the target of therapeutic intervention.

The brief overview of invention

In a kind of feature, the present invention relates to the method for anticancer, this method comprises makes cell, tissue or organ with cancer cell contact with the formula I compound of treatment effective dose.Formula I compound has following general formula:

Formula I

Among the formula I, R1 and R2 include but not limited to following functional group: hydroxyl ,-NH ₂With-the SH group.

Among the formula I, R3 includes but not limited to following functional group: hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, C1-C6-alkoxyl, halogen, NO ₂, NH ₃And COOCH ₃

Among the formula I, R4 includes but not limited to following functional group: hydrogen, C1-C6-alkoxyl, glucose, B-D-glucopyranose, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, halogen, NO ₂, NH ₃, carbohydrate, amino acid, nucleotide and lipid.

Among the formula I, R5 includes but not limited to following functional group: hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, C1-C6-alkoxyl, halogen, NO ₂, NH ₃And CH ₃

Among the formula I, X1-X3 includes but not limited to following functional group: oxygen, sulphur ,-CH ₂-or carboxyl, they can be different, but preferably identical functional group.

In preferred embodiment of the present invention, formula I compound is the oleuropein (oleuropein) of formula C25H32O13, is described in the tenth edition of THE MERK INDEX, and special topic is numbered 6709, shown in II.

Formula II

Can obtain plant extracts, any alcohol, acetone, inorganic and organic acid or alkali and/or aqueous extract that it can comprise plant extracts will contain oleuropein, i.e. compound shown in the formula II.Plant extracts is the following plant part acquisition that includes but not limited to from olive tree Olea europaea L., glossy privet tree Ligustrumobtusifolium (Oleaceae) etc.: leaf, bud, fruit, trunk, bark and root.

On the other hand, the present invention relates to the strong method that suppresses cancer cell in tissue or the organ, this method comprises makes this cell contact with pharmaceutical composition, described pharmaceutical composition contains oleuropein, is its derivative or its hydrolysate shown in compound shown in the formula II or the formula I, this can include but not limited to Oleuropeine aglycone, elenolic acid, β-3,4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate or its pharmaceutical composition, content is enough to suppress cancer.In presently preferred embodiments, this cell is arranged in organism alive.This based composition can comprise alcohol, acetone, inorganic and organic acid or the alkali and/or the aqueous solution of plant extracts, perhaps the derivative of its alcohol, acetone, inorganic and organic acid or alkali and/or the aqueous solution or component.

On the other hand, the present invention relates to treat by not needing and growth of cancer cells out of control, survival, duplicate, dynamic role, invasion and attack with shift mediate or the method for relevant with it Cancerous disease, this method comprises gives the effective dose of medicine compositions to the animal that lives, described pharmaceutical composition contains oleuropein, be its derivative or its hydrolysate shown in compound shown in the formula II or the formula I, this can include but not limited to Oleuropeine aglycone, elenolic acid, β-3,4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate or its pharmaceutical composition, dosage are enough to the anticancer survival, duplicate, growth, dynamic role, invasion and attack and transfer.These methods can be used for improving with unusual or worthless cancer cell multiplication and/or divide a word with a hyphen at the end of a line is the illness consequence of feature.This based composition can comprise alcohol, acetone, inorganic and organic acid or the alkali and/or the aqueous solution of plant extracts X, perhaps the derivative of its alcohol, acetone, inorganic and organic acid or alkali and/or the aqueous solution or component.

On the other hand, the present invention relates to cancer cell by the method for the glucose moiety selectivity target of oleuropein, oleuropein is its derivative or its hydrolysate shown in compound shown in the formula II or the formula I, this can include but not limited to Oleuropeine aglycone, elenolicacid, β-3,4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate or its pharmaceutical composition, consumption is enough to suppress cancer.According to such method, oleuropein enters described cell by the diffusion that is promoted by glucose transporter (GLUT).Have 12 kinds of GLUT albumen at present, they have different glucose compatibilities and tissue specificity distributes.It is feature that existing report human malignancies raises with glucose uptake and utilization, and this expression that is based on multiple GLUT isoform strengthens.For example, find that GLUT1 and/or GLUT 3mRNA increase in breast cancer, cancer of the esophagus and colon cancer.Cancer cell overexpression GLUT albumen, thereby more likely absorb the oleuropein that contains glucose molecule.

On the other hand, the method that the present invention relates to destroy cytoskeleton and prevent its regeneration, zooblast is mellow and full thus, can not break up, move or attack, this method comprises makes this cell contact with pharmaceutical composition, described pharmaceutical composition contains oleuropein, is its derivative or its hydrolysate shown in compound shown in the formula II or the formula I, this can include but not limited to Oleuropeine aglycone, elenolic acid, β-3,4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate or its pharmaceutical composition, content is enough to suppress cancer.In presently preferred embodiments, described cell is arranged in organism alive.This based composition can comprise alcohol, acetone, inorganic and organic acid or the alkali and/or the aqueous solution of plant extracts, perhaps the derivative of its alcohol, acetone, inorganic and organic acid or alkali and/or the aqueous solution or component.

On the other hand, the present invention relates to destroy the method for cytoskeleton, the infection of zooblast resistant bacteria, virus or parasite thus, this method comprises makes this cell contact with pharmaceutical composition, described pharmaceutical composition contains oleuropein, is its derivative or its hydrolysate shown in compound shown in the formula II or the formula I, this can include but not limited to Oleuropeine aglycone, elenolic acid, β-3,4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate or its pharmaceutical composition, content are enough to make described cell tolerance to infect.This based composition can comprise alcohol, acetone, inorganic and organic acid or the alkali and/or the aqueous solution of plant extracts X, perhaps the derivative of its alcohol, acetone, inorganic and organic acid or alkali and/or the aqueous solution or component.According to such method, believe the zooblast skeleton, comprise human cell's skeleton between by various viruses, bacterium and parasite infection period by target.For example, the unicellular gene of pathogene listeria spp is a kind of by the bacterium of host cell internalization, it induces actin (a kind of cytoskeletal protein) at its surface aggregate, and is used in the energy drives cell of auto polymerization effect dynamic role and cell to the propagation of cell.Oleuropein suppresses the cytoskeleton regeneration of zooblast, can protecting from infection property disease spread.In presently preferred embodiments, described cell is arranged in organism alive.

Other features, purpose and the advantage of the present invention and preferred implementation thereof will become apparent because of following detailed description.

Brief description of drawings

With reference to accompanying drawing, these and other feature of the present invention will become more apparent.

Fig. 1: matrigel invasion and attack determination method.Research most of invasive carcinomas (colon) on matrigel, we find the effect of oleuropein.The cell of handling with 0.1% oleuropein has stopped the invasion and attack gel fully, attaches to the filter on the bottom side.The diagram of matrigel invasion and attack chamber is shown in (A).Pair cell layer and filter plane photographing in experimentation.Pair cell and filter plane photographing are distinguished as (B) with (C) in the invasion and attack chamber of containing untreated cell.From photo, obviously see, can see the cell shade, show them, and move through the matrix glue-line near filter at the focal plane of filter.On the contrary, filter plane, the invasion and attack chamber photo (F) that contains the cell that useful oleuropein handled does not contain the cell shade.As if in fact, cell is taken a picture on the much higher plane in matrigel, be not movably (E) in matrigel.At the experiment terminal point, thoroughly remove matrigel.Filter is fixed, and dyeing is taken a picture.Divided a word with a hyphen at the end of a line the cell that passes the matrix glue-line and attach to the filter downside as (D) with (G).Resulting filter lacks cell from the invasion and attack chamber (G) of containing oleuropein, and showing does not have cell to arrive filter.Filter from the chamber of being untreated contains the cell (D) that penetrates matrigel and attach to filter.

Fig. 2: cell migration determination method.In this determination method, the Grawitz's tumor cell culture to converging, is scratched with aseptic batten then, form the appearance on a road.Make cell culture then and divide a word with a hyphen at the end of a line and pass this road, to repair the wound in the culture.(A), untreated cell has successfully sealed wound area shown in (B), (C), (D).By comparison, (E), the cell of handling with 0.01% oleuropein shown in (F), (G), (H) does not have wound closure effectively.Arrow shows the direction of wound.

Fig. 3: describe the variation of above-mentioned Fig. 2 cell migration determination method.Wound in this determination method is more extensive, forms corner or curved edge.In this experiment, malignant melanoma cell is grown to converge, scratch with batten then, mode as mentioned above.(A) be depicted as untreated cell, all directions are divided a word with a hyphen at the end of a line in dull and stereotyped upper edge.The dividing a word with a hyphen at the end of a line of cell (B) of crossing with 0.01% oleuropein solution-treated stopped fully.In this wound determination method, can clearly see edge of wound, be used to assess advancing of migratory cell.

Fig. 4: utilize the cell identical to describe the variation of above-mentioned experiment, except plating becomes circle with Fig. 3.(A) having handled cell (0.01% oleuropein) does not travel to beyond the appointment circle.But (B) untreated cell outward radial is divided a word with a hyphen at the end of a line, and covers dull and stereotyped.

Fig. 5: growth measurement method.The concrete cancerous cell line of five kinds of most of invasive cancers of representative is seeded on the flat board that contains the variable concentrations oleuropein with the number that equates.After 5 days, utilize MTS determination method assessment cell number, display light density (490nm) value on picture y axle.The x axle is represented the concentration of oleuropein.Do not carry out the processing of data.True and the original statistics that these representatives are carried out on micro-plate reader.In whole cancers of studying, oleuropein suppresses growth effectively.

Fig. 6: the tracheal rupture determination method on the matrigel.Cell is placed on the matrigel type tubulose network, and the latter disintegrated in 24-48 hour and is clump (Fig. 6 A, top line).The feature that pipe is disintegrated process proves that it takes place because of shrinking back of pipe, and tubercle (pipe infall) more approaches to merge each other (Fig. 6 A, end row) thus.Detailed information born of the same parents still prolong and spread all over everywhere.This cell slowly moves and contraction process (24-48 hour) involves initiatively cytoskeleton regeneration.Add 0.1% oleuropein " mellow and full " or induce each cell to form sphere (Fig. 6 B) on the spot to the pipe that is carried out, destroy the tubulose network by the quick process that occurs in 2 hours.Can not move based on forming sphere or mellow and full cell, rest on a kind of like this understanding of original position since die, thereby by preventing that cytoskeleton regeneration from obstructing the cell mobility, ends the process of shrinking back.

Fig. 7: the tracheal rupture determination method on the matrigel.Add oleuropein, it is mellow and full that the downtrod cell of glucose takes place.

Fig. 8: cell migration determination method.Make rabbit skin fibroblastic growth to the monolayer that converges, scratch with aseptic batten subsequently.Cultivate cell then, make it in cultivation, repair wound.Untreated cell is successfully divided a word with a hyphen at the end of a line and is passed injured area (A).Shown in (C), the cell of handling with oleuropein (0.01%) has effectively been suppressed.The feature of the cell that the process oleuropein is handled shows the discontinuity (D and E) of monolayer, and this is that cell forms spherical or mellow and full because be subjected to the influence of oleuropein pair cell skeleton.On the contrary, untreated cell seem smooth (B).The oleuropein of flush away cell reverses mellow and full process.Cell flattens, and the discontinuity (H) of compensation in the individual layer begins from edge of wound divide a word with a hyphen at the end of a line (F and G).

Detailed description of the invention

Following detailed description is intended as the explanation of current preferred invention embodiment, does not plan Represent unique form that the present invention can be implemented or utilize. This explanation is set forth and is implemented the present invention's step Rapid function and order. But be appreciated that different embodiments also can finish identical or Function of equal value and order, the function of described equivalence and order also plan to covered in of the present invention In the scope.

A. definition

The biology that term " animal " expression has the sealing blood vessels circulatory system comprises bird, lactation Animal, amphibian, fish and reptile. Terminology used here " animal " also comprises the people Class.

A kind of character that term " anti-infection property " expression oleuropein is given zooblast makes this A little cell resistance infectious agent, bacteria-like, virus and parasite. Give this property by oleuropein Matter and its direct effect to microorganism itself are irrelevant, the existing detailed record of document.

Out of control and the imbalance of term " cancer " expression cell in tissue, organ or human body background Growth.

Term " transfer " expression tumour cell diffuses to the process of the remote part of body. This art Language also is used for the tumour that expression forms by transfer process in this article.

Term " contact " in this article with the following term Alternate: merge, add, mix, Pass through, cultivate, flow through etc. And The compounds of this invention can " be given by any conventional method Medicine ", for example parenteral, oral, part and inhalation route are as described herein.

The compound salt that term " pharmaceutically acceptable salt " expression is such, they keep free The biological effectiveness of alkali and character, by getting with inorganic acid reaction, for example hydrochloric acid, hydrobromic acid, Sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethyl sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid etc. Pharmacy Upper acceptable salt for example comprises alkali metal salt, for example the salt of sodium and potassium, alkali salt and ammonium Salt.

" capacity ", " effective dose ", " treatment effective dose " or " anti-angiogenic growing amount " table Show that compound or composition effectively reduce, suppress or suppress Angiogenesis or cause with blood vessel living The amount of the doing well,improving that the disease that becomes second nature is relevant. Results needed can be the subjectivity sx↓ or Objectively identifiablely accept that administration person takes a turn for the better, endothelial cell vascularization minimizing or Angiogenesis Rate reduces, and carries out record by clinician or other titular observers.

The general expression such as term " treatment cancer ", " therapy " suffers from animal any of cancer Take a turn for the better, wherein this improvement can be owing to treating with The compounds of this invention. This improvement can be main Seeing property or objectivity. For example, if animal is the people, the patient can be good energy or vigor Turn to or pain relief is recorded as that the subjectivity symptom takes a turn for the better or the therapy response. Select as an alternative, The clinician can send out based on physical examination, laboratory parameters, tumor markers or radiography Now notice the minimizing of tumor size or tumor load. Some can observe therapy for the clinician The laboratory sign of response comprises the normalization of test, for example leukocyte count, red blood cell number, blood Platelet number, erythrocyte sedimentation rate (ESR) and various enzyme level. In addition, the clinician can observe can The tumor markers that detects reduces. Select as an alternative, can utilize other to test to estimate the visitor Sight improvement, for example audiograph, nuclear magnetic resonance test and positron emission test.

" inhibition tumor cell growth " can be by any generally acknowledged measurement growth of tumour cell The no method that has been delayed or eliminated is estimated. This comprises direct observation and Indirect evaluation, example Such as subjective symptom as discussed above or objective sign.

Term " alkyl " is used for expression branch or unbranched, saturated or unsaturated in this article , monovalence hydrocarbon atomic group with 1-12 carbon, preferred 1-6 carbon. When alkyl has 1 During-6 carbon atoms, it is called as " low alkyl group ". The alkyl atomic group that is fit to for example wraps Draw together methyl, ethyl, n-pro-pyl, isopropyl, 2-acrylic (or pi-allyl), normal-butyl, The tert-butyl group, isobutyl group (or 2-methyl-propyl) etc. As used herein, this term is contained " alkyl of replacement ".

" alkyl of replacement " expression as described alkyl just now comprise one or more functional groups, for example low alkyl group, aryl, acyl group, halogen (be alkyl halide, CF for example₃), hydroxyl, ammonia Base, alkoxyl, alkylamine, acyl amino, acyloxy, aryloxy group, aryloxy alkyl, mercapto Basic, saturated and undersaturated cyclic hydrocarbon, heterocycle etc. These groups can be connected in moieties Any carbon.

Term " S-alkyl " is used for expression group-SR in this article, and wherein R decides such as this paper The low alkyl group of justice or the low alkyl group of replacement.

Term " aryl " is used for the expression aromatic substituent in this article, and it can be single virtue Family ring or a plurality of aromatic ring, they condense together, covalently bound or connect with public group Connect, for example methylene or ethylidene part. Public linking group can be carbonyl also, such as two In the benzophenone. Aromatic ring can comprise phenyl, naphthyl, biphenyl, benzhydryl and benzophenone. Term " aryl " is contained " aryl alkyl ".

" aryl of replacement " expression as described aryl just now comprise one or more functional groups, for example low alkyl group, acyl group, halogen, alkyl halide (CF for example₃), hydroxyl, amino, alcoxyl Base, alkylamine, acyl amino, acyloxy, sulfydryl, saturated and undersaturated cyclic hydrocarbon, it Condense with aromatic ring, covalently bound or be connected with public group, for example methylene or Asia The ethyl part. Linking group also can be carbonyl, for example in the cyclohexyl-phenyl ketone. Term " is got The aryl in generation " contain " aryl alkyl of replacement ".

Term " halogen " is used for expression fluorine, bromine, chlorine and iodine atom in this article.

Term " hydroxyl " is used for expression group-OH in this article.

Term " amino " is used to represent group-NRR ' in this article, and wherein R and R ' can be alkyl, the aryl of hydrogen, alkyl, replacement, the aryl or the acyl group of replacement independently.

Term " nitro " is used to represent group-NO in this article ₂

Term " alkoxyl " is used for expression-OR group in this article, wherein R is the aryl alkyl of aryl, aryl alkyl or replacement of low alkyl group, aryl, the replacement of low alkyl group, replacement, and wherein the aryl alkyl of the aryl of this alkyl, aryl, replacement, aryl alkyl and replacement is as described herein.The alkoxyl group that is fit to for example comprises the phenoxy group, benzyloxy, benzene ethyoxyl, tert-butoxy of methoxyl group, ethyoxyl, phenoxy group, replacement etc.

Term " alkenyl " is used to represent undersaturated side chain, straight chain or ring-type monovalence hydrocarbon atomic group in this article, has at least one carbon-to-carbon double bond.This atomic group can be about the cis of two keys or anti-configuration.The alkenyl atomic group that is fit to for example comprises vinyl, acrylic, isopropenyl, cyclopropanyl, cyclobutenyl, isobutenyl, cyclobutane base, uncle's cyclobutenyl, pentenyl, hexenyl etc.

Term " alkynyl " is used to represent undersaturated side chain, straight chain or ring-type monovalence hydrocarbon atomic group in this article, has at least one carbon-to-carbon, three key.The alkynyl atomic group that is fit to for example comprises acetenyl, propinyl, butynyl, isobutyl alkynyl, pentynyl, hexin base etc.

The term enantiomer be used in this article represent molecular entity to one of, they are mirror images each other, and can not be overlapping.

B. compound

The present invention relates to such discovery, the growth of formula I compound anticancer can be used for treating Cancerous disease.Formula I compound has following general formula:

Formula I

Wherein R1, R2, R3, R4, R5 and X1, X2 and X3 are as defined above.In preferred embodiment of the present invention, formula I compound is an oleuropein, shown in II:

Compound oleuropein used in this invention can easily obtain from plant, includes but not limited to olive tree Olea europaea L., glossy privet tree Ligustrum obtusifolium (Oleaceae).

Utilize in the external and body Screening test method can easily differentiate the compound that is suitable for use in the inventive method.This class determination method can screen specific compound external with body in anticancer growth, move the ability with invasion and attack or tumour generation.Select as an alternative, this class determination method can be screened the ability that specific compound suppresses to cause the cytoskeleton that cell is mellow and full.For example, the matrigel invasion and attack determination method that has a detailed description below can be used to screen the ability that given compound suppresses the cell invasion extracellular matrix.Such cell invasion is the essential of cancer foundation and growth.In as the matrigel invasion and attack determination method that the present invention carried out, cancer cell is seeded on the matrigel, the latter is an extracellular matrix.Make cell dissociation and invasion and attack gel.The cell adhesion of successfully attacking matrigel is in the filter downside, and filter contains porose.Utilize microscope to focus on to contain each plane of the chamber of cell, can carry out the monitoring of cell invasion matrigel.Measuring terminal point, remove matrigel fully, filter to be fixed, dyeing is to appear the cell that successfully penetrates the matrix glue-line.Based on a large amount of cell number on the filter, can assess the invasive inhibitory action result of compound pair cell.This determination method can be used to assess the anticancer character of The compounds of this invention.

In another kind of determination method, the cell of some types when be grown in high concentration Ranvier's membrane matrix (when matrigel ＞10mg/ml) goes up in 48 hours a series of metamorphosis of experience.This makes the researcher can be by joining compound in the culture to their influence of the effect of these variations analysis.During this process, the form of cell becomes three-dimensional pipe sample network from smooth bidimensional cell.But, the pipe that is generated is unsettled, finally shrinks back and disintegrates 48 hours internal causes.Join and cause cell to be considered to act on cytoskeleton in the pipe that is generated because of the mellow and full compound that breaks.This determination method can be used to assess the compound destruction of pair cell skeleton in the present invention.To be it is evident that The compounds of this invention can be taked the form of pharmaceutically acceptable salt and/or pharmaceutical composition by independent administration by those skilled in the art.

C. the purposes of The compounds of this invention

Just as explained above, the present invention relates to such discovery, formula I compound or its preferred implementation oleuropein or its hydrolysate can be used for anticancer survival, growth, move and invasion and attack, can be used for then treating and cancer growth diseases associated out of control and imbalance.Therefore, in one embodiment, the invention provides and suppress method out of control and the imbalance cancer cell, this method comprises makes cell and effective dose, be that the formula I compound of anticancer amount or its preferred implementation oleuropein or its hydrolysate contact.In another embodiment, the invention provides the method for anticancer, this method comprises contacts the cell, tissue or the organ that contain cancer cell and effective dose formula I compound or its preferred implementation oleuropein or its hydrolysate.In presently preferred embodiments, described cell is arranged in animal subject.

The present invention relates to treat with the method for disease of related to cancer unwanted and out of control, this method comprises gives anticancer formula I compound or its preferred implementation oleuropein or its hydrolysate to animal, consumption, is that dosage is enough to suppress cancer.Formula I compound or its preferred implementation oleuropein or its hydrolysate suppress cancer and/or the required given dose of cancer disease will depend on the seriousness of illness, the approach and the related factors of administration, and this will depend on the attending doctor.Generally speaking, dosage will be the amount that is enough to effectively suppress cancer and/or cancer disease putative and effective every day.In this, method of the present invention will be very similar to the applicant's unsettled U.S. Patent application No.10/153, and 003, be entitled as the method that suppresses angiogenesis, submit on May 22nd, 2002, now be in the promulgation process.In this, the applicant is specially with U.S. Patent application No.10/153, and 003 instruction is incorporated herein by reference.

By methods of treatment provided by the present invention is to implement like this, the animal of needs is given formula I compound or its preferred implementation or its hydrolysate (perhaps its pharmaceutically acceptable salt or solvate) of doses, and described dosage effectively suppresses cancer and/or cancer disease.Term " inhibition " is used to comprise its putative implication in this article, and this comprises that the preventive disposal human subject avoids obtaining cancer and/or cancer disease, prevents and/or existing cancer of treatment and/or cancer disease.Therefore, the present invention includes therapeutic treatment and/or preventive disposal, depend on the circumstances.

Method of the present invention can be used for the treatment of multiple cancer disease.The Cancerous disease of the enough the inventive method treatments of known energy includes but not limited to the acute lymphoblastic leukemia of being grown up; The children acute lymphoblastic leukemia; Adult's acute myelogenous leukemia; Adrenocortical carcinoma; Children's adrenocortical carcinoma; AIDS correlation lymphoma; AIDS correlation malignant tumour; Cancer of anus; Children's cerebellar astrocytoma; The big cerebral astrocytoma of children; Cholangiocarcinoma; Carcinoma of urinary bladder; Children's carcinoma of urinary bladder; Osteosarcoma/MFH type osteocarcinoma; Children's brain stem glioma; Adult's brain tumor; Children's brain stem glioma type brain tumor; Children's cerebellar astrocytoma type brain tumor; The big cerebral astrocytoma of children/glioblastoma type brain tumor; Children's ependymoma type brain tumor; Children's medulloblastoma type brain tumor; Former neuroectodermal tumors type brain tumor on children's curtain; Children look road and hypothalamus glioma type brain tumor; Children's brain tumor (other); Breast cancer; Breast cancer and gestation; Children's breast cancer; Male breast carcinoma; Children's bronchial adenoma/carcinoid; Children's carcinoid tumor; The stomach and intestine carcinoid tumor; Adrenocortical carcinoma; Islet-cell carcinoma; Unknown primary cancer; Primary central nervous system lymphoma; Children's cerebellar astrocytoma; Big cerebral astrocytoma/the glioblastoma of children; Cervical carcinoma; Children's cancer; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic spinal cord hyperplasia disease; The stndon sheath clear cell sarcoma; Colon cancer; Children's colorectal cancer; Skin T-cell lymphoma; Carcinoma of endometrium; Children's ependymoma; Epithelial ovarian cancer; Cancer of the esophagus; Children's cancer of the esophagus; Ewing's tumor family; The outer enblastoma of children's cranium; The outer enblastoma of gonad; Cholangiocarcinoma; Intraocular melanoma type cancer eye; Retinoblastoma type cancer eye; Carcinoma of gallbladder; Cancer of the stomach; Children's cancer of the stomach; The stomach and intestine carcinoid tumor; The outer enblastoma of children's cranium; The outer enblastoma of gonad; Germinal tumor of ovary; The gestation trophoblastic tumor; Children's brain stem glioma; Children look road and hypothalamus glioma; Hairy cell leukemia; Head and neck cancer; Adult (primary) liver cell (liver) cancer; Children's (primary) liver cells (liver) cancer; Adult He Jiejin lymphomas; Children He Jiejin lymphomas; Pregnancy duration He Jiejin lymphomas; The hypopharynx cancer; Children's hypothalamus with look the road glioma; Intraocular melanoma; Islet-cell carcinoma (endocrine pancreas); Kaposi; Kidney; Laryngocarcinoma; Children's laryngocarcinoma; Adult's acute lymphoblastic leukemia; The children acute lymphoblastic leukemia; Adult's acute myelogenous leukemia; The children acute myelomatosis; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Hairy cell leukemia; Lip and carcinoma of mouth; Adult's (primary) liver cancer; Children's (primary) liver cancer; Lung cancer in non-cellule type; Small cell lung cancer; Adult's acute lymphoblastic leukemia; The children acute lymphoblastic leukemia; Chronic lymphocytic leukemia; AIDS correlation lymphoma; Central nervous system (primary) lymphoma; Skin T-cell lymphoma; Adult He Jiejin lymphomas; Children He Jiejin lymphomas; Pregnancy duration He Jiejin lymphomas; Adult's non_hodgkin lymphoma; Children's non_hodgkin lymphoma; Pregnancy duration Fei Hejiejinshi disease; Primary central nervous system lymphoma; Walden Si Telunshi macroglobulinemia; Male breast carcinoma; Become the HMM; Children's malignant mesothelioma; Children's medulloblastoma; Melanoma; Intraocular melanoma; Mei Keer Schwann Cells cancer; Malignant mesothelioma; Recessive primary metastatic squamous neck cancer; The multiple internal secretion neoplasia of children syndrome; Huppert's disease/plasmacytoma forms; Mycosis fungoides; Myelodysplastic syndrome; Chronic myelogenous leukemia; The children acute myelomatosis; Huppert's disease; Chronic spinal cord hyperplasia disease; Nasal cavity and nasal sinus cancer; Nasopharyngeal carcinoma; Children nasopharyngeal carcinoma; Neuroblastoma; Adult's non_hodgkin lymphoma; Children's non_hodgkin lymphoma; The pregnancy duration non_hodgkin lymphoma; Lung cancer in non-cellule type; Children's mouth cancer; Oral cavity and lip cancer; The oropharynx cancer; Osteosarcoma/pernicious bone fibres histocytoma; Children's oophoroma; Epithelial ovarian cancer; Germinal tumor of ovary; Ovary hangs down pernicious potential tumor; Cancer of pancreas; Children's cancer of pancreas; The islet cells cancer of pancreas; Paranasal sinus and CARCINOMA OF THE NASAL CAVITY; Parathyroid carcinoma; Carcinoma of penis; Pheochromocytoma; Children's pineal gland is gone up former PNET with curtain; Hypophysoma; Plasmacytoma formation/Huppert's disease; The pleura pulmonary blastoma; Gestation and breast cancer; Gestation and He Jiejin lymphomas; Gestation and non_hodgkin lymphoma; Primary central nervous system lymphoma; Become the human primary liver cancer; Children's primary carcinoma of liver; Prostate cancer; The carcinoma of the rectum; Nephrocyte (kidney) cancer; Children's clear-cell carcinoma; Renal plevis and ureter transitional-cell carinoma; Retinoblastoma; Children's rhabdomyosarcoma; Salivary-gland carcinoma; Children's salivary gland cancer; Ewing's tumor family sarcoma; Kaposi; The sarcoma of bone (osteosarcoma)/MFH; Children's rhabdomyosarcoma; Adult soft tissue sarcoma; Children soft tissue sarcoma; Sezary syndrome; Cutaneum carcinoma; Children's cutaneum carcinoma; Cutaneum carcinoma (melanoma); Mei Keer Schwann Cells property cutaneum carcinoma; Small cell lung cancer; Carcinoma of small intestine; Adult soft tissue sarcoma; Children soft tissue sarcoma; Recessive primary metastatic squamous neck cancer; Cancer of the stomach; Children's cancer of the stomach; Former PNET on children's curtain; Skin T-cell lymphoma; Carcinoma of testis; Children's thymoma; Thymoma and thymic carcinoma; Thyroid cancer; Pediatric thyroid carcinomas; Renal plevis and ureter transitional-cell carinoma; The gestation trophoblastic tumor; The unknown primary of being grown up position cancer; The unknown primary of children position cancer; The unusual cancer of children; Ureter and renal plevis transitional-cell carinoma; Carcinoma of urethra; Carcinoma of endometrium; Sarcoma of uterus; Carcinoma of vagina; Children look road and hypothalamus glioma; Carcinoma of vulva; Walden Si Telunshi macroglobulinemia; Wilms' tumor.

Method of the present invention also can be used to give zooblast with anti-infection property.By destroying the cytoskeleton of zooblast, oleuropein is given the resistance of cell to virus, bacterium and parsitism.

Also can treat with blister, ulcer formation, incrustation and cicatrization diseases associated with method of the present invention.Cause that ulcer forms and synulotic disease includes but not limited to chemicals, heat and radiation burn, scratch and incised wound, wound, fibroma, tumour, keloid, acne, gastritis, vagina, uterine neck, the uterus, ovary, stomach, cornea, retina, diabetes, the cicatrization of AIDS correlation, ileum and colonic ulcer, interstitial lung disease, people's fibre modification lung disease, people's kidney disease, glomerulonephritis, the ephritis relevant with systemic lupus, peritoneal fibrosis, cystic fibrosis, hepatic fibrosis, myofibrosis cordis, pnemnofibrosis, GraveShi illness in eye, drug-induced ergotism, angiocardiopathy, cancer, Alzheimer's, cicatrization, chorionitis, spongioblastoma in the Li-Fraumeni syndrome, sporadic spongioblastoma, myelomatosis, acute myelogenous leukemia, myelodysplastic syndrome, spinal cord hyperplasia syndrome, the gynaecology cancer, Kaposi, hansen's disease, or do not comprise the inflammatory bowel disease of collagenous colitis, the kidney fibre modification, the belly adhesion, radiation induced fibre modification, bronchiolitis obliterans, the silicosis damage, or ocular capsule fibroplasia, perhaps blood vessel birthmark, tatoo and traumatic synulotic laser disposal.

In particularly preferred embodiments, the present invention relates to unite the method for giving formula I compound or its preferred implementation oleuropein with active immne therapy (for example tumor vaccine inoculation).

And according to the method described above, the animal subject that treat includes but not limited to people, laboratory animal, pet and livestock.

D. pharmaceutical preparation/method of administration

In the method for the invention, animal be sent or be administered to formula I compound or its preferred implementation oleuropein or its hydrolysate can separately, human patients for example, take the form of pharmaceutically acceptable salt or pharmaceutical composition, the compound that to treat effective dose in described composition and the carrier or the mixed with excipients that are fit to, described effective dose for example effectively reduce, suppress or suppress cancer or cause the dosage of the doing well,improving relevant with the cancer disease.

Can be incorporated in the several formulations with in the methods of the invention formula I compound or its preferred implementation oleuropein or its hydrolysate, for the therapeutic administration.More properly, formula I compound or its preferred implementation oleuropein or its hydrolysate can become pharmaceutical composition with suitable pharmaceutically acceptable carrier or thinner co-formulated, can be mixed with the prepared product of solid, semisolid, liquid or gaseous form, for example tablet, capsule, pill, pulvis, granule, lozenge, gel, slurries, ointment, solution, suppository, injection, inhalant and aerosol.Therefore, the administration of compound can realize by different way, comprise in mouth, cheek, rectum, parenteral, the peritonaeum, in the corium, transdermal, intracheal administration etc.And, described compound can be with the part but not the systemic fashion administration for example this compound directly is injected in the entity tumor, in be everlasting Drug Storage or sustained release preparation.In addition, described compound can administration in the target drug delivery system, for example scribbles the liposome of tumor specific antibody.This lipoid plastid will be by target in tumour, and is absorbed by tumor-selective.

In addition, formula I compound or its preferred implementation oleuropein or its hydrolysate can be prepared with common excipient, diluent or carrier, and compacting perhaps is mixed with elixir or solution in flakes, to make things convenient for oral administration, perhaps by intramuscular or intravenous route administration.This compound can be mixed with slow release formulation etc. by cutaneous penetration.

Formula I compound or its preferred implementation oleuropein or its hydrolysate can be by independent or the administrations that combines with one another, perhaps they can unite use (for example other anticarcinogens or other drug, for example AZT, antiinflammatory, antibiotic, corticosteroid, vitamin etc.) with other known compounds.For example, formula I compound or its preferred implementation oleuropein or its hydrolysate can be used in (for example catharanthus alkaloid, antibiotic, antimetabolite, platinum coordination complex etc.) in the conjoint therapy with other known angiogenesis inhibitor chemotherapeutics or anti-neoplasia agent.For example, formula I compound or its preferred implementation oleuropein or its hydrolysate can be used in the conjoint therapy with following medicine: catharanthus alkaloid compound, for example vinblastine, vincristine, taxol etc.; Antibiotic, for example adriamycin (adriamycin), actinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C) etc.; Antimetabolite, for example methotrexate (MTX), cytarabine (AraC), azauridine, azaribine, fluorodeoxyuridine, deoxycoformycin, purinethol etc.; Perhaps platinum coordination complex, for example cis-platinum (cis-DDP), carboplatin etc.In addition, those skilled in the art will figure out, and The compounds of this invention can be used in the conjoint therapy with other known angiogenesis inhibitor chemotherapeutics or anti-neoplasia compound.In pharmaceutical dosage form, described compound can be with they pharmaceutically acceptable salt form administrations, and perhaps they also can be used separately or suitably, and unite use with other pharmaceutically active compounds.

Pharmaceutical composition described herein can be made in the manner known to persons skilled in the art, just by mixing, dissolving, granulation, system ingot, grinding, the emulsification of routine, seal, embedding or freeze-drying process.Following method and excipient only supply illustration, by no means restriction.

With regard to injection, compound can be formulated into prepared product like this, their is dissolved, suspends or is emulsified in water-based or the non-aqueous solvent, for example vegetable oil or other similar oil, synthctic fat acid glyceride, high-grade aliphatic ester or propane diols; If necessary, also have conventional additives, for example solubilizer, isotonic agent, suspending agent, emulsifier, stabilizing agent and preservative.Preferably, The compounds of this invention can be formulated in the aqueous solution, compatible buffer solution, for example Han Kesishi solution, Ringer's mixture or normal saline buffer solution on the preferred physiology.With regard to transmucosal administration, in preparation, use the bleeding agent of the barrier that is suitable for the infusion of wanting.This class bleeding agent is well known in the art.

With regard to oral administration,, can easily prepare formula I compound or its preferred implementation oleuropein or its hydrolysate by mixing with pharmaceutically acceptable carrier well known in the art.This class carrier makes described compound can be formulated into tablet, pill, lozenge, capsule, emulsion, lipophilicity and hydrophily suspension, liquid, gel, syrup, slurries, suspension etc., supplies patient's orally ingestible of being treated.Mouth can obtain like this with pharmaceutical preparations, and described compound is mixed with solid excipient, grinds the gained mixture alternatively, and process mixture is a particle, if necessary, obtains tablet or lozenge core after adding the auxiliary agent that is fit to.The excipient that is fit to has filler definitely, and for example carbohydrate comprises lactose, sucrose, mannitol or sorbitol; Preparation of cellulose thing, for example corn starch, wheaten starch, rice fecula, potato starch, gelatin, bassora gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP).If necessary, can add disintegrant, for example crospolyvinylpyrrolidone, agar or alginic acid or its salt, for example mosanom.

The lozenge core has suitable dressing.For this reason, priming be can use, gum Arabic, talcum, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture wherein can be contained alternatively.Can add dyestuff or pigment to tablet or lozenge dressing, for the various combination of differentiating or distinguish active compound doses.

Pharmaceutical preparations that can mouthful usefulness comprises that described plasticizer is glycerine or sorbitol for example by pushing of making of the gelatin-formula capsule and the soft seal capsule agent of being made by gelatin and plasticizer.Described pushing-formula capsule can contain the mixture of active component and following ingredients: filler, for example lactose; Adhesive, for example starch; And/or lubricant, for example talcum or dolomol; With optional stabilizing agent.In soft capsule, reactive compound can be dissolved or suspended in the suitable liquid, for example fat oil, atoleine or liquid macrogol.In addition, can add stabilizing agent.The dosage of all oral Preparations all should be suitable for this class administration.With regard to the cheek administration, described composition can be taked the form of tablet or dragee, in the usual way preparation.

With regard to inhalation, being used for the suitable mode with aerosol spray of compound of the present invention delivers from pressurized package or atomizer, use the propellant that is fit to, for example dicholorodifluoromethane, trichlorine fluomethane, dichlorotetra-fluoroethane, carbonic acid gas or other gas that is fit to perhaps deliver from the Diskus of no propellant.Under the situation of pressurised aerosol, provide the valve of metered delivery can determine dosage device.The for example gelatine capsule and the cartridge case that are used in inhalator or the insufflator can be formulated into the mixture that contains compound and suitable powder matrix, and described matrix is lactose or starch for example.

Described compound can be formulated into by injection and supply parenteral, for example bolus injection or continuous infusion.Ejection preparation can the display unit formulation, for example in ampoule or multi-agent container, contains the preservative that adds to some extent.Described composition can be taked the form of suspension, solution or emulsion in oiliness or aqueous carrier, can contain preparaton, for example suspending agent, stabilizing agent and/or dispersant.

The aqueous solution that comprises the reactive compound of active water-soluble form for the pharmaceutical preparation of parenteral.In addition, the suspension of reactive compound can be made suitable oily injection suspension.The lipophilic solvent or the carrier that are fit to comprise fat oil, for example sesame oil, or Acrawax, for example ethyl oleate or triglycerides, perhaps liposome.The water-based injection suspension can contain the material that increases suspension viscosity, for example sodium carboxymethylcellulose, sorbitol or glucan.Alternatively, described suspension also can contain suitable stabilizing agent or increase the reagent of described compound dissolution degree, so that the solution that preparation highly concentrates.Select as an alternative, described active component can be a powder type, for using the carrier regeneration that is fit to, for example aseptic pyrogen-free water before using.

Described compound also can be formulated in the rectal compositions, and for example suppository or enema,retention for example contain conventional suppository bases, for example cocoa butter, carbowax, polyethylene glycol or other glyceride, all they all under body temperature, melt, at room temperature solidify.

Except previous formulations, described compound also can be formulated into the Drug Storage prepared product.This class durative action preparation can be by implanting (for example subcutaneous or intramuscular) or intramuscular injection administration.Thereby for example, this compound can be prepared with the polymerism that is fit to or hydrophobic material (for example being mixed with emulsion in acceptable oil) or ion exchange resin, perhaps is mixed with the slightly solubility derivative, for example indissoluble salt.Select as an alternative, can adopt other delivery systems of hydrophobic pharmaceutical compounds.Liposome and emulsion are the examples of the hydrophobic drug delivery vector known.

In addition, can adopt the target of mark on unusual tumor vascular system.Targeting moiety will play the effect of concentrating medicine as required when with drug toxicity or radioisotope coupling.Also can use the part of tumour associativity vessel landmarks thing.For example, can adopt cell adhesion molecule, it combines with tumor vessel element surface mark.Also can use liposome and other drug delivery system, if especially the part that makes carrier preferentially point to tumor vascular system is contained on their surface.Liposome provides additional advantage, promptly shields medicine and most of normal structure, reduces the intrinsic toxicity of a lot of compounds thus.So that phagocyte when absorbing minimized polyethylene glycol (PEG) (being stealthy liposome) and the selectively targeted part of tumor vascular system, liposome provides longer plasma half-life, lower non-target tissue's toxicity and is better than the not effect of targeted drug when bag.Other target strategies include but not limited to ADEPT (pointing to the enzyme precursor medicinal treatment of antibody), GDEPT (pointing to the EPT of gene) and VDEPT (pointing to the EPT of virus).In ADEPT, the inactive precursor drug targeting is subjected to the influence of antitumor associativity mark antibody in tumor mass.Among the tumour or near enzyme environment pro-drug is converted into active toxic agents, the latter acts on tumor tissues then.Similarly, in GDEPT and VDEPT, utilizing different genes expression or viral target on the tumor locus respectively is its active toxic forms with prodrug activation.Other strategies comprise different gene, enzyme or the surface markers of expressing of target, and they appear on the tumour associativity vascular system, to realize the control of tumor growth.Utilize said method, formula I compound or its preferred implementation oleuropein or its hydrolysate can be by target in tumor vascular systems, and with the control of realization tumour progression, or target is in other relevant positions (for example endothelial cell).

Also can adopt some organic solvent, for example dimethyl sulfoxide (DMSO).In addition, can utilize slow-released system to send described compound, for example contain the semipermeability matrix of the solid hydrophobic polymer of therapeutic agent.Various types of slow-release materials all have been ripe, are well-known to those skilled in the art.According to their chemical attribute, Duracaps can discharge described compound and reach several thoughtful more than 100 days.

Described pharmaceutical composition also can contain suitable solid or gel phase carrier or excipient.The example of this class carrier or excipient includes but not limited to calcium carbonate, calcium phosphate, various carbohydrate, starch, cellulose derivatives, gelatin and polymer, for example polyethylene glycol.

The pharmaceutical composition that is suitable for use among the present invention comprises such composition, wherein contains the treatment effective amount of actives.The dosage of composition will depend on the curee who is treated, curee's body weight, the seriousness of sufferer, the mode of administration and attending doctor's judgement certainly.Determining fully in those skilled in the art's limit of power of effective dose is especially in view of detailed content that this paper provided.

Current, the treatment effective dose that expectation is used in the compound in the invention process anywhere can comprise every kilogram of curee's body weight of this compounds of about 0.030g to 20.000g.

F. embodiment

The present invention will be described in detail by specific embodiment.The following example only supplies purposes of illustration, does not plan to limit by any way the present invention.Those skilled in the art will recognize the multiple nonessential parameter that can change or revise easily, to obtain identical in essence result.

Embodiment 1

This example is set forth the anticancer character of oleuropein in matrigel invasion and attack determination method.The terminal point of matrigel invasion and attack determination method is invasion and attack and digestion matrigel and arrive the mensuration of the cell number of filter on the chamber other end.A kind of like this quantification will be set forth their aggressive by the ability of cancer cell digestion and invasion and attack extracellular matrix.Find the cell number on the filter because of using of oleuropein and reduce, promptly show its anticancer character.

Utilize Boyden chamber (Figure 1A) as shown in drawings, we deposit the top layer of matrigel, concentration 10mg/ml.Utilize maximum invasive carcinoma (colon) cell-line, we with cell inoculation at the matrigel top layer that contains or do not have oleuropein.Allow cell migration to pass gel and reach 3 days, during this period pair cell layer and filter plane photographing.At the experiment terminal point, remove the matrix glue-line, will be attached to the cell fixation of filter downside, dyeing is taken a picture.The cell of handling with 0.1% oleuropein stops to attack gel fully and attaches to the filter bottom side.The invasion and attack chamber that contains untreated cell is distinguished pair cell and filter plane photographing shown in Figure 1B and Fig. 1 C.Obviously find out from photo, can see the cell shade, show that they are near filter with move through the matrix glue-line at the focal plane of filter.On the contrary, the filter plane photo (Fig. 1 F) that contains in the invasion and attack chamber of the cell that useful oleuropein handled does not contain the cell shade.As if in fact, pair cell is to take a picture on the plane much higher in matrigel, be not movably (Fig. 1 E).Divided a word with a hyphen at the end of a line the cell that passes the matrix glue-line and attach to the filter downside shown in Fig. 1 D and Fig. 1 G.Lack cell from the chamber gained filter (Fig. 1 G) that contains oleuropein, showing does not have cell to arrive filter.Filter from the chamber that is untreated contains the cell (Fig. 1 D) that penetrates matrigel and attach to filter.This class presentation of results, oleuropein are effective cancer cell invasion and attack inhibitor, thereby are regarded as anticancer compound.

Embodiment 2

This example is set forth the anticancer character of oleuropein in the cell migration determination method.The terminal point of cell migration determination method is the mensuration of cell number of the wound that produced to be enclosed in the culture dish of dividing a word with a hyphen at the end of a line.A kind of like this quantification will be set forth their mobility by the ability of a kind of like this wound of cancer cell complete closed.Find the cell number in the wound area because of using of oleuropein and reduce, promptly show its anticancer character.

In classical wound determination method, cell culture to converging, is scratched with aseptic batten then, form the appearance on a road.With cell culture, divide a word with a hyphen at the end of a line and pass this road then, to repair the wound in the culture.In this experiment, we use the Grawitz's tumor cell.Untreated cell wound closure zone successfully shown in figure (2A), (2B), (2C), (2D).By contrast, the cell of handling with 0.01% oleuropein shown in figure (2E), (2F), (2G), (2H) does not have wound closure effectively.The mobility that shows the oleuropein anticancer once more has anticancer character.

A kind of variation of above-mentioned cell migration determination method is to utilize widely injured area to carry out the wound determination method, forms corner or curved edge (Fig. 3).In this experiment, malignant melanoma cell is grown to converge, utilize batten to scratch then, mode as mentioned above.Fig. 3 A is depicted as untreated cell, and all directions are divided a word with a hyphen at the end of a line in dull and stereotyped upper edge.Divide a word with a hyphen at the end of a line (Fig. 3 B) of the cell of crossing with 0.01% oleuropein solution-treated stopped fully.In this wound determination method, can clearly see edge of wound, be used to assess advancing of migratory cell.This shows the anticancer character of oleuropein once more.

It is that the melanoma cells plating is become circle (Fig. 4) that the another kind of above-mentioned experiment changes.Handle cell (0.01% oleuropein) and do not travelled to the outside (Fig. 4 A) of specifying circle.But, the untreated cell outward radial is divided a word with a hyphen at the end of a line, and covers flat board (Fig. 4 B).This class result points out the anticancer character of oleuropein strongly.

Embodiment 3

The cell growth of imbalance is the mark of cancer.This example is set forth the anticancer character of oleuropein in cell growth measurement method.The cancer cell equivalent of the maximum invasive carcinoma of representative is seeded on the flat board that contains the variable concentrations oleuropein.After 5 days, utilize MTS determination method assessment cell number, optical density (490nm) is worth shown in picture y axle (Fig. 5).The x axle is represented the concentration of oleuropein.True and the original statistics that these representatives are carried out on micro-plate reader.In whole cancers of studying, oleuropein suppresses growth effectively.

Embodiment 4

This example is set forth the influence of oleuropein pair cell skeleton by the tracheal rupture determination method.In this determination method, on matrigel type tubulose network, the latter was broken down into clump (Fig. 6 A, top line) in 24-48 hour with the cell plating.The feature that pipe is disintegrated process proves that it takes place because of shrinking back of pipe, and tubercle (pipe infall) more approaches to merge each other (Fig. 6 A, end row) thus.Each cell still prolongs and spreads all over everywhere.This cell slowly moves and contraction process (24-48 hour) involves initiatively cytoskeleton regeneration.Add oleuropein by occurring in mellow and full on the spot each cell of quick process (Fig. 6 B) in 2 hours to the pipe that is carried out, destroy the tubulose network.Mellow and full cell can not move, and rests on original position since die.Thereby, by destroying cytoskeleton and preventing that its regeneration from obstructing the cell mobility, the process of shrinking back is ended.This character of oleuropein is subjected to the inhibition of glucose, shows that oleuropein enters cell by glucose transporter, and glucose is competed this transhipment (Fig. 7).

Embodiment 5

This example is set forth oleuropein influence to wound healing in the cell migration determination method.In this example, oleuropein pair cell skeleton and the mellow and full influence of cell have been proved.This example is also set forth the invertibity of oleuropein pair cell influence, has pointed out small side effect.

Make rabbit skin fibroblastic growth to the monolayer that converges, scratch with aseptic batten subsequently.Cultivate cell then, allow to repair the wound in the culture.Untreated cell is successfully divided a word with a hyphen at the end of a line and is passed injured area (Fig. 8 A).The cell of handling with oleuropein (0.01%) shown in Fig. 8 C has effectively been suppressed.The feature of the cell that the process oleuropein is handled shows the discontinuity (Fig. 8 D and 8E) of monolayer, and this is that cell is mellow and full because be subjected to the influence of oleuropein pair cell skeleton.On the contrary, untreated cell seem smooth (Fig. 8 B).The oleuropein of flush away cell reverses mellow and full process.Cell flattens, and the discontinuity (H) of compensation in the individual layer begins from edge of wound divide a word with a hyphen at the end of a line (Fig. 8 F and 8G).

Other changes and improvements of the present invention also can be apparent for those of ordinary skills institute.Thereby the described herein and part set forth and the particular combinations of step plan only to represent some embodiment of the present invention, do not plan to serve as the replacement device that belongs to spirit and scope of the invention and the restriction of method.In this, expect that method of the present invention can further find to exceed disclosed herein and other the applicant's unsettled U.S. Patent application No.10/153,003 disclosed and claimed treatment is used.

Claims

1, the treatment curee involves the method for the medical conditions of cancer, this method comprises the pharmaceutical composition with chemoprophylaxis activity of the curee of this class of needs treatment being given to treat effective dose, and described composition contains the following formula: compound for the treatment of effective dose or its enantiomer as active component:

Wherein R1 and R2 be selected from hydroxyl ,-NH ₂Functional group with-SH group;

R3 is selected from hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, C1-C6-alkoxyl, halogen, NO ₂, NH ₃And COOCH ₃Functional group;

X1-X3 be selected from oxygen, sulphur ,-CH ₂-or the functional group of carboxyl;

R4 is selected from hydrogen, C1-C6-alkoxyl, glucose, B-D-glucopyranose, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, halogen, NO ₂, NH ₃, carbohydrate, amino acid, nucleotide and lipid functional group; With

R5 is selected from hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, C1-C6-alkoxyl, halogen, NO ₂, NH ₃And CH ₃Functional group.

2, the process of claim 1 wherein that this pharmaceutical composition comprises pharmaceutically acceptable carrier or thinner.

3, anticancer growth, move, invasion and attack and the method that shifts, comprise described cell contacted with the pharmaceutical composition that is enough to suppress cancer or its recurrence amount, described pharmaceutical composition contains following formula: compound or its enantiomer of effective dose:

4, the method for claim 3, wherein this pharmaceutical composition comprises pharmaceutically acceptable carrier or thinner.

5, the method for claim 3, wherein said cancer cell survival, growth, inhibition mobile, that attack and shift take place in vivo.

6, the method for claim 3, wherein said cancer cell survival, growth, inhibition mobile, that attack and shift occur in external.

7, the treatment curee involves the method for the medical conditions of cancer, described method comprises the pharmaceutical composition that curee to the treatment of this class of needs gives long, anti-mobile, anti-invasion of effective realization of therapeutic dose and anti-survival, the antibiosis of described related to cancer and antimetastatic activity, and this pharmaceutical composition contains at least a composition that is generated by following formula: compound or its enantiomer hydrolysis as active component:

8, the method for anticancer growth, comprise cancer cell is contacted with the pharmaceutical composition that is enough to suppress its amount of growth, described pharmaceutical composition contains the compound of effective dose, and described compound is selected from the group of being made up of at least a composition that is generated by following formula: compound or its enantiomer hydrolysis:

9, the process of claim 1 wherein that described composition comprises following formula: compound or its enantiomer:

10, the method for claim 3, wherein said composition comprise following formula: compound or its enantiomer:

11, treatment needs the animal method for cancer of this class treatment, comprises the compound with following formula structure or its enantiomer of described patient being given to treat effective dose:

R5 is selected from hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, aryl, hydroxyl, C1-C6-alkoxyl, halogen, NO ₂, NH ₃And CH ₃Functional group;

Perhaps its pharmaceutically acceptable salt, pro-drug or hydrate.

12, according to the treatment method for cancer of claim 11, wherein said cancer is selected from down group: adult's acute lymphoblastic leukemia; The children acute lymphoblastic leukemia; Adult's acute myelogenous leukemia; Adrenocortical carcinoma; Children's adrenocortical carcinoma; AIDS correlation lymphoma; AIDS correlation malignant tumour; Cancer of anus; Children's cerebellar astrocytoma; The big cerebral astrocytoma of children; Cholangiocarcinoma; Carcinoma of urinary bladder; Children's carcinoma of urinary bladder; Osteosarcoma/MFH type osteocarcinoma; Children's brain stem glioma; Adult's brain tumor; Children's brain stem glioma type brain tumor; Children's cerebellar astrocytoma type brain tumor; The big cerebral astrocytoma of children/glioblastoma type brain tumor; Children's ependymoma type brain tumor; Children's medulloblastoma type brain tumor; Former neuroectodermal tumors type brain tumor on children's curtain; Children look road and hypothalamus glioma type brain tumor; Children's brain tumor (other); Breast cancer; Breast cancer and gestation; Children's breast cancer; Male breast carcinoma; Children's bronchial adenoma/carcinoid; Children's carcinoid tumor; The stomach and intestine carcinoid tumor; Adrenocortical carcinoma; Islet-cell carcinoma; Unknown primary cancer; Primary central nervous system lymphoma; Children's cerebellar astrocytoma; Big cerebral astrocytoma/the glioblastoma of children; Cervical carcinoma; Children's cancer; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic spinal cord hyperplasia disease; The stndon sheath clear cell sarcoma; Colon cancer; Children's colorectal cancer; Skin T-cell lymphoma; Carcinoma of endometrium; Children's ependymoma; Epithelial ovarian cancer; Cancer of the esophagus; Children's cancer of the esophagus; Ewing's tumor family; The outer enblastoma of children's cranium; The outer enblastoma of gonad; Cholangiocarcinoma; Intraocular melanoma type cancer eye; Retinoblastoma type cancer eye; Carcinoma of gallbladder; Cancer of the stomach; Children's cancer of the stomach; The stomach and intestine carcinoid tumor; The outer enblastoma of children's cranium; The outer enblastoma of gonad; Germinal tumor of ovary; The gestation trophoblastic tumor; Children's brain stem glioma; Children look road and hypothalamus glioma; Hairy cell leukemia; Head and neck cancer; Adult (primary) liver cell (liver) cancer; Children's (primary) liver cells (liver) cancer; Adult He Jiejin lymphomas; Children He Jiejin lymphomas; Pregnancy duration He Jiejin lymphomas; The hypopharynx cancer; Children's hypothalamus with look the road glioma; Intraocular melanoma; Islet-cell carcinoma (endocrine pancreas); Kaposi; Kidney; Laryngocarcinoma; Children's laryngocarcinoma; Adult's acute lymphoblastic leukemia; The children acute lymphoblastic leukemia; Adult's acute myelogenous leukemia; The children acute myelomatosis; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Hairy cell leukemia; Lip and carcinoma of mouth; Adult's (primary) liver cancer; Children's (primary) liver cancer; Lung cancer in non-cellule type; Small cell lung cancer; Adult's acute lymphoblastic leukemia; The children acute lymphoblastic leukemia; Chronic lymphocytic leukemia; AIDS correlation lymphoma; Central nervous system (primary) lymphoma; Skin T-cell lymphoma; Adult He Jiejin lymphomas; Children He Jiejin lymphomas; Pregnancy duration He Jiejin lymphomas; Adult's non_hodgkin lymphoma; Children's non_hodgkin lymphoma; Pregnancy duration Fei Hejiejinshi disease; Primary central nervous system lymphoma; Walden Si Telunshi macroglobulinemia; Male breast carcinoma; Become the HMM; Children's malignant mesothelioma; Children's medulloblastoma; Melanoma; Intraocular melanoma; Mei Keer Schwann Cells cancer; Malignant mesothelioma; Recessive primary metastatic squamous neck cancer; The multiple internal secretion neoplasia of children syndrome; Huppert's disease/plasmacytoma forms; Mycosis fungoides; Myelodysplastic syndrome; Chronic myelogenous leukemia; The children acute myelomatosis; Huppert's disease; Chronic spinal cord hyperplasia disease; Nasal cavity and nasal sinus cancer; Nasopharyngeal carcinoma; Children nasopharyngeal carcinoma; Neuroblastoma; Adult's non_hodgkin lymphoma; Children's non_hodgkin lymphoma; The pregnancy duration non_hodgkin lymphoma; Lung cancer in non-cellule type; Children's mouth cancer; Oral cavity and lip cancer; The oropharynx cancer; Osteosarcoma/pernicious bone fibres histocytoma; Children's oophoroma; Epithelial ovarian cancer; Germinal tumor of ovary; Ovary hangs down pernicious potential tumor; Cancer of pancreas; Children's cancer of pancreas; The islet cells cancer of pancreas; Paranasal sinus and CARCINOMA OF THE NASAL CAVITY; Parathyroid carcinoma; Carcinoma of penis; Pheochromocytoma; Children's pineal gland is gone up former PNET with curtain; Hypophysoma; Plasmacytoma formation/Huppert's disease; The pleura pulmonary blastoma; Gestation and breast cancer; Gestation and He Jiejin lymphomas; Gestation and non_hodgkin lymphoma; Primary central nervous system lymphoma; Become the human primary liver cancer; Children's primary carcinoma of liver; Prostate cancer; The carcinoma of the rectum; Nephrocyte (kidney) cancer; Children's clear-cell carcinoma; Renal plevis and ureter transitional-cell carinoma; Retinoblastoma; Children's rhabdomyosarcoma; Salivary-gland carcinoma; Children's salivary gland cancer; Ewing's tumor family sarcoma; Kaposi; The sarcoma of bone (osteosarcoma)/MFH; Children's rhabdomyosarcoma; Adult soft tissue sarcoma; Children soft tissue sarcoma; Sezary syndrome; Cutaneum carcinoma; Children's cutaneum carcinoma; Cutaneum carcinoma (melanoma); Mei Keer Schwann Cells property cutaneum carcinoma; Small cell lung cancer; Carcinoma of small intestine; Adult soft tissue sarcoma; Children soft tissue sarcoma; Recessive primary metastatic squamous neck cancer; Cancer of the stomach; Children's cancer of the stomach; Former PNET on children's curtain; Skin T-cell lymphoma; Carcinoma of testis; Children's thymoma; Thymoma and thymic carcinoma; Thyroid cancer; Pediatric thyroid carcinomas; Renal plevis and ureter transitional-cell carinoma; The gestation trophoblastic tumor; The unknown primary of being grown up position cancer; The unknown primary of children position cancer; The unusual cancer of children; Ureter and renal plevis transitional-cell carinoma; Carcinoma of urethra; Carcinoma of endometrium; Sarcoma of uterus; Carcinoma of vagina; Children look road and hypothalamus glioma; Carcinoma of vulva; Walden Si Telunshi macroglobulinemia; Wilms' tumor.

Thereby 13, give the zooblast resistance and give the method for cell with antiviral, bacterium and parsitism, comprise described cell is contacted with the pharmaceutical composition that is enough to give its resistance amount, described pharmaceutical composition contains following formula: compound or its enantiomer of effective dose:

14, according to the method for the treatment disease of claim 13, wherein said cell is infected by HIV, malaria, helicobacter pylori and vagina yeast.

The method of the medical conditions that 15, treatment and blister, ulcer formation, incrustation are relevant with cicatrization, this method comprises the pharmaceutical composition of the curee of this class of needs treatment being given to treat effective dose, and described composition contains following formula: compound or its enantiomer as its active component:

16, according to the method for the treatment disease of claim 15, wherein said disease is selected from down group: burn, scratch, incised wound, wound, fibroma, tumour, keloid, acne, gastritis, vagina, official's neck, the uterus, ovary, stomach, cornea, retina, diabetes, the cicatrization of AIDS correlation, ileum (iliac) and colonic ulcer, interstitial lung disease, people's fibre modification lung disease, people's kidney disease, glomerulonephritis, the ephritis relevant with systemic lupus, peritoneal fibrosis, cystic fibrosis, hepatic fibrosis, myofibrosis cordis, pnemnofibrosis, GraveShi illness in eye, drug-induced ergotism, angiocardiopathy, cancer, Alzheimer's, cicatrization, chorionitis, spongioblastoma in the Li-Fraumeni syndrome, sporadic spongioblastoma, myelomatosis, acute myelogenous leukemia, myelodysplastic syndrome, spinal cord hyperplasia syndrome, the gynaecology cancer, Kaposi, hansen's disease, the inflammatory bowel disease that does not comprise collagenous colitis, the kidney fibre modification, the belly adhesion, radiation induced fibre modification, bronchiolitis obliterans, the silicosis damage, ocular capsule fibroplasia, the blood vessel birthmark, tatoo and traumatic synulotic laser disposal, vagina yeast infection and helicobacter pylori ulcer.

17, the process of claim 1 wherein that described composition is formulated into tablet for oral administration or elixir.

18, the process of claim 1 wherein described composition via a kind of administration, described approach is selected from the group of being made up of intramuscular or intravenous administration.

19, the process of claim 1 wherein that described composition is via inhalation.

20, the method for claim 11, wherein said compound be via a kind of administration, described approach be selected from by in mouth, cheek, rectum, parenteral, the peritonaeum, in the corium, the group formed of transdermal and intracheal.

21, the method for claim 11, wherein said composition is formulated into tablet for oral administration or elixir.

22, the method for claim 11, wherein said composition are via a kind of administration, and described approach is selected from the group of being made up of intramuscular or intravenous administration.

23, the method for claim 11, wherein said composition is via inhalation.

24, according to the treatment method for cancer of claim 11, wherein said composition comprises following formula: compound or its enantiomer:

25, the method for claim 7, wherein said at least a composition are selected from by Oleuropeine aglycone, elenolic acid, β-3, the group that 4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate forms.

26, the method for claim 8, wherein said at least a composition are selected from by Oleuropeine aglycone, elenolic acid, β-3, the group that 4-dihydroxyphenyl ethanol and methyl-o-methyl elenolate forms.

27, the method for claim 3, wherein said zooblast survival, growth, inhibition mobile, that attack and shift take place in vivo.

28, the method for claim 3, wherein said zooblast survival, growth, inhibition mobile, that attack and shift occur in external.

29, by the R4 part selectivity target of medical compounds and the method for sending the effective dose compound, described amount is enough to suppress the cancerous growths or the recurrence of described cell, and described compound has following formula or its enantiomer:

30, the method for claim 27, wherein this R4 partly is the B-D-glucopyranose, described composition comprises following formula: compound or its enantiomer:

31, the method for claim 27, wherein said cell is a zooblast.

32, destroy and prevent cytoskeleton regeneration, zooblast presents the method that sphere can not be broken up, moves or be attacked thus, this method comprises makes this cell contact with following formula: compound or its enantiomer of effective dose:

33, the method for claim 30, wherein said composition comprise following formula: compound or its enantiomer:

34, the method for claim 32, wherein said cell is arranged in live animal.

35, the method for claim 32, wherein said cell is arranged in the cancer patient.

36, the method for claim 32, wherein said cell is arranged in AIDS patient.