CN101568332A

CN101568332A - Methods of inhibiting angiogenesis and treating angiogenesis-associated diseases

Info

Publication number: CN101568332A
Application number: CNA2006800561122A
Authority: CN
Inventors: N·P·德塞; V·特里优
Original assignee: Abraxis Bioscience Inc USA
Current assignee: Abraxis Bioscience LLC
Priority date: 2006-08-31
Filing date: 2006-08-31
Publication date: 2009-10-28
Also published as: CA2662140A1; BRPI0622006A2; AU2006347740A1; EP2056812A1; WO2008027055A1; MX2009002054A; IL197276A0; JP2010502603A

Abstract

The invention provides methods of inhibiting angiogenesis in an individual by administering a composition (such as protein containing composition) comprising colchicine or thiocolchicine dimer. The composition is in an amount that is effective in inhibiting angiogenesis but is in some embodiments insufficient to induce significant cytotoxicity in the individual. The methods described herein are useful for treating angiogenesis-associated diseases, such as age-related macula degeneration, diabetic retinopathy, rheumatic arthritis, psoriasis, and cancer.

Description

The method that suppresses angiogenesis and treatment angiogenesis-associated diseases

Technical field

[0001] the application relates to the method that suppresses angiogenesis and treatment angiogenesis-associated diseases.Particularly, the application relates to by giving the method that colchicine or the dimeric compositions of muscoril (thiocolchicine) suppress angiogenesis and treat angiogenesis-associated diseases that contains of effective dose.

Background technology

[0002] angiogenesis is the bioprocess of highly regulating and control, and new blood vessel forms by this process.Not controlled angiogenesis causes multiple disease.A kind of such disease is age-related macular degeneration (age-related macular degeneration) (" AMD "), it is characterized in that the neovascularity invasion enters in the different structure such as macula lutea and retinal pigment epithelium of eye.The another kind of disease of angiogenesis influence is a rheumatic arthritis, wherein the experience of the blood vessel in the synovial membrane lining in joint (synovial lining) angiogenesis.Except forming new blood vessel network, endotheliocyte discharges the factor and the reactive oxygen species that causes pannus (pannus) growth and cartilage destruction (cartilage destruction).Not controlled angiogenesis also with disease association such as diabetic retinopathy, psoriasis, cardiovascular restenosis (restenosis) and neovascular glaucoma.

[0003] in addition, angiogenesis also participates in tumor formation and shifts.For example, show that diameter increases to the blood supply that must obtain them greater than about 2 millimeters tumor, and by inducing new capillary vessel growth to finish this.After these new vesselses embedded in the tumor, they provided necessary nutrition of tumor growth and somatomedin, and promoted the transfer of tumor cell.

[0004] particular target has been developed to the anti-angiogenic agent of angiogenesis and has been used for the treatment of angiogenesis-associated diseases.Referring to, for example, U.S. Patent number 6,919,309; U.S. Patent Application Publication No. 2006/0009412; With PCT application publication number WO04/027027 and WO05/117876.In addition, targeting has formed the medicament (so-called " blood-vessels target agent " or VTA) also be developed of blood vessel.These medicaments it is believed that such performance function: optionally make the microtubule cytoskeleton instability of endotheliocyte, cause the great change of cell shape, this finally causes blood vessel blockage and blood flow to stop.Referring to, for example, WO 2005/113532.

[0005] the muscoril dimer is a hydrophobic compound, and it is former to be described.Referring to, for example, U.S. Patent number 6,627,774.These chemical compounds have dual function mechanism, that is, described chemical compound has the active and topoisomerase I inhibition activity of anti-microtubule.Raspaglio etc., Biochem.Pharmacol.2005,69 (1): 113-21.The muscoril dimer preparation Nab-5404 of nano-particle albumin bound and Nab-5676 have been developed as the cytotoxicity chemotherapeutant and have been used for the treatment of cancer.Referring to, for example, Bernacki etc., Proc.Amer.Assoc.CancerRes., vol.46,2005#2390 and PCT number of patent application PCT/US2006/006167.Have been found that: when with 24mg/kg, when the qdx5 vein was given approximately, Nab-5404 can induce the tumor in the A121 ovarian tumor allotransplant to degenerate fully and cure.

[0006] disclosure of all publications mentioned in this article, patent, patent application and published is incorporated herein by reference with its integral body at this.

Summary of the invention

[0007] the present invention is provided at the method that suppresses angiogenesis in the individuality on the one hand, comprise and give the compositions that this individuality contains colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of effective inhibition angiogenesis, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in this individuality.In some embodiments, a kind of method that suppresses angiogenesis in individuality is provided, comprise the compositions that gives this individual effective dose, described compositions comprises colchicine or muscoril dimer (as IDN5404), wherein said colchicine or muscoril dimer (as IDN5404) as described in amount (being the amount of each administration) in the compositions about 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15mg/m ²Below body surface arbitrary.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the compositions that gives this individual effective dose, described compositions contains colchicine or muscoril dimer (as IDN5404), wherein colchicine or muscoril dimer (as IDN5404) as described in amount in the compositions about 0.05,0.08,0.1,0.2,0.3,0.4,0.5 or below 0.6mg/kg arbitrary.In some embodiments, the following administration of described compositions: at least about per three weeks once, every biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day any.In some embodiments, described compositions by administration (interrupt or do not interrupt) at least about 1,2,3,4,5,6,7,8,9,10,11,12 or any of more a plurality of middle of the month.In some embodiments, described compositions is by any administration in intravenous, ophthalmic, intra-arterial, oral, part or the inhalation route.

[0008] compositions that is used in the methods described herein can further comprise biocompatible polymer, for example carrier protein.For example, in some embodiments, provide and suppress the method that individual medium vessels generates, comprise and give the compositions that described individuality comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), the amount of wherein said compositions effectively suppresses angiogenesis, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in this individuality.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the compositions that comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404) that gives this individual effective dose, the amount of colchicine or muscoril dimer (as IDN5404) is about 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15mg/m in the wherein said compositions ²Below body surface arbitrary.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the compositions that comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404) that gives this individual effective dose, the amount of colchicine or muscoril dimer (as IDN5404) is about 0.05,0.08,0.1,0.2,0.3,0.4,0.5 or below 0.6mg/kg arbitrary in the wherein said compositions.In some embodiments, the described compositions that contains carrier protein is substantially free of (as not containing) surfactant.In some embodiments, the following administration of described compositions: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day in any in some embodiments, described compositions by administration (interrupt or do not interrupt) at least about 1,2,3,4,5,6,7,8,9,10,11,12 or more a plurality of middle of the month any.In some embodiments, described compositions is by any administration in intravenous, ophthalmic, intra-arterial, oral, part or the inhalation route.

[0009] compositions that is used in the methods described herein can comprise granule (as microgranule or nano-particle), and it comprises carrier protein (as albumin) and colchicine or muscoril dimer.For example, in some embodiments, provide and suppress the method that individual medium vessels generates, comprise and give the compositions that this individuality comprises nano-particle, described nano-particle contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of effective inhibition angiogenesis, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in individuality.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the compositions that comprises nano-particle that gives this individual effective dose, described nano-particle contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), and the amount of colchicine or muscoril dimer (as IDN5404) is about 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15mg/m in the wherein said compositions ²Below body surface arbitrary.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the compositions that comprises nano-particle that gives this individual effective dose, described nano-particle contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), and the amount of colchicine or muscoril dimer (as IDN5404) is about 0.05,0.08,0.1,0.2,0.3,0.4,0.5 or below 0.6mg/kg arbitrary in the wherein said compositions.In some embodiments, the following administration of described compositions: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day in any.In some embodiments, described compositions by administration (interrupt or do not interrupt) at least about 1,2,3,4,5,6,7,8,9,10,11,12 or more a plurality of middle of the month any.In some embodiments, described compositions is by any administration in intravenous, ophthalmic, intra-arterial, oral, part or the inhalation route.

[0010] in some embodiments, the granule in the described compositions has the average diameter that is not more than about 200nm.In some embodiments, contain grains of composition and do not contain (as not containing) surfactant substantially.In some embodiments, the albumen in the described compositions (as albumin) is about 18: 1 or littler with colchicine or the dimeric weight ratio of muscoril, 9: 1 or littler according to appointment.In some embodiments, colchicine or muscoril dimer wrap quilt with carrier protein (as albumin).In some embodiments, the granule in the described compositions has the average diameter that is not more than about 200nm, and described compositions does not contain (as not containing) surfactant substantially.In some embodiments, the granule in the described compositions (especially, nano-particle) has the average diameter that is not more than about 200nm, and colchicine or muscoril dimer albumen (as albumin) bag quilt.Other combination of above-mentioned feature is also taken into account.In some embodiments, described particulate composition is Nab-5404.Comprise the dimeric particulate composition of other colchicine or muscoril and also can comprise one or more above-mentioned features.

[0011] colchicine as herein described or muscoril dimer comprise two (identical or different) subunits of colchicine, muscoril or derivatives thereof.In some embodiments, described colchicine or muscoril dimer comprise at least one muscoril subunit.In some embodiments, described colchicine or muscoril dimer comprise two muscoril subunits.In some embodiments, described colchicine or muscoril dimer are the chemical compounds of formula (I):

[0012] wherein the B in each subunit is methoxyl group or methyl mercapto, R ₂When with R ₃When choosing together methoxyl group, hydroxyl or methylene-dioxy (methylenedioxy); R ₃When with R ₂When choosing together methoxyl group, hydroxyl or methylene-dioxy.In some embodiments, X contains at least one carbon atom.

[0013] in some embodiments, described colchicine or colchicine dimer are the chemical compounds of formula (II):

[0014] B wherein ₁Be methoxyl group or methyl mercapto, B ₂Be methoxyl group or methyl mercapto, n is from 0 to 8 integer, and Y is CH ₂Group, perhaps when n was 1, Y also can be the group of formula NH.In some embodiments, n is any (for example being selected from them) in 0,1,2,3,4,5,6,7 or 8.In some embodiments, n is 1.In some embodiments, n be 1 and Y be NH.In some embodiments, n is 2.

[0015] in some embodiments, B ₁And B ₂It all is methoxyl group.In some embodiments, B ₁And B ₂It all is methyl mercapto.In some embodiments, B ₁Be methoxyl group and B ₂It is methyl mercapto.In some embodiments, B ₁Be methyl mercapto and B ₂It is methoxyl group.In some embodiments, described colchicine or muscoril dimer are following any (and being selected from some embodiments): IDN5404, IDN5676, IDN5800 and IDN5801.In some embodiments, described colchicine or muscoril dimer are IDN5404.In some embodiments, described colchicine or muscoril dimer are IDN5676.

[0016] in some embodiments, provide and suppress the method that individual medium vessels generates, comprise giving the compositions that this individuality contains nano-particle that described nano-particle comprises albumin and IDN5404, IDN5676, IDN5800 and IDN5801 any (following called after " Nab-5404 ", " Nab-5676 ", " Nab-5800 " and " Nab-5801 " respectively).In some embodiments, provide and suppress the method that individual medium vessels generates, comprise and give this individuality Nab-5404 (or among Nab-5676, Nab-5800 and the Nab-5801 any), wherein said Nab-5404 (or among Nab-5676, Nab-5800 and the Nab-5801 any) is in the amount of effective inhibition angiogenesis, and wherein the quantity not sufficient of Nab-5404 (or among Nab-5676, Nab-5800 and the Nab-5801 any) to induce significant cytotoxicity in the individuality.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the Nab-5404 that gives this individual effective dose (or among Nab-5676, Nab-5800 and the Nab-5801 any), wherein the amount of Nab-5404 (or among Nab-5676, Nab-5800 and the Nab-5801 any) is about 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15mg/m ²In the body surface arbitrary below.In some embodiments, provide and suppress the method that individual medium vessels generates, comprise the Nab-5404 that gives this individual effective dose (or among Nab-5676, Nab-5800 and the Nab-5801 any), wherein the amount of Nab-5404 (or among Nab-5676, Nab-5800 and the Nab-5801 any) is about 0.05,0.08,0.1,0.2,0.3,0.4,0.5 or below 0.6mg/kg arbitrary.In some embodiments, the following administration of described compositions: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day in any.In some embodiments, described compositions by administration (interrupt or do not interrupt) at least about 1,2,3,4,5,6,7,8,9,10,11,12 or more a plurality of middle of the month any.In some embodiments, described compositions is by any administration in intravenous, ophthalmic, intra-arterial, oral, part or the inhalation route.

[0017] method as herein described is generally used for the treatment of angiogenesis-associated diseases.In some embodiments, described angiogenesis-associated diseases is non-tumprigenicity angiogenesis-associated diseases, for example comprise oculopathy (as degeneration of macula, diabetic retinopathy or neovascular glaucoma), cardiovascular disease (as restenosis and atherosclerosis), dermatosis (as psoriasis) and arthritis (as rheumatic arthritis).

[0018] for example, in some embodiments, the method of non-tumprigenicity angiogenesis-associated diseases in the treatment individuality is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of muscoril, wherein said compositions is in the amount of the non-tumprigenicity angiogenesis-associated diseases of effective treatment.In some embodiments, the quantity not sufficient of described compositions is to induce significant cytotoxicity in this individuality.In some embodiments, the method of non-tumprigenicity angiogenesis-associated diseases in the treatment individuality is provided, comprise giving the compositions that this individuality contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of non-tumprigenicity angiogenesis-associated diseases described in effective treatment individuality.In some embodiments, the quantity not sufficient of described compositions is to induce significant cytotoxicity in this individuality.In some embodiments, the method of non-tumprigenicity angiogenesis-associated diseases in the treatment individuality is provided, comprise and give the compositions that this individuality contains granule (as nano-particle), described granule comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), and wherein said compositions is in the amount of the non-tumprigenicity angiogenesis-associated diseases of effective treatment.In some embodiments, the quantity not sufficient of described compositions is to induce significant cytotoxicity in this individuality.In some embodiments, the method of non-tumprigenicity angiogenesis-associated diseases in the treatment individuality is provided, comprise giving this individuality Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801), wherein Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801) is in the amount of the non-tumprigenicity angiogenesis-associated diseases of effective treatment.In some embodiments, the quantity not sufficient of described Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801) is to induce significant cytotoxicity in this individuality.

[0019] in some embodiments, described angiogenesis-associated diseases is the cancer-related disease, comprises for example cancer and benign tumor.Can include, but are not limited to by the cancer of methods described herein treatment: breast carcinoma, colorectal carcinoma (colorectal cancer), rectal cancer, nonsmall-cell lung cancer (non-small cell lung cancer), non Hodgkin lymphoma (non-Hodgkinslymphoma) (NHL), the kidney cell cancer, carcinoma of prostate, hepatocarcinoma, cancer of pancreas, soft tissue sarcoma, kaposi sarcoma (Kaposi ' s sarcoma), benign tumor (carcinoid carcinoma), head and neck cancer (head and neck cancer), melanoma, ovarian cancer, mesothelioma, glioma, glioblastoma multiforme, neuroblastoma and multiple myeloma.In some embodiments, described cancer is an entity tumor (solid tumor) (as the transitivity entity tumor).By way of example, the treatment method for cancer is further described below.

[0020] for example, in some embodiments, method for cancer in the treatment individuality is provided, comprise that giving this individuality comprises colchicine or the dimeric compositions of muscoril, wherein said compositions is in the amount of effective treatment cancer, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in this individuality.In some embodiments, method for cancer in the treatment individuality is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of muscoril, wherein said compositions is in the amount of effective treatment cancer, and in the wherein said compositions colchicine or the dimeric amount of muscoril colchicine or the corresponding maximum tolerated dose of colchicine dimer (" MTD ") about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, 14% or 15% in arbitrary below.In some embodiments, method for cancer in the treatment individuality is provided, comprise and give the compositions that this individuality contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of effective treatment cancer, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in this individuality.In some embodiments, method for cancer in the treatment individuality is provided, comprise and give the compositions that this individuality contains carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of effective treatment cancer, and in the wherein said compositions colchicine or the dimeric amount of muscoril colchicine or muscoril dimer corresponding M TD about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, 14% or 15% in arbitrary below.In some embodiments, the way of cancer in the treatment individuality is provided, comprise and give the compositions that this individuality contains granule (as nano-particle), described granule comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), wherein said compositions is in the amount of effective treatment cancer, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in individuality.In some embodiments, method for cancer in the treatment individuality is provided, comprise and give the compositions that this individuality contains granule (as nano-particle), described granule comprises carrier protein (as albumin) and colchicine or muscoril dimer (as IDN5404), and in the described compositions amount of colchicine or muscoril dimer (as IDN5404) at about 1% of colchicine or muscoril dimer corresponding M TD, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, in 14% or 15% arbitrary below.In some embodiments, method for cancer in the treatment individuality is provided, comprise and give this individuality Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801), wherein Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801) is in the amount of effective treatment cancer, and wherein the quantity not sufficient of Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801) in this individuality, to induce significant cytotoxicity.In some embodiments, method for cancer in the treatment individuality is provided, comprise and give this individuality Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801), Nab-5404 (or Nab-5676 wherein, Nab-5800 or Nab-5801) be in the amount of effective treatment cancer, and Nab-5404 (or Nab-5676 wherein, Nab-5800 or Nab-5801) amount at Nab-5404 (or Nab-5676, Nab-5800 or Nab-5801) corresponding M TD about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, in 14% or 15% arbitrary below.

[0021] also is provided for pharmaceutical composition, unit dose, test kit and the goods of methods described herein.

Should be appreciated that [0022] one of various embodiments as herein described, some or all of character can be combined, to form other embodiment of the present invention.

The accompanying drawing summary

[0023] Figure 1A shows use MX-1 breast cancer cell, the effect that Nab-5404 forms microtubule.Figure 1B shows use MX-1 breast cancer cell, the effect that Nab-5676 forms microtubule.

[0024] Fig. 2 A-2C show with control vector (PBS, 2A), after 0.6 μ g/ml Nab-5404 (2B) and μ g/ml Nab-5676 (2C) handle, the microphotograph of the microtubule network that in the MX-1 cell, dyes.

[0025] Fig. 3 demonstration formation is assessed according to blood capillary/tubule (microvessel/tubule) Nab-5404, Nab-5676 and the anti-angiogenesis activity of CA4P.Cell dyeed with different chemical compound incubations and at the 12nd day at the 1st day.Fig. 3 A-3D provides with control vector (3C), 0.01 μ g/ml Nab-5404 (3A), 0.01 μ g/ml Nab-5676 (3B) and 0.01 μ g/ml CA4P (3D) and handles the microphotograph that the back tubule forms.

[0026] Fig. 4 shows with concentration range to be the comparison of the little length of tube of Nab-5404, the Nab-5676 of 0.0 to 100 μ g/ml and the cell that CA4-P handles.

[0027] Fig. 5 shows according to blood capillary/tubule and forms or destroy Nab-5404 that (disruption) assess and the anti-angiogenesis activity of Nab-5676.Cell dyeed with different chemical compound incubations and at the 11st day at the 8th day.Fig. 5 A-5C provides the microphotograph of handling tubule formation afterwards with control vector (5A), 0.01 μ g/mlNab-5404 (5B) and 0.01 μ g/ml Nab-5676 (5C).Fig. 5 D shows with concentration range to be the comparison of the little length of tube of the Nab-5404 of 0.0 to 10 μ g/ml and the cell that Nab-5676 handles.

[0028] Fig. 6 shows the Nab-5404 anti-angiogenesis activity according to blood capillary/tubule forms or destruction is assessed.Cell dyeed with the chemical compound incubation of various concentration and at the 12nd day at the 11st day.Fig. 6 A provides with control vector (6A), 0.001 μ g/ml Nab-5404 (6B), 0.01 μ g/ml Nab-5404 (6C), 0.1 μ g/ml Nab-5404 (6D), 1 μ g/ml Nab-5404 (6E) and 10.0 μ g/ml Nab-5404 (6F) to 6F and handles back tubule formation and destructive microphotograph.

[0029] Fig. 7 shows the Nab-5676 anti-angiogenesis activity according to blood capillary/tubule forms or destruction is assessed.Cell dyeed with the chemical compound incubation of various concentration and at the 12nd day at the 11st day.Fig. 7 A shows that to 7F handling the back tubule with control vector (7A), 0.001 μ g/ml Nab-5676 (7B), 0.01 μ g/ml Nab-5676 (7C), 0.1 μ g/ml Nab-5676 (7D), 1 μ g/ml Nab-5676 (7E) and 10.0 μ g/ml Nab-5676 (7F) forms and destructive microphotograph.

[0030] Fig. 8 shows the anti-angiogenesis activity that forms or destroy the CA4P that assesses according to blood capillary/tubule.Cell dyeed with the chemical compound incubation of various concentration and at the 12nd day at the 11st day.Fig. 8 A shows respectively with tubule formation and destructive microphotograph after the CA4P processing of 0.01,0.1 and 1.0 μ g/ml to 8C.

[0031] Fig. 9 shows the comparison that forms or destroy the anti-angiogenesis activity of the Nab-5404, the Nab-5676 that assess and CA4P according to blood capillary or tubule.Cell dyeed with the chemical compound incubation and at the 12nd day at the 11st day.This figure shows the little length of tube with the cell of the every kind compound treatment of concentration range in 0.0 to 10 μ g/ml.

[0032] Figure 10 demonstration utilizes 2 cycles low dosage/high dose scheme, and Nab-5404 is to the effect of HT-29 tumor growth in the xenotransplantation Muridae model.Period 1 dosage is 1.7,2.5 or 3.4mg/kg, 0-14 days; Second round, dosage was 20,30 or 40mg/kg, 15-30 days.Figure 10 A shows the mean tumour volume (n=10) from the 0th day to the 40th day.Figure 10 B shows the average percentage that mice loses weight in the 0th day to the 40th day.

[0033] Figure 11 demonstration utilizes 2 cycles low dosage/high dose scheme, and Nab-5676 is to the effect of HT-29 tumor growth in the xenotransplantation Muridae model.Period 1 dosage is 1.7,2.5 or 3.4mg/kg, 0-14 days; Second round, dosage was 20,30 or 40mg/kg, 15-30 days.Figure 11 A shows the mean tumour volume (n=10) from the 0th day to the 40th day.Figure 11 B shows the 0th day to the 40th day average percentage that mice loses weight.

[0034] Figure 12 shows the effect of CA4P to HT-29 tumor growth in the xenotransplantation Muridae model.Reach 900mm in the HT-29 tumor ³Handle them with 100mg/kg behind the volume.Figure 12 A shows the mean tumour volume (n=10) from the 28th day to the 41st day; Square=vehicle Control, rhombus=CA4P handles.Figure 12 B shows the average percentage that mice loses weight in the 28th day to the 41st day; Square=vehicle Control, rhombus=CA4P handles.

Detailed Description Of The Invention

[0035] the present invention part is based on our observation: contain the dimeric composition of muscoril, especially, muscoril is dimeric to contain albuminous nanoparticle formulations, more particularly, IDN-5404 contain albuminous nanoparticle formulations (" Nab-5404 ") and IDN-5676 contain albuminous nanoparticle formulations (" Nab-5676 ") suppress that capilary forms and the established capilary of external destruction aspect effective. Trieu etc., 97^thAACR Annual Meeting, Abstract No. 3823. These active IC50 values significantly are lower than the outer desired value of cellular cytoxicity activity of described composition. We further observe: contain the dimeric composition of muscoril, especially, the dimeric albuminous nano particle (such as Nab-5404 and Nab-5676) that contains of muscoril effectively stops tumor growth in vivo with the dosage of the corresponding maximum tolerated dose (MTD) that significantly is lower than described composition. These observations show: the composition that contains muscoril dimer or its analog (such as the colchicin dimer) has anti-angiogenic generation and blood-vessels target activity, and described activity and their cytotoxic effect are irrelevant. When with the amount administration of effective and no cytotoxicity, described composition is growth and the blocking blood flow of the new blood vessel of target optionally, and does not cause significantly cell death in the target tissue.

[0036] colchicin or muscoril dimer (namely being not enough to induce obvious Cytotoxic amount) effective and the no cytotoxicity amount are that treatment Non-cancerous angiogenesis-associated diseases is expected in order to minimize the cytotoxic effect of described compound. Amount effective and no cytotoxicity also is favourable to the treatment cancer. The traditional chemical therapy of utilizing the cytotoxicity medicament is usually to equal or to carry out near the dosage of medicament maximum tolerated dose, to maximize the cytotoxic effect of this medicament. But, during the nothing treatment that this high dose scheme need to prolong, to allow the normal host cellular-restoring. Simultaneously, tumour cell also may recover growth during without treatment. This can increase the risk of development antiradiation drug tumour cell. The colchicin of no cytotoxicity dosage (being quantity) or muscoril dimer allow in the treatment for the treatment of cycle without obvious interruption, have therefore reduced the risk of development drug resistance. In addition, the dosage of the no cytotoxicity possibility that will develop drug-induced obvious general toxicity (as losing weight) and side effect reduces to minimum.

[0037] therefore, one aspect of the present invention provides and suppresses the method (comprising the vascularization that target has occured) that individual medium vessels generates, comprise that giving this individuality contains colchicin or the dimeric composition of muscoril, wherein said composition is in the amount of establishment Angiogenesis, and the quantity not sufficient of wherein said composition is to induce obvious cytotoxicity in this individuality. In some embodiments, described method comprises that giving this individuality contains carrier protein (such as albumin) and colchicin or the dimeric composition of muscoril, wherein said composition is in the amount of establishment Angiogenesis, and the quantity not sufficient of wherein said composition is to induce obvious cytotoxicity in this individuality.

[0038] on the other hand, the invention provides the method for Non-cancerous angiogenesis-associated diseases in the treatment individuality, comprise that giving described individuality contains colchicin or the dimeric composition of muscoril, wherein said composition is in the amount of effective treatment Non-cancerous angiogenesis-associated diseases. In some embodiments, described method comprises that giving this individuality contains the dimeric composition of carrier protein and colchicin or muscoril, and wherein said composition is in the method for effective treatment Non-cancerous angiogenesis-associated diseases.

[0039] on the other hand, the invention provides the method for Tumor-assaciated disease in the treatment individuality, comprise that giving this individuality contains colchicin or the dimeric composition of muscoril, wherein said composition is in the amount of effective treatment Tumor-assaciated disease, and the quantity not sufficient of wherein said composition is to induce obvious cytotoxicity in this individuality. In some embodiments, described method comprises that giving this individuality contains the dimeric composition of carrier protein and colchicin or muscoril, wherein said composition is in the amount of effective treatment cancer, and the quantity not sufficient of wherein said composition is to induce obvious cytotoxicity in this individuality.

[0040] also is provided for pharmaceutical composition, UD, kit and the goods of methods described herein.

[0041] " the described composition " generally mentioned or " composition " comprise and applicable composition of the present invention. The present invention also provides the pharmaceutical composition that contains composition described herein.

[0042] term " individuality " is mammal, comprises the mankind. Individuality includes but not limited to people, ox, pig, cat, dog, mouse or primate. In some embodiments, described individuality is the people. In some embodiments, described individuality is for being used for the experimental animal model of research Angiogenesis or angiogenesis-associated diseases (one or more).

[0043] disclosure comprises all stereoisomers of compound mentioned in this article, comprises enantiomter and diastereoisomer. Unless spatial chemistry structurally has clear and definite expression, disclosed structure is intended to comprise all possible spatial chemistry modification. The disclosure comprises all enantiomters of disclosed any chipal compounds, they or substantially pure left-handed or dextrorotatory form, or in the optically-active mixture, or the enantiomter of arbitrary ratio. The disclosure comprises any diastereoisomer of the compound of mentioning in the following formula, and they are in the form of the mixture of substantially pure diastereo-isomerism form and all ratios. The disclosure also comprises all solvates of compound mentioned in this article, comprises all hydrates of compound mentioned in this article. The disclosure also comprises all polymorphs (polymorph), comprises crystal and the amorphous form of compound mentioned in this article. The disclosure also comprises all salt, particularly pharmaceutically acceptable salt of compound mentioned in this article. Metabolite and the pro-drug of compound disclosed herein are also comprised by the disclosure. In all purposes of compound disclosed herein, the disclosure also comprises the purposes of any or whole spatial chemistry, enantiomter, diastereoisomer, solvate, hydrate, polymorph, crystal of described compound, noncrystal, salt, pharmaceutically acceptable salt, metabolite and pro-drug modification.

Should be appreciated that [0044] aspect of the present invention as herein described and embodiment comprise by aspect and embodiment and " form (consisting of) " and/or " basic composition (consisting essentially of) ".

The method that suppresses Angiogenesis

[0045] the present invention provide on the one hand by use contain colchicin or the dimeric composition of muscoril (as, contain the composition of carrier protein) suppress the method that individual medium vessels generates. Described composition is in the amount of establishment Angiogenesis. But the quantity not sufficient of the described composition of using is to induce obvious cytotoxicity in this individuality.

[0046] " Angiogenesis " is with referring to the process that produces new blood vessel in tissue or organ here. Angiogenesis starts from the erosion of the vascular basement membrane (basement membrane) that caused by the enzyme that endothelial cell and leucocyte discharge usually. The endothelial cell of small pieces of cloth used for patches intravascular space passes subsequently basement membrane and stretches out. The basement membrane that is etched is passed in the migration of Angiogenesis stimulus inducing endothelial cell. The cell of migration forms " sprouting (sprout) " outside the parental generation blood vessel, endothelial cell carries out mitosis and propagation there. The sprouting of endothelium is merged the formation capillary loops each other, produces new blood vessel.

[0047] " inhibition Angiogenesis " refers to minimizing, stops or suppresses one or more in-house Angiogenesiss in the individuality, comprises for example ocular tissue, cardiovascular organization, skin histology, joint tissue and tumor tissues. The inhibition of Angiogenesis can realize by the one or more steps that affect angiogenesis, for example, by migration and the survival that weakens the endothelial cell that is activated, stops cell " to be sprouted " from the parental generation blood vessel, and/or stops the formation of new blood vessel. The variation of microvessel density is also contained in the term " inhibition Angiogenesis ". Term " inhibition Angiogenesis " also comprises the vascular system that destruction has been set up. " destroy the vascular system of having set up " and refer to obstruction, dissolve or otherwise affect the ability of the already present vascular system that is formed by Angiogenesis. The destruction of vascular system can be reversible or irreversible, part or whole.

[0048] therefore, method provided herein comprises one or more following aspects: suppress (as, reduce, stop or prevent) endothelial cell migration, suppress (as, reducing, stop or prevent) endothelial cell " sprouts " from the parental generation blood vessel, suppress (as, reduce, stop or prevent) formation of new blood vessel, and target (as, block, interrupt or destroy) the built vertical vascular system that formed by Angiogenesis. In some embodiments, provide inhibition (as, reduce, stop or prevent) method that forms of individual medium vessels. In some embodiments, provide interruption (as, block, dissolving or otherwise impact) method of the vascular system set up. In some embodiments, provide the method that reduces individual Microvessel Density.

[0049] after " effective dose " refers to single dose or multiple dose and give individuality, in this individuality, provides amount or the dosage of the composition of desired effects. For example, if the amount of composition is enough in one or more tissues of individuality to reduce, stop or to prevent Angiogenesis (aspect Angiogenesis one or more), described composition is in " amount of establishment Angiogenesis ". Be used in the amount that the term " effective dose " for the treatment of in the content refers to the compound or the composition that are enough to treat particular disorder, situation or disease, as improving, relax, alleviate and/or postponing its one or more symptoms. Effective dose can external and/or in vivoassay. Measure to be used for suppress the method for effective dose of composition of Angiogenesis known in the art.

[0050] in some embodiments, the amount of described composition is effective to the Angiogenesis that suppresses in the ocular tissue. In some embodiments, the amount of described composition is effective to the Angiogenesis that suppresses in the cardiovascular organization. In some embodiments, the amount of described composition is effective to the Angiogenesis that suppresses in the skin histology. In some embodiments, the amount of described composition is effective to the Angiogenesis that suppresses in the joint tissue. In some embodiments, the amount of described composition is effective to the Angiogenesis that suppresses in the tumor tissues. In some embodiments, the amount of described composition is effective to suppressing neovascularization. In some embodiments, the amount of described composition " is sprouted " effectively from the parental generation blood vessel to suppressing endothelial cell. In some embodiments, the amount of described composition is effective to the migration that suppresses endothelial cell. In some embodiments, the amount of described composition is effective to destroying the vascular system of having set up. In some embodiments, the amount of described composition to suppress at least about 5%, 10%, 20%, 40%, 50% or above Angiogenesis (such as any one or many aspects of Angiogenesis) in arbitrary effectively.

[0051] if the quantity not sufficient of composition is so that colchicin or muscoril dimer cause significant cell death in the individuality, said composition is in " being not enough to induce remarkable Cytotoxic amount " (also being known as " amount of no cytotoxicity "). Cytotoxicity can be measured by one or more following manners. For example, the amount of no cytotoxicity can be based on the test determination of cell in vitro survival rate. The amount of no cytotoxicity can be to be not enough to cause in cell in vitro survival rate test about 50% or the amount of many cells death more. In some embodiments, the quantity not sufficient of described composition with in cell in vitro survival rate test, cause about 40% or more, 30% or more, 20% or more, 10% or more, 5% or more, 4% or more, 3% or more, 2% or more 1% or more in arbitrary cell death. In some embodiments, the quantity not sufficient of described composition is to cause any measurable cell death in the test of cell in vitro survival rate. The suitable cell that is used for the test of Motility rate includes but not limited to tumour cell (such as MX-1 breast cancer cell line, HepG2 SMMC-7721 and HT-29 colon carcinoma cell line) and normal cell (such as primary rat hepatocyte). The amount of no cytotoxicity also can be determined based on the in vivo studies of drug toxicity. For example, the amount of no cytotoxicity can be to be not enough in vivo to kill about 50% or the amount of more testing population in the cell toxicity test. In some embodiments, the dimeric amount of colchicin or muscoril be not enough to kill in the cell toxicity test in vivo about 40% or more, 30% or more, 20% or more, 10% or more, 5% or more, 4% or more, 3% or more, 2% or more or 1% or more test arbitrary amount in the population. In some embodiments, the quantity not sufficient of described composition is to cause any death of testing population in the toxicity trial in vivo. The amount of no cytotoxicity also can be based on inducing in the individuality obviously required colchicin or the dimeric amount of muscoril of general toxicity (as losing weight) to determine, that is to say, if the amount of medicine does not cause any obvious general toxicity, then the amount of this medicine is no cytotoxicity. For example, in some embodiments, the amount of no cytotoxicity be cause about 15% (comprise, for example about 10%, 8%, 5% or still less in arbitrary amount) below the amount that loses weight.

[0052] in some embodiments, during each administration in the described compositions colchicine or the dimeric amount of muscoril about 2,3,4,5,6,7,8,9,10,11,12,13,14 and 15mg/m ²Below arbitrary.For example, the dimeric amount of colchicine or muscoril can be at about 0.25mg/m ²Body surface is to about 15mg/m ², about 0.25mg/m ²To about 10mg/m ², about 0.25mg/m ²To about 8mg/m ², about 0.25mg/m ²To about 4mg/m ²And about 0.25mg/m ²To about 2mg/m ²Scope in.In some embodiments, during each administration in the described compositions colchicine or the dimeric amount of muscoril about 0.05,0.08,0.1,0.2,0.3,0.4,0.5 or 0.6mg/kg in arbitrary below.For example, the dimeric amount of colchicine or muscoril can arrive about 0.3mg/kg and about 0.1mg/kg at about 0.05mg/kg and arrives in the scope of about 0.2mg/mg to about 0.5mg/kg, about 0.08mg/kg.

[0053] in some embodiments, colchicine and muscoril dimer be in interrupt or unbroken prolongation administration during do not cause the amount of drug resistance in (as 6 months or longer).In some embodiments, colchicine and muscoril dimer are in and do not cause the apparent side effect amount of (as following chemotherapeutic side effect usually) in the individuality.Usually follow chemotherapeutic side effect for example to comprise and dewater, suffer from diarrhoea, feel sick, vomit, lose one's sight or visual disorder and anemia.

[0054] administration frequency of described compositions includes but not limited to following arbitrary: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day.In some embodiments, the interval between each administration below an about week, according to appointment in 6,5,4,3,2 or 1 days arbitrary below.In some embodiments, the interval between each administration is constant.For example, administration can every day, per two days, per three days, per four days, per five days or is carried out weekly.In some embodiments, administration can twice of every day, every day three times or is carried out more continually.

[0055] using of described compositions can be extended in the time period that prolongs, for example from about one month to about 3 years.For example, dosage regimen can prolong in arbitrary time period in about 2,3,4,5,6,7,8,9,10,11,12,18,24,30 and 36 middle of the month.In some embodiments, free of discontinuities in dosage regimen.In some embodiments, the no more than about week of interval between each administration.

[0056] compositions as herein described can give individuality by any approach in this area, described approach includes but not limited to intravenous, intraperitoneal, ophthalmic, intra-arterial, in the lung, oral, in the vesicle (intravesicular), intramuscular, in the trachea, subcutaneous, ophthalmic, in the sheath, transdermal, saturating pleura, intra-arterial, local, suck (as, as the aerosol of spraying), saturating mucosa (as passing through nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, in the brain pond, in the ventricle, (promptly the passing through suppository) of rectum, (promptly by the vaginal suppository (pessary)) of vagina, intracranial, in the urethra, in the liver and in the tumor.In some embodiments, described compositions is by the whole body administration.In some embodiments, described compositions is by topical.In some embodiments, described compositions is by any administration in intravenous, ophthalmic, intra-arterial, oral, part or the inhalation route.

[0057] relate to when suppressing in the ocular tissue angiogenesis when described method, described compositions can be applied directly to eye or ocular tissue.Described compositions can locally apply to eye, as in eye drop.Described compositions also can be by being expelled to eye or relevant with eye organizing administration.Described compositions can be passed through intraocular injection, periocular injections, subretinal injection, intravitreal injection, saturating barrier film (trans-septal) injection, (subscleral) injection under the sclera, injection in the choroid, (intracameral) injection in the iris, subconjunctival injection, fascia bulbi injection down (sub-Tenon ' sinjection), retrobulbar injection, the nearly sclera in (peribulbar) injection or back is carried (posterior juxtascleral delivery) administration around the eyeball.Described compositions can be applied to, for example, and near in the zone between vitreous body, aqueous humor (aqueous humor), sclera, conjunctiva, the CSC, retina tela chorioidea, macula lutea or the individual eye or other zone the eye.For the description of the exemplary approach near the eyes that is used for the retina drug conveying, referring to Periocular routes forretinal drug delivery, Raghava etc. (2004), Expert Opin.Drug Deliv.1 (1): 99-114.Described compositions also can be used as implant and gives individuality.Preferred implant is biocompatible and/or the Biodegradable sustained-release preparation, and it discharges described chemical compound gradually in a period of time.Be used for that the ocular implants of drug conveying is known in the art.Referring to for example, U.S. Patent number 5,501,856,5,476,511 and 6,331,313.Described compositions also can use ionotherapy to give individuality, include, but are not limited at U.S. Patent number 4,454,151 and U.S. Patent Application Publication No. 2003/0181531 and 2004/0058313 described in the ion means of delivery.

The method of treatment angiogenesis-associated diseases

[0058] method as herein described is normally used for the treatment of angiogenesis-associated diseases." angiogenesis-associated diseases " refers to disease or the disease that angiogenesis is an aspect of described disease.Angiogenesis-associated diseases can be caused by unusual angiogenesis.In some embodiments, described angiogenesis-associated diseases to small part is mediated by angiogenesis.In some embodiments, angiogenesis is necessary to the development of angiogenesis-associated diseases.Angiogenesis-associated diseases is known in the art, and comprises for example non-tumprigenicity angiogenesis-associated diseases such as degeneration of macula, diabetic retinopathy, rheumatic arthritis and other disease as herein described.In some embodiments, described angiogenesis-associated diseases is the tumor relevant disease, as cancer or benign tumor.

[0059] as used herein, " treatment " is the method that is used to obtain the clinical effectiveness of useful or expectation.For purposes of the present invention, clinical effectiveness useful or expectation include, but are not limited to following any one or multiple: the delay of the generation of stable (promptly not the worsening) of the alleviating of the alleviation of one or more symptoms, disease degree, morbid state, the prevention of disease's spread or delay, disease or the prevention of recurrence or delay, disease progression or slow down, the mitigation of morbid state and elimination (no matter be part or all).What also comprised by " treatment " is the alleviating of pathological examination of angiogenesis-associated diseases.Method of the present invention consider these treatment aspects any or a plurality of.

The treatment of non-tumprigenicity angiogenesis-associated diseases

[0060] method as herein described is used for the treatment of non-tumprigenicity angiogenesis-associated diseases.

[0061] in some embodiments, described method is used for the treatment of non-tumprigenicity angiogenesis-associated diseases in ocular tissue such as cornea, retina, macula lutea and the choroid.Described method is generally used for preventing losing one's sight, visual loss (as the forfeiture in the visual acuity or the visual field) and/or other result of being caused by multiple oculopathy.In some embodiments, provide treatment to comprise the method for the degeneration of macula of age-related macular degeneration (AMD).The Clinical symptoms of AMD is the progressively forfeiture of central vision, and the progressively forfeiture of described central vision is to take place owing to result to the damage of the photoreceptor cell that is known as macula lutea in the retinal area.AMD broadly is divided into two clinical states: wet form (wet form) and dry form (dry form), wherein dry form constitutes the 80-90% of whole cases.Described dry form is clinically with the feature that exists for of the little wart of macula lutea retina (macular drusen), the little wart of described retina is the local deposits that is positioned between retinal pigment epithelium (RPE) and the Bu Luheshi film, and be feature with map shape atrophy (geographic atrophy), the atrophy of described map shape is a feature with the RPE cell death of (overlying) photoreceptor atrophy of covering on following.Wet AMD, it causes about 90% severe visual forfeiture, forms relevant with the seepage of these neovascularity with new vessels in the macular region.Accumulating of blood and liquid can cause retina shedding, then is photoreceptor degeneration rapidly and visual loss.The AMD that it has been generally acknowledged that wet form and is produced by dry form after dry form.

[0062] method provided herein is used in particular for the treatment or the inhibition of the degeneration of macula of wet form.The inhibition that the ocular tissue medium vessels generates also prevents or postpones the transformation of the dry form of degeneration of macula to the wet form of degeneration of macula.Therefore the present invention also provides the Therapeutic Method of degeneration of macula dry form.The present invention also comprises treatment or the one or more aspects of prevention degeneration of macula or the method for symptom, and described aspect or symptom include, but are not limited to forfeiture, vision (comprising for example visual acuity and the visual field) forfeiture and the retina shedding of photoreceptor cell.Other related fields are also comprised as the forfeiture and the photoreceptor atrophy of cell function in the forfeiture of the organizational structure integrity of the cell of photoreceptor degeneration, RPE degeneration, retinal degeneration, chorioretinal degeneration, the cone (cone) degeneration, retinal function obstacle, retina injury, Bu Luheshi membrane damage, RPE afunction, normal macula lutea and/or extracellular matrix, the macula lutea.

[0063] can include but not limited to by other non-tumprigenicity angiogenesis associated ophthalmopathy of method treatment as herein described: amphiblestroid new vessels forms, the new vessels of cornea forms (as by sand holes, infect, inflammation, the new vessels that transplanting or wound cause forms), diabetic retinopathy, the diabetic retinal edema, diabetic macular edema, the ischemic retinal disease, the hypertension retinopathy, occlusive retinopathy (occlusive retinopathy), retinopathy of prematurity (retinopathy of prematurity), post-traumatic neovascularization, metainfective new vessels forms, new vessels after the transplanting forms, new vessels after retina shedding or the retinal degeneration forms, neovascular glaucoma, anterior chamber and/or angle of anterior chamber new vessels form, choroidal neovascularization forms (CNV), new vessels forms under the retina, retrolental fibroplasia, ocular histoplasmosis syndrome (ocular histoplasmosis syndrome), the myopia degeneration, angioid streaks, uveitis, rubescent, retrolental fibroplasia, ocular histoplasmosis and constitutional central serous chorioretinopathy.In some embodiments, described oculopathy is diabetic retinopathy.In some embodiments, described oculopathy is neovascular glaucoma.

[0064] in some embodiments, provide the method for treatment angiopathy, described angiopathy comprises the cardiovascular diseases, as medicated porridge sample arteriosclerosis, restenosis, atheroma and hemangioma.Medicated porridge sample arteriosclerosis is a kind of chronic blood vessel injury of form, and wherein some normal blood vessels smooth muscle cell (VSMC) change its character and the intensive blood capillary network of development in atheromatous plaque in the arterial wall.These fragile blood capillaries can cause bleeding, and cause blood clotting, are that the blood flow that arrives cardiac muscle reduces and heart attack subsequently.Restenosis usually occurs in after coronary artery bypass surgery, endarterectomy and the heart transplantation, and occurs in especially after heart sacculus angioplasty, ATH (atherectomy), laser ablation or endovascular stent insert.It relates to microvascular extensive growth.By suppressing the angiogenesis in the cardiovascular organization, method provided herein is used for the treatment of these cardiovascular disease.

[0065] in some embodiments, described method is used for the treatment of rheumatic arthritis, and wherein the blood vessel in the joint experiences angiogenesis, forms the tissue of extensive vascularization, and its intrusion also destroys cartilage.By suppressing the angiogenesis in the joint tissue, method provided herein is used for the treatment of rheumatic arthritis.The method in treatment hemophiliac joint (hemophiliacjoint) also is provided.

[0066] in some embodiments, described method is used for the treatment of the relevant dermatosis of angiogenesis, and it includes but not limited to psoriasis, scleroderma, forms (as cat scratch disease, antibacterial ulcer etc.) and other dermatosis as the new vessels of result of infection.In some embodiments, described method is used for the treatment of psoriasis.Psoriasis is a kind of chronic dermatosis, and it occurs in the population in the whole world about 3%.The Histological research that comprises electron microscope technique has determined that the change that skin heart forms is psoriatic marked feature.The inhibition that the skin histology medium vessels generates is therefore useful to the treatment psoriasis.

[0067] Ao-Wei two syndromes, hereditary hemorrhagic capillary hemorrhage, speckle new vessels be can include, but are not limited to by other angiogenesis-associated diseases of the method for the invention treatment and (plaque neovascularization), telangiectasis, fibrohemangioma, hemangioma, wound granulate (wound granularization), endometriosis etc. formed.In addition, the present invention also is used for the nasal polyp of polypous treatment, particularly cystic fibrosis sufferers.

[0068] except the treatment angiogenesis-associated diseases, method as herein described also can be used to regulate and control or prevent the normal physiological situation incident relevant with angiogenesis.For example, method of the present invention can be used to weaken the new vessels relevant with ovulation, embryo's implantation, placentation etc. and form, and therefore is used for the purpose of birth control.

[0069] this paper provides the method for treatment multiple disease described herein.In some embodiments, the method of non-tumprigenicity angiogenesis-associated diseases in the treatment individuality is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of the muscoril compositions of carrier protein (as contain), wherein said compositions is in the amount of the non-tumprigenicity angiogenesis-associated diseases of effective treatment.In some embodiments, provide the individual medium vessels of treatment to generate the method for associated ophthalmopathy (comprising for example degeneration of macula, diabetic retinopathy or neovascular glaucoma), comprise that giving this individuality contains colchicine or the dimeric compositions of the muscoril compositions of carrier protein (as contain), wherein said compositions is in the amount of the described oculopathy of effective treatment.In some embodiments, the method of treatment cardiovascular disease (as restenosis and medicated porridge sample arteriosclerosis) is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of the muscoril compositions of carrier protein (as contain), wherein said compositions is in the amount of the described cardiovascular disease of effective treatment.In some embodiments, the method of the relevant dermatosis (as psoriasis) of treatment angiogenesis is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of the muscoril compositions of carrier protein (as contain), wherein said compositions is in the described dermopathic amount of effective treatment.In some embodiments, the method of treatment of arthritis (as rheumatic arthritis) is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of the muscoril compositions of carrier protein (as contain), wherein said compositions is in the amount of effective treatment of arthritis.

[0070] in some embodiments, following any administration of colchicine or muscoril dimer compositions: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day.In some embodiments, the interval between each administration below an about week, according to appointment in 6,5,4,3,2 or 1 days arbitrary below.In some embodiments, the interval between each administration is constant.For example, administration can every day, per two days, per three days, per four days, per five days or is carried out weekly.In some embodiments, administration can twice of every day, every day three times or is carried out more continually.Using of described compositions can be extended (interrupt or do not interrupt) in the time period that prolongs, for example from about one month to about 3 years.For example, dosage regimen can prolong (interrupt or do not interrupt) in arbitrary time period in about 2,3,4,5,6,7,8,9,10,11,12,18,24,30 and 36 middle of the month.In some embodiments, free of discontinuities in dosage regimen.In some embodiments, the interval between each administration is not more than an about week.

By suppressing the method for angiogenesis treatment tumor relevant disease

[0071] method as herein described also is used for the inhibition that the tumor tissues medium vessels generates, and is used for the treatment of the tumor relevant disease, as cancer and benign tumor.

[0072] suppresses angiogenesis (for example blood vessel) and prevent that prescribing adequate nutrition and oxygen are supplied to tumor and support its growth to surpass specific size by suppressing neovascularization or having generated by targeting.Because angiogenesis relates to the primary tumor growth and shifts, method provided herein can suppress the tumor growth of tumor in the constitutional position, and prevents that tumor is in the Secondary cases position transfer.In some embodiments, colchicine or muscoril dimer suppress neovascularization.In some embodiments, colchicine or muscoril dimer compositions are destroyed the tumor vascular system of having set up.For example, described compositions can be in reversible or irreversible, partly or entirely selectively blocking, decomposition or otherwise influence aspect the tumor vascular system (be sometimes propagation tumor vascular system) effective.

[0073] the invention provides by giving the method that contains colchicine or the dimeric combination treatment tumor of muscoril relevant disease of effective dose, the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity (also being known as " no cytotoxicity dosage ").The traditional chemical therapy of utilizing the cytotoxicity medicament usually equal or dosage near the maximum tolerated dose of this medicament under carry out so that maximize the cytotoxic effect of this medicament.But, during the nothing treatment that this high dose scheme need prolong, to allow the normal host cellular-restoring.Simultaneously, tumor cell also may recover growth during not having treatment.This can increase the risk of development antiradiation drug tumor cell.The colchicine of no cytotoxicity dosage or muscoril dimer allow not have in treatment cycle the treatment of cancer of obvious interruption, have therefore reduced the risk of development drug resistance.

[0074] for example is provided, the treatment method for cancer is further described at this.Should be appreciated that this description is suitable for all cancer-related diseases usually, comprises benign tumor.

[0075] in some embodiments, method for cancer in this individuality of treatment is provided, comprise that giving this individuality contains colchicine or the dimeric compositions of muscoril, wherein said compositions is in the amount of effective treatment cancer, and wherein each give colchicine in the described compositions approximately or the dimeric amount of muscoril in according to identical (or similar) preparation of same or similar dosage regimen or traditional dosage regimen identical (or similar) colchicine or the dimeric corresponding MTD of colchicine about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, in 14% or 15% arbitrary below.In some embodiments, the colchicine in each administration composition or the amount of muscoril corresponding MTD about 1% to about 15% between, comprise for example following arbitrary amount: about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 1% to about 5%, about 1% to about 3% of corresponding MTD.The dimeric MTD of colchicine or muscoril is known or can easily be measured by those skilled in the art.For example, be about 90-100mg/m according to the MTD of the Nab-5404 of scheme weekly ²Body surface.

[0076] in some embodiments, the following arbitrary mode administration of colchicine or muscoril dimer compositions: at least about per three weeks once, whenever biweekly, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day.In some embodiments, the interval between each administration below an about week, according to appointment in 6,5,4,3,2 or 1 days arbitrary below.In some embodiments, the interval between each administration is constant.For example, administration can every day, per two days, per three days, per four days, per five days or is carried out weekly.In some embodiments, administration can twice of every day, every day three times or is carried out more continually.Using of described compositions can be extended (interrupt or do not interrupt) in the time period that prolongs, for example from about one month to about 3 years.For example, dosage regimen can prolong (interrupt or do not interrupt) in arbitrary time period in about 2,3,4,5,6,7,8,9,10,11,12,18,24,30 and 36 middle of the month.In some embodiments, free of discontinuities in dosage regimen.In some embodiments, the interval between each administration is not more than an about week.

[0077] can include, but are not limited to cancer, lymphoma, blastoma, sarcoma and leukemia by the cancer of method treatment of the present invention.These tumors example more specifically include, but are not limited to squamous cell carcinoma, pulmonary carcinoma (comprises small cell lung cancer, nonsmall-cell lung cancer, the adenocarcinoma of lung and the squamous cell carcinoma of lung), peritoneal cancer, hepatocarcinoma, stomach (gastric) or stomach (stomach) cancer (comprising human primary gastrointestinal cancers), cancer of pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatocarcinoma (heptoma), breast carcinoma, colon cancer, endometrium or uterus carcinoma, salivary-gland carcinoma, kidney (kidney) or kidney (renal) cancer, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma (hepaticcarcinoma), head and neck cancer, the B-cell lymphoma (comprises minuent/folliculus non_hodgkin lymphoma (NHL) (low grade/follicular non-Hodgkin ' s lymphoma), small lymphocyte (SL) NHL, moderate/folliculus NHL, moderate dispersivity NHL, hyperimmunization blast cell NHL, height lymphoblast NHL, highly little non-cleaved cell NHL, huge tumor (bulky disease) NHL, lymphoma mantle cell, relevant lymphoma of AIDS-and waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and transplanting back lymphocytic hyperplasia disorder (PTLD), and with scar nevus property hamartomatosis, edema (as the edema relevant) and the Meigs's syndrome (abnormal angiogenesis that Meigs ' syndrome) is relevant with cerebroma.Method as herein described is particularly useful to cancer such as squamous cell carcinoma that treatment relates to extensive angiogenesis.

The method of treatment primary tumor is provided [0078] in some embodiments.The method of treatment metastatic cancer (that is the cancer that has shifted from primary tumor) is provided in some embodiments.The method of treatment terminal cancer is provided in some embodiments.In some embodiments, provide the method for treatment breast carcinoma (it can be the HER2 positive or HER2 feminine gender), described breast carcinoma comprises for example advanced breast cancer, IV primary breast cancer, local advanced breast cancer and metastatic breast cancer.In some embodiments, described cancer is a pulmonary carcinoma, comprises for example nonsmall-cell lung cancer (NSCLC is as advanced NSCLC), small cell lung cancer (SCLC is as SCLC in late period) and lung middle and advanced stage malignant solid tumor.In some embodiments, described cancer is ovarian cancer, head and neck cancer, stomach malignant tumor, melanoma (comprising metastatic melanoma), colorectal carcinoma, cancer of pancreas and solid tumor (as advanced solid tumor).In some embodiments, described cancer is following any (and being selected from following in some embodiments): breast carcinoma, colorectal carcinoma, rectal cancer, nonsmall-cell lung cancer, non Hodgkin lymphoma (NHL), renal cell carcinoma, carcinoma of prostate, hepatocarcinoma, cancer of pancreas, soft tissue sarcoma, kaposi sarcoma (Kaposi ' s sarcoma), benign tumor (carcinoidcarcinoma), head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioma, glioblastoma multiforme, neuroblastoma and multiple myeloma.In some embodiments, described cancer is a solid tumor.

[0079] method as herein described can be implemented auxiliary the setting in (adjuvant setting)." the auxiliary setting " refers to so clinical setting: wherein individuality has the cancer medical history, and (but nonessential) responds to treatment usually, and described treatment includes but not limited to operation (as excision), radiotherapy and chemotherapy.Yet because their cancer medical history, these individualities considered to be under the risk of the described disease of development.Treatment in " the auxiliary setting " or administration refer to treatment pattern subsequently.Degree of risk (that is, when individuality is considered to " excessive risk " or " low-risk " in auxiliary the setting) depends on a number of factors, and modal is disease degree when treating the first time.Method provided herein also can be provided with middle enforcement new auxiliary, and promptly described method can be carried out before main/treatment of determining.In some embodiments, described individuality was treated in the past.In some embodiments, described individuality was not treated in the past.In some embodiments, described treatment is first-line treatment (primary treatment, first line therapy).

[0080] method as herein described can be carried out (for example, in therapeutic alliance is provided with) jointly with other method of treatment of cancer.For example, in some embodiments, described method comprises first treatment and second treatment, and described first treats and to comprise using and contain colchicine or the dimeric compositions of muscoril.In some embodiments, described second therapy is a chemotherapy.In some embodiments, described second therapy is an X-ray therapy.In some embodiments, described second therapy is operation.First and second therapies (that is, first therapy was carried out before or after second therapy) are simultaneously or sequentially carried out.In some embodiments, described method comprises that using (comprising simultaneously or sequentially) jointly to individuality contains colchicine or the dimeric compositions of muscoril and second chemotherapeutant, wherein said compositions is effective to the angiogenesis that suppresses in this individuality, and wherein said compositions is in the amount that is not enough to inducing cytotoxic in this individuality.In some embodiments, described second chemotherapeutant is a cytotoxic agent.

Contain the dimeric compositions of colchicine or muscoril

Colchicine or muscoril dimer

[0081] method as herein described comprises and contains using of colchicine or muscoril dimer compositions." colchicine or muscoril dimer " uses the chemical compound that refers to two (identical or different) subunits that contain colchicine, muscoril or derivatives thereof here." derivant " of colchicine or muscoril include but not limited to and colchicine or muscoril structural similarity, or with colchicine and muscoril the chemical compound in identical total chemical classes.In general, the derivant of colchicine or muscoril or analog maintenance biology, pharmacy, chemistry and/or the physical property (comprise for example degree of functionality) similar to colchicine or muscoril.In some embodiments, described colchicine or muscoril dimer comprise at least one muscoril subunit.In some embodiments, described colchicine or muscoril dimer comprise two muscoril subunits (being called " muscoril dimer " hereinafter).In some embodiments, described colchicine or muscoril dimer comprise two colchicine subunits (being called " colchicine dimer " hereinafter).

[0082] in some embodiments, described colchicine or muscoril dimer are the chemical compounds of formula (I):

[0083] wherein the B in each subunit is methoxyl group or methyl mercapto, R ₂When with R ₃When choosing together methoxyl group, hydroxyl or methylene-dioxy (methylenedioxy); R ₃When with R ₂Be methoxyl group, hydroxyl or methylene-dioxy when choosing together, and X is a linking group.

[0084] multiple crosslinked group can be used to introduce linking group X.Those of ordinary skill in the art will recognize that dimeric colchicine or muscoril monomer component have single reactive amino; If there is any other reaction (nucleophilic) group to be present on the intermediate, they can use group well known in the art easily to be protected.The example of blocking group referring to, for example, Greene, T.W. and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3 ^RdEdition, Wiley:New York, 1999, its content so integral body are incorporated herein by reference.Therefore, the multiple crosslinked group with the reaction of amine degree of functionality can be used.

[0085] in some embodiments, linking group X comprises at least one carbon atom.(Sigma-Aldrich) reagent malonyl chloride Cl-C (the O)-CH that for example, can buy ₂-C (O)-Cl can be used for forming the colchicine dimer, and wherein the X group is-CH ₂-.Similarly, other diacyl chlorine of all lengths can be used to form the X group of desired length.For example, in formula (II), when n=8 and Y are CH ₂The time, (Sigma-Aldrich) reagent dodecane diacyl dichloro Cl-C (O)-(CH that can buy ₂) ₁₀-C (O)-Cl can be used to synthetic described dimer, and wherein the X group is-(CH ₂) ₁₀-.For Y is the group of NH and n=1, reagent 3-isocyanato-propionyl chloride (Organic Syntheses, Coll.Vol.6, p.715 (1988); Vol.59, p.195 (1979)) can be used to synthetic linking group X, this moment, X was-NH-CH ₂CH ₂-.Other cross-linking reagent of knowing can be used to produce the X connector.Those of ordinary skills are referring to Wong, Shan S., Chemistry of Protein Conjugation and Cross Linking, CRC Press:Boca Raton, 1991, particularly 33-38 page or leaf the 2nd chapter IV (B) part relates to the amino group reactant; 75-103 page or leaf the 4th chapter part ii relates to amino group cross-linking reaction connector; With 209-220 page or leaf the 7th chapter, relate to the step and the analysis of reagent cross-linking reaction and be fit to the crosslinked step that contains amino-compound.Therefore the full content of the list of references of aforementioned Wong and particularly cited specific part are introduced into this paper as a reference.

[0086] in some embodiments, described colchicine or colchicine dimer are the chemical compounds of formula (II):

[0087] B wherein ₁Be methoxyl group or methyl mercapto, B ₂Be methoxyl group or methyl mercapto, n is from 0 to 8 integer, and Y is CH ₂Group, perhaps when n was 1, Y also can be the group of formula NH.

[0088] in some embodiments, n is in (and being selected from some embodiments) 0,1,2,3,4,5,6,7 or 8 any.In some embodiments, n is 1.In some embodiments, n be 1 and Y be NH.In some embodiments, n is 2.

[0089] in some embodiments, B ₁And B ₂It all is methoxyl group.In some embodiments, B ₁And B ₂It all is methyl mercapto.In some embodiments, B ₁Be methoxyl group and B ₂It is methyl mercapto.In some embodiments, B ₁Be methyl mercapto and B ₂It is methoxyl group.In some embodiments, described colchicine or muscoril dimer are following arbitrary (and being selected from some embodiments): IDN5404, IDN5676, IDN5800 and IDN5801.

[0090] in some embodiments, described chemical compound is muscoril dimer IDN5404.IDN5404 is the chemical compound of formula (III):

[0091] in some embodiments, described chemical compound is muscoril dimer IDN5676.IDN5676 is the chemical compound of formula (IV):

Biocompatible polymer and carrier protein

[0092] in some embodiments, colchicine as herein described or muscoril dimer compositions further comprise biocompatible polymer, as carrier protein.

[0093] with here, the material that does not obviously change or influence introducing biosystem wherein in any disadvantageous mode described in term " biocompatible ".Biocompatible polymer comprises natural appearance or synthetic biocompatible substance, as protein, polynucleotide, polysaccharide (as, starch, cellulose, glucosan, alginate, chitosan, colloid, hyaluronic acid etc.) and lipid.Suitable biocompatible polymer comprises, for example, natural appearance or synthetic protein, as albumin, insulin, hemoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fiber adhesion albumen, vitronectin, Fibrinogen, casein etc., and their two or more combinations arbitrarily.Synthetic polymer comprises, for example, poly alkylene glycol (as linearity or side chain), polyvinyl alcohol, polyacrylate, poly hydroxy ethyl acrylate, polyacrylic acid, Ju ethyl oxazoline (polyethyloxazoline), polyacrylamide, PNIPAM, polyvinylpyrrolidone, polyactide/Acetic acid, hydroxy-, bimol. cyclic ester (polylactide/glycolide) etc. and their combination.

[0094] term " protein " refers to the random length amino acid whose polypeptide or the polymer of (comprising total length or fragment), and it can be linearity or branching, comprises the aminoacid of modification and/or the aminoacid that is interrupted by non-aminoacid.This term also comprises natural modifications or by inserting the amino acid polymer of modifying; For example, disulfide bond formation, glycosylation, fatization, acetylation, phosphorylation or any other operation or modification.Being also included within this term for example is to contain one or more amino acid analogues (comprise, for example alpha-non-natural amino acid etc.), and other modified polypeptides known in the art.Protein as herein described can be natural appearance, promptly from natural origin (as blood) acquisition or deutero-; Or synthetic (as chemosynthesis or synthetic) by recombinant DNA technology.

[0095] the proteic example of suitable carriers comprises the protein of usually finding in blood or blood plasma, and it includes but not limited to albumin, comprises the immunoglobulin of IgA, lipoprotein, apolipoprotein B, alpha-acid glycoprotein, β-2-macroglobulin, Elityran, transferrins (transferin), fiber adhesion albumen, factor VII, Factor IX, factors IX, factor X etc.In some embodiments, described carrier protein is non-blood protein, as casein, alpha lactalbumin and beta lactoglobulin.Described carrier protein can be natural origin or synthetic preparation.In some embodiments, described carrier protein is an albumin, as the human serum albumin.Human serum albumin (HSA) is M _rBe the soluble globular preteins of the height of 65K, and form by 585 aminoacid.HSA is an albumen maximum in the blood plasma, and constitutes the 70-80% of human plasma colloid osmotic pressure.The aminoacid sequence of HSA contains 17 disulfide bond, a free mercaptan (thiol) (Cys 34) and tryptophans (Trp 214) altogether.The vein of HSA solution use be shown as prevention and treatment hypovolemic shock (hypovolumic shock) (referring to, Tullis for example, JAMA, 237,355-360,460-463, (1977) and Houser etc., Surgery, Cynecology and Obstetrics, 150,811-816 (1980)), and with exchange transfusion unite the treatment that is used for hyperbilirubinemia of newborn (referring to, Finlayson for example, Seminars in Thrombosis and Hemostasis, 6,85-120, (1980)).Other albumin also is considered, as bovine serum albumin.These inhuman albuminous uses can be suitable under the situation of these compositionss of use in non-human mammal for example, as veterinary animal (comprising domestic pets and agricultural animal).

[0096] human serum albumin (HSA) has a plurality of hydrophobic binding sites (to add up to 8 of fatty acid, described fatty acid is the endogenous ligands of HSA) and combine with a lot of groups of medicines, particularly neutral and electronegative hydrophobic compound (Goodman etc., The Pharmacological Basisof Therapeutics, 9 ^ThEd, McGraw-Hill New York (1996)).Proposed two high-affinity binding sites in the subdomain IIA of HSA and IIIA, they are highly extended hydrophobic pockets (hydrophobic pocket) and have charged lysine and arginine residues near surface, described residue as the attachment point of polar ligand feature play a role (referring to, Fehske etc. for example, Biochem.Pharmcol., 30,687-92 (1981), Vorum, Dan.Med.Bull., 46,379-99 (1999), Kragh-Hansen, Dan.Med.Bull., 1441,131-40 (1990), Curry etc., Nat.Struct.Biol., 5,827-35 (1998), Sugio etc., Protein.Eng., 12,439-46 (1999), He etc., Nature, 358,209-15 (1992) and Carter etc., Adv.Protein.Chem., 45,153-203 (1994)).

[0097] for example is provided, carrier protein is further described hereinafter.Should be appreciated that this is described and is suitable for biocompatible polymer at large.

[0098] carrier protein (as albumin) in the described compositions is usually as colchicine or the dimeric carrier of muscoril, promptly compare with the compositions that does not contain carrier protein, the carrier protein in the described compositions makes that colchicine or muscoril dimer are easier to be suspended in the aqueous medium or to help to keep suspension.This can be avoided being used to dissolve colchicine or the dimeric toxic solvent of muscoril, and therefore can reduce one or more side effect that caused by those toxic solvents.In some embodiments, described compositions does not contain surfactant substantially, and promptly when described compositions was given individuality, the quantity not sufficient of surfactant was to cause one or more side effect in the described compositions in individuality.In some embodiments, described compositions does not contain surfactant.

[0099] in some embodiments, described carrier protein and colchicine or muscoril associate, and promptly described compositions contains associating colchicine of carrier protein or muscoril dimer." associate (Association) " or " associating (associated) " is used in here with common implication, and refers to influence the carrier protein of colchicine in the waterborne compositions or behavior of muscoril dimer and/or character.For example, compare with the compositions that does not have carrier protein, be suspended in the aqueous medium if described carrier protein makes colchicine or the muscoril dimer is easier, described carrier protein and described colchicine or muscoril dimer are considered to " associating ".As another example, if described carrier protein makes colchicine or muscoril in the water slurry stable, described carrier protein and described colchicine or muscoril dimer are associating.For example, described carrier protein and described colchicine or muscoril dimer may reside in granule or the nano-particle, and it is further described at this paper.

[0100] if colchicine or muscoril dimer keep being suspended in the aqueous medium (visiblely separate out or precipitate as not having) for a long time, as at least about arbitrary time in 0.1,0.2,0.25,0.5,1,2,3,4,5,6,7,8,9,10,11,12,24,36,48,60 or 72 hour, then it is " stablizing " in water slurry.The common still also nonessential individuality (as the people) that is fit to give of described suspension.The stability of suspension (but and nonessential) is usually assessed under storage temperature (as room temperature (as 20-25 ℃) or frozen state (as 4 ℃)).For example, if after suspension preparation about 15 minutes the time, with the naked eye or when observing under 1000 times of optical microscopes, it does not demonstrate and visiblely coagulates wadding or granule gathers, and then this suspension is stable under storage temperature.Stability also can be assessed under the test condition of accelerating, as assessing being higher than under about 40 ℃ temperature.

[0101] carrier protein in the described compositions and colchicine or muscoril dimer can associate in a variety of forms.For example, in some embodiments, described carrier protein mixes with colchicine or muscoril dimerization bulk phase.In some embodiments, year a colchicine or a muscoril dimer are sealed or wrapped to described carrier protein.In some embodiments, described carrier protein combines (as non-covalent combination) with colchicine or muscoril dimer.In some embodiments, described compositions can be showed one or more above-mentioned aspects.

[0102] in some embodiments, described compositions comprises the granule (as microgranule or nano-particle) that contains (in numerous embodiments, being made up of it substantially) colchicine or muscoril dimer and carrier protein (as albumin).In some embodiments, colchicine or muscoril dimer wrap quilt with carrier protein (as albumin).In some embodiments, described compositions comprises nano-particle (that is, average or average diameter are not more than the granule of about 1000 nanometers (nm)), as be not more than about 900,800,700,600,500,400,300,200 and 100nm in arbitrary.In some embodiments, the average of nano-particle or average diameter are not more than about 200nm in the described compositions.In some embodiments, the average of nano-particle or average diameter arrive between about 400nm about 20 in the described compositions.In some embodiments, the average of described nano-particle or average diameter are between about 40 to about 200nm.In some embodiments, but described nano-particle is aseptic filtration.

[0103] granule described herein (as microgranule or nano-particle) can be present in the dry preparation (as freeze dried compositions) or be suspended in the biocompatible media.Suitable biocompatible media include but not limited to optional buffer solution, the synthetic polymer of optional buffer solution, the vitamin of water, buffered water medium, saline, buffer saline, amino acid whose optional buffer solution, proteinic optional buffer solution, saccharide optional buffer solution, contain the emulsion of lipid etc.

[0104] amount of carrier protein depends on that described combination of Chinese medicine thing reagent changes with other component in the compositions described herein.In some embodiments, the amount of the carrier protein that described compositions contains is enough to colchicine or the muscoril dimer in the stable aqueous suspension, for example, is in the form (as the stable suspension of microgranule or nano-particle) of stable colloidal suspension liquid.In some embodiments, described carrier protein is in the amount that reduces colchicine in the aqueous medium or muscoril dimer rate of deposition.For containing grains of composition, the amount of described carrier protein also depends on colchicine or particulate size of muscoril dimer and density.

[0105] in some embodiments, described carrier protein exists with the amount that is enough to the colchicine in the water slurry or muscoril dimer are stabilized in specific concentrations.For example, the dimeric concentration of colchicine or muscoril is about 0.01 to about 100mg/ml in the described compositions, comprises for example following arbitrary: about 0.01 arrives about 50mg/ml, about 0.1 to about 50mg/ml, about 1 arrives about 8mg/ml, about 4 to about 6mg/ml, about 5mg/ml to about 10mg/ml, about 2mg/ml.In some embodiments, the dimeric concentration of colchicine or muscoril is at least about following arbitrary: 0.01mg/ml, 0.03mg/ml, 0.05mg/ml, 0.08mg/ml, 0.1mg/ml, 0.3mg/ml, 0.5mg/ml, 0.8mg/ml, 1mg/ml, 1.3mg/ml, 1.5mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 40mg/ml and 50mg/ml.In some embodiments, described carrier protein is to avoid the using amount of surfactant to exist, so that described compositions does not contain or do not contain surfactant substantially.

[0106] in some embodiments, the compositions of liquid form comprises about 0.1% to about 50% (w/v) (0.5% (w/v), about 5% (w/v), about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v), about 40% (w/v) or about 50% (w/v) according to appointment) carrier protein.In some embodiments, the compositions of liquid form comprises about 0.5% carrier protein to about 5% (w/v).

[0107] in some embodiments, carrier protein such as albumin make the colchicine of capacity or muscoril dimer combine with cell or be transported by cell with colchicine or the dimeric weight ratio of muscoril.In some embodiments, the dimeric weight ratio of carrier protein such as albumin and colchicine or muscoril (w/w) is about 0.01: 1 to about 100: 1, about 0.02: 1 to about 50: 1, about 0.05: 1 to about 20: 1, about 0.1: 1 to about 20: 1, about 1: 1 to about 18: 1, about 2: 1 to about 15: 1, about 3: 1 to about 12: 1, about 4: 1 to about 10: 1, about 5: 1 to about 9: 1 or about 9: 1.In some embodiments, carrier protein (as albumin) and colchicine or the dimeric weight ratio of muscoril are for approximately following arbitrarily: 18: 1 or littler, 15: 1 or littler, 14: 1 or littler, 13: 1 or littler, 12: 1 or littler, 11: 1 or littler, 10: 1 or littler, 9: 1 or littler, 8: 1 or littler, 7: 1 or littler, 6: 1 or littler, 5: 1 or littler, 4: 1 or littler and 3: 1 or littler.

[0108] in some embodiments, described compositions comprises and contains (in numerous embodiments, being made up of it substantially) muscoril dimer (as IDN5404 or IDN5676) and albuminous granule (as microgranule or nano-particle).Described granule (as microgranule or nano-particle) can have and is not more than about 10,9,8,7,6,5,4,3,2 or 1 microns average or average diameter.In some embodiments, described granule is a nano-particle, the granule that promptly about 1000 nanometers (nm) are following.For example, described nano-particle can be not more than about 900,800,700,600,500,400,300,200 and 100nm in arbitrary.In some embodiments, the average of described nano-particle or average diameter are not more than about 200nm.In some embodiments, the average of described nano-particle or average diameter are between about 20 to about 400nm.In some embodiments, the average of described nano-particle or average diameter are between about 40 to about 200nm.In some embodiments, but described granule is aseptic filtration.

[0109] in some embodiments, described muscoril dimer (as IDN5404 or IDN5676) is with albumin bag quilt.In some embodiments, the weight ratio (w/w) of albumin and muscoril dimer (as IDN5404 or IDN5676) is following arbitrary: about 0.01: 1 to about 100: 1, about 0.02: 1 to about 50: 1, about 0.05: 1 to about 20: 1, about 0.1: 1 to about 20: 1, about 1: 1 to about 18: 1, about 2: 1 to about 15: 1, about 3: 1 to about 12: 1, about 4: 1 to about 10: 1, about 5: 1 to about 9: 1 and about 9: 1.In some embodiments, the dimeric weight ratio of albumin and muscoril following arbitrary below: about 18: 1 or littler, 15: 1 or littler, 14: 1 or littler, 13: 1 or littler, 12: 1 or littler, 11: 1 or littler, 10: 1 or littler, 9: 1 or littler, 8: 1 or littler, 7: 1 or littler, 6: 1 or littler, 5: 1 or littler, 4: 1 or littler and 3: 1 or littler.

[0110] in some embodiments, contain muscoril dimer (as IDN5404 or IDN5676) and albuminous granule (as microgranule or nano-particle) and be suspended in (as containing albuminous aqueous medium) in the aqueous medium.For example, described compositions can be the colloidal suspension liquid that contains muscoril dimer (as IDN5404 or IDN5676) granule (as microgranule or nano-particle).In some embodiments, described compositions is dry composition (as a freeze-dried composition), and it can be reconfigured as and contain the particulate water slurry of sulfo-colchicine dimer (as IDN5404 or IDN5676).In some embodiments, the concentration of muscoril dimer in the described compositions (as IDN5404 or IDN5676) comprises for example following arbitrary between about 0.1 to about 100mg/ml: about 0.1 arrives about 50mg/ml, about 0.1 to about 20mg/ml, about 1 arrives about 8mg/ml, about 4 to about 6mg/ml, about 5mg/ml to about 10mg/ml, about 2mg/ml.In some embodiments, the concentration of IDN5404 or IDN5676 is at least about following arbitrary: 1.3mg/ml, 1.5mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 40mg/ml and 50mg/ml.

[0111] in some embodiments, the nanoparticle formulations of described composition I DN5404 or IDN5676 (hereinafter being called Nab-5404 or Nab-5676).Nab-IDN5404 and Nab-5676 are respectively the nanoparticle formulations by human serum albumin's steady I DN5404 and IDN5676.These nanoparticle formulations can produce by U.S. Patent number 5,916,596 and U.S. Patent Application Publication No. 2005/0004002 described method.When being dispersed in suitable aqueous medium, as in 0.9% sodium chloride injection or the 5% glucosan injection time, Nab-5404 (or Nab-5676) forms the dimeric stable colloidal suspension liquid of muscoril.Particulate size in the described colloidal suspension liquid (being average or average diameter) can be at 20nm in 8 micrometer ranges, and preferred range is about 20-400nm.Because HSA freely is dissolved in the water, Nab-5404 (or Nab-5676) can be with wide range of concentrations reconstruct, from (0.1mg/ml IDN5404 or IDN5676) to spissated (20mg/mlIDN5404 or IDN5676) concentration of dilution, comprise that for example about 2mg/ml is to about 8mg/ml, about 5mg/ml.In some embodiments, the concentration of IDN5404 or IDN5676 is about following arbitrary: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 and 20mg/l.

Pharmaceutical composition, unit dose and test kit

[0112] this paper also provides and contains colchicine or the dimeric pharmaceutical composition of muscoril.Described pharmaceutical composition can be fit to multiple administering mode as herein described, comprises for example whole body or topical.Described pharmaceutical composition can be the form of eye drop, Injectable solution, or is fit to suck the form of (through port or nose) or oral administration.Pharmaceutical composition as herein described can be with single unit dose or with the multiple dose packaged.In some embodiments, described compositions is fit to be applied to the people.In some embodiments, described compositions is fit to be applied to mammal, as, under veterinary's environment, domestic pets and agricultural animal.Have multiple described compositions appropriate formulation (referring to, for example U.S. Patent number 5,916,596 and 6,096,331).

[0113] in some embodiments, described pharmaceutical composition comprises the pharmaceutically acceptable carrier of colchicine or muscoril dimer, carrier protein and suitable intraocular injection.In some embodiments, described pharmaceutical composition comprises colchicine or muscoril dimer, carrier protein and is fit to be locally applied to the pharmaceutically acceptable carrier of eye.In some embodiments, described pharmaceutical composition comprises the pharmaceutically acceptable carrier of colchicine or muscoril dimer, carrier protein and suitable intra-arterial injection.

[0114] described pharmaceutical composition is usually with aseptic and isoosmotic substantially compositions preparation.For injection, described pharmaceutical composition can be the form of liquid solution, for example, and in physiology compatible buffers such as hanks' solution or Ringer's solution (Ringer ' s solution).Described pharmaceutical composition also can be solid form, and dissolves immediately before use or resuspending again.Also comprise freeze-dried composition.

[0115] for oral formulations, described pharmaceutical composition can adopt and for example utilize tablet or the capsular form of pharmaceutically acceptable excipient by the traditional method preparation, described pharmaceutically acceptable excipient such as binding agent (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl emthylcellulose); Filler (for example, lactose, microcrystalline Cellulose or calcium hydrogen phosphate); Lubricant (for example, magnesium stearate, Talcum or silicon dioxide); Disintegrating agent (for example, potato starch or sodium starch glycol); Or wetting agent (for example, sodium lauryl sulphate).The liquid preparation of oral administration can adopt for example form of solution, syrup or suspension, or they can present with dryed product, constitutes with water or other suitable carrier before being used for using.These liquid preparations can utilize pharmaceutically acceptable additive preparation by traditional method, described additive such as suspending agent (for example, Sorbitol syrup, cellulose derivative or hydrogenant edible fat); Emulsifying agent (for example, lecithin or arabic gum); Non-aqueous carrier (for example, ationd oil, oiliness esters, ethanol or fractionated vegetable oil); And antiseptic (for example, methyl parahydroxybenzoate or propyl ester, or sorbic acid).Described preparation also can contain buffer salt, flavoring agent, coloring agent and sweeting agent according to circumstances.

[0116] the present invention provides the compositions that comprises colchicine or muscoril dimer, carrier protein and be fit to be applied to the pharmaceutically acceptable carrier of eye in some embodiments.Such pharmaceutical carrier can be a sterile liquid, and Ru Shui and oil comprise oil, animal, plant or synthetic those of originating, as Oleum Arachidis hypogaeae semen, soybean oil, mineral wet goods.Saline solution and glucan aqueous solution, Polyethylene Glycol (PEG) and glycerite also can be used as liquid-carrier, are used in particular for Injectable solution.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, sodium stearate, glyceryl monostearate, glycerol, propylene, water etc.Described pharmaceutical composition if wish, also can contain a spot of wetting agent or emulsifying agent, or the pH buffer reagent.The component of described compositions can be enclosed in polymer or the Fibrin Glue, discharges so that controlled molecule to be provided.These compositionss can adopt the form of solution, suspension, Emulsion, ointment, gel or other solid or semi-solid combination etc.Described compositions has the pH in 4.5 to 8.0 scopes usually.Described compositions also must be formulated into the compatible osmotic value of aqueous humor that has with eye and ocular tissue.Such osmotic value arrives in the scope of about 400 milli osmole/kg water (mOsm/kg) about 200 usually, but is preferably about 300mOsm/kg.

[0117] in some embodiments, described compositions is configured to the pharmaceutical composition of suitable intravenous, intraperitoneal or intravitreal injection according to conventional steps.Typically, composition for injection is the solution in solutions in sterile isotonic aqueous buffer.In case of necessity, described compositions also can comprise solubilizing agent and local anesthetic such as lignocaine to alleviate the pain of injection position.In general, described composition is supplied separately or is mixed with unit dosage forms, for example, and at the gas-tight seal container such as the dry freeze-dried powder among ampoule or the sachette of lined out activity dose or there is not aqueous concentrate.When described compositions will be used by infusion, it can be dispersed in and contain in aseptic pharmaceutical grade water or the brinish infusion bottle.When described compositions is passed through drug administration by injection, can provide Injectable sterile water or brinish ampoule, so that component is being used preceding mixing.

[0118] described compositions can further comprise other component, for example antiseptic, buffer agent, tonicity agent, antioxidant and stabilizing agent, nonionic wetting agent or clarifier, viscosifier etc.

[0119] the suitable antiseptic that is used in the solution comprises polyquaternary ammonium salt-1, benzalkonium chloride, thiomersalate, methaform, methyl butex, propylparaben, phenylethanol, disodiumedetate, sorbic acid, benzethonium chloride etc.Typically (but not necessarily), these antiseptic are being used with 0.001% to 1.0% amount by weight.

[0120] suitable reducing comprises boric acid, sodium bicarbonate and potassium bicarbonate, sodium borate and potassium borate, sodium carbonate and potassium carbonate, sodium acetate, sodium dihydrogen phosphate etc., present in an amount at least sufficient to keep pH between about pH6 and pH8, and preferably, between about pH7 and pH7.5.

[0121] suitable tonicity agent is Gentran 40, macrodex, glucose, glycerol, potassium chloride, propylene glycol, sodium chloride etc., and the sodium chloride equivalent of feasible eye solution is in 0.9 ± 0.2% scope.

[0122] suitable antioxidant and stabilizing agent comprise sodium sulfite, sodium pyrosulfite, sodium thiosulfite, thiourea etc.Suitable wetting agent and clarifier comprise polysorbate80, polysorbate20, poloxamer 282 and tyloxapol (tyloxapol).Suitable viscosifier comprise Gentran 40, macrodex, gelatin, glycerol, hydroxyethyl-cellulose, hydroxymethyl-propyl cellulose, lanoline, methylcellulose, Dormant oils, Polyethylene Glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose etc.

[0123] uses viscosity intensifier so that provide greater than the viscosity of simple solution viscosity and can expect,, or be increased in the retention time of eye with the ocular absorption of increase target tissue to reactive compound as topical composition.Such viscosity synergist (viscosity building agent) for example comprises, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropyl emthylcellulose, hydroxyethyl-cellulose, carboxymethyl cellulose, hydroxypropyl cellulose or other material well known by persons skilled in the art.These materials are typically to be used on 0.01% to 2% the amount by weight.

[0124] compositions as herein described also can comprise other reagent, excipient or stabilizing agent, to improve the character of described compositions.For example, increase stability by the negative zeta potential that improves nano-particle, some electronegative component can be added.These electronegative components comprise, but be not limited to the cholate of cholic acid, described cholic acid comprises glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholate, Taurochenodeoxycholic Acid, lithocholic acid, ursodesoxycholic acid, dehydrocholic acid and other cholic acid; Phospholipid; comprise the phospholipid of lecithin (egg yolk) base, it comprises following phosphatidylcholine: palmityl oleoyl phosphatidylcholine, the inferior oleoyl phosphatidylcholine of palmityl, the inferior oleoyl phosphatidylcholine of hard ester acyl, hard ester acyl oleoyl phosphatidylcholine, hard ester acyl Semen arachidis hypogaeae phosphatidyl choline (stearoylarachidoylphosphatidylcholine) and dipalmitoyl phosphatidyl choline.Other phospholipid comprises L-α-dimyristoyl phosphatidyl choline (DMPC), dioleoyl phospholipid phatidylcholine (DOPC), two hard ester phosphatidyl cholines (DSPC), oil with hydrogenated soybean phosphatidylcholine (HSPC) and other related compound.Electronegative surfactant or emulsifying agent also are suitable as additive, as cholesteryl sodium sulfate etc.

[0125] also provides the unit dosage forms of colchicine or muscoril dimer compositions, for example, contain the unit dose that the 0.1mg that has an appointment arrives about 50mg, comprise that for example about 0.2mg arrives about 20mg or about 15mg colchicine or muscoril dimer to about 50mg, about 0.5mg to about 30mg, about 1mg.Term " unit dosage forms " refers to and is suitable as the physically separated unit that is used for individual single dose, and each unit contains and the bonded active substance that can produce the scheduled volume of expectation therapeutic effect as calculated of suitable pharmaceutical carrier, diluent or excipient.These unit dosage forms can single or multiple unit dose be stored in the suitable packing, and also can further be sterilized and seal.In some embodiments, provide the unit dosage forms of colchicine or muscoril dimer and carrier protein, its suitable intraocular injection.In some embodiments, provide the unit dosage forms of colchicine or muscoril dimer and carrier protein, it is fit to be applied topically to eye.In some embodiments, provide the unit dosage forms of colchicine or muscoril dimer and carrier protein, its suitable intra-arterial injection.

[0126] also is provided at the goods that contain compositions described herein in the suitable package.The suitable package that is used for compositions described herein (as ophthalmic composition) is known in the art, and comprises for example bottle (vial) (as the bottle of sealing), tubule (vessel), ampoule, bottle, jar, flexible package (as polyester film or the plastic bag that seals) etc.These goods can further be sterilized and/or be sealed.

[0127] the present invention also provides the test kit that contains compositions described herein (or unit dosage forms and/or goods), and it can further comprise the described method for compositions description of use, usage as described herein.Test kit as herein described can further comprise other material of expecting from commercial and user's position, comprises other buffer agent, diluent, filter, syringe needle, syringe and has the description of the explanation that is used to implement any method described herein.For example, in some embodiments, described test kit comprises colchicine or muscoril dimer, carrier protein, is fit to the pharmaceutically acceptable carrier of intraocular injection, and one or more: buffer agent, diluent, filter, syringe needle, syringe and having is used to implement the description of the explanation of intraocular injection.In some embodiments, described pharmaceutical composition comprises colchicine or muscoril dimer, carrier protein, is fit to the pharmaceutically acceptable carrier of intra-arterial injection, and one or more: buffer agent, diluent, filter, syringe needle, syringe and have the description that is used to implement the intra-arterial injection explanation.

[0128] although foregoing invention, by setting forth and quite at length being described for example, is apparent that for those of ordinary skills that variation that some are little and change can be implemented for the clear purpose of understanding.Therefore, the scope that should not be construed as limiting the invention of described description and example.

Embodiment

The vitro cytotoxicity activity of embodiment 1.Nab-5404 and Nab-5676

[0129] cellular cytoxicity activity of Nab-5404 and Nab-5676 uses MX-1 breast carcinoma, HepG2 hepatoma, HT-29 colon carcinoma cell line and normal primary rat hepatocyte to carry out external assessment.Make Nab-5404 that cellular exposure increases in a series of concentration and Nab-567672 hour at 37 ℃.Survival rate uses Cell Titer Blue cell survival rate to detect (cell viability assay), and (Promega, Madison WI) analyze.(GraphPad, San Diego CA) utilize single-phase exponential damping formula to calculate IC50 to use Prism software.

[0130] Nab-5404 demonstrates the cellular cytoxicity activity of antagonism HepG2 hepatoma cells and primary rat hepatocyte, and IC50 is respectively 16 and 9 μ g/ml.Nab-5404 and Nab-5676 demonstrate medium cytotoxicity to the MX-l cell, and IC50 is respectively 43 and 54 μ g/ml.Nab-5404 and Nab-5676 antagonism HT-29 cell activity are low, and IC50 is respectively 1l0 and 149 μ g/ml.Nab-5676 demonstrates very little in HepG2 cell or primary rat hepatocyte or showed cell toxicity activity not.The result is summarized in table 1.

Table 1

Cell line	Nab-5404IC50(μg/ml)	Nab-5676IC50(μg/ml)
Cell line	Nab-5404IC50(μg/ml)	Nab-5676IC50(μg/ml)	MX-1 breast carcinoma	43	54
The HepG2 hepatoma	16	125	MX-1 breast carcinoma	43	54
The HepG2 hepatoma	16	125	The HT-29 colon cancer	110	149
Primary rat hepatocyte	9	Non-activity	The HT-29 colon cancer	110	149

The anti-microtubule activity of embodiment 2.Nab-5404 and Nab-5676

[0131] the active MX-1 breast cancer cell line that uses of the microtubule depolymerization of Nab-5404 and Nab-5676 is tested.The MX-1 cell is inoculated on coverslip and was handled 2 hours at 37 ℃ with the Nab-5404 of 0.01 to 100 μ g/ml concentration range or Nab-5676.Behind the incubation, cell is fixed and tubulin and actin is dyeed.Actin dyes with fluorescein-labeled virotoxins (phalloidin) tubulin with the dyeing of monoclonal anti tubulin antibody.The microtubule network uses ImagePro software (MediaCybernetics, Inc., Silver Spring MD) to observe and analyze.Nab-5404 and Nab-5676 go the IC50 of stabilizing active to use Prism software to microtubule, and (GraphPad, SanDiego CA) calculate.

[0132] Nab-5404 and Nab-5676 demonstrate effective microtubule depolymerization activity, and the IC50 of calculating is respectively 0.06 μ g/ml and 0.12 μ g/ml (Figure 1A and 1B).Even at the least concentration 0.6 μ g/ml of test, the microtubule network is being destroyed (Fig. 2 A-2C) with Nab-5404 or Nab-5676 incubation after 2 hours fully.On the contrary, the actin bundle is not subjected to Nab-5404 under any drug level or the influence of Nab-5676.

The anti-angiogenesis activity of embodiment 3.Nab-5404 and Nab-5676

[0133] for studying Nab-5404 and Nab-5676 effect to angiogenesis, described chemical compound uses TCS CellWorks AngioKit model (TCS CellWorks Ltd., BotolphClaydon, Buckingham UK) in detecting, blood capillary formation assesses.The AngioKit model uses the human endothelial cell of cultivating altogether with other human archeocyte.Endotheliocyte forms island at first in culture matrix, begin propagation subsequently and then enter the migration phase, migration interim they move through substrate, form the little tubular construction of wire.These couple together gradually, form little managed network, itself and capillary bed closely similar (at 9-11 days).Tubule is to Feng's von willebrand's factor, CD31 (PECAM-1) and ICAM-2 stained positive.

[0134] except Nab-5404 and Nab-5676, and known blood-vessels target agent combretastatin 4-phosphate ester (combretastatin 4-phosphate, CA4P), tested external anti-angiogenesis activity.TCS CellWorks AngioKit model is used according to the explanation of manufacturer.As indicated above, human endothelial cell and people's fibroblast are cultivated in 24 orifice plates altogether, and are exposed to Nab-5404, Nab-5676 or the CA4P of a series of concentration (0.01 to 100 μ g/ml).Incubation 11 or after 12 days is by fixed cell and use monoclonal antibody, two anti-conjugates and the painted substrate staining of anti-CD31 to observe tubule.The length of tubule uses the IC50 of ImagePro software analysis and every kind of chemical compound to use the Prism computed in software.

[0135] Nab-5404 and Nab-5676 suppress tubule formation and decompose established tubule, demonstrate anti-angiogenesis activity.In first test, cell was used described compositions-treated at the 1st day, and in the inhibition of dyeing in the 12nd day with the formation of analysis tubule.In second test, cell was handled at the 8th day and was dyeed at the 11st day, and in the 3rd test, cell was handled at the 11st day and dyeing in the 12nd day, this allowed to analyze the decomposition that has formed tubule.

[0136] Nab-5404 and Nab-5676 can both suppress newborn microvascular formation (Fig. 3 and 4).In second test, Nab-5404 and Nab-5676 can both suppress tubule formation and/or decompose established tubule (Fig. 5).Nab-5404 is decomposing aspect the established tubule than Nab-5676 more effective (Fig. 6,7 and 9).The IC50 of Nab-5404 is calculated as 0.002 μ g/ml, and it is that the Nab-5676 of 0.02 μ g/ml renders a service big 10 times than IC50.Nab-5404 is aspect the established tubule of decomposition the same with CA4P (IC50=0.003 μ g/ml) effective (Fig. 6,8 and 9).

The anti-tumor activity of embodiment 4.Nab-5404 and Nab-5676

[0137] anti-tumor activity of Nab-5404 and Nab-5676 is assessed in the xenotransplantation mouse model.In two cycles at the HT-29 colon cell tumor that has generated, body build-in test chemical compound, the period 1 is the low dosage scheme, then is the second high dose scheme.Irinotecan (Irinotecan) is used as the positive control in this research.Mice (n=10) is divided into 8 groups, and wherein Nab-5404 and Nab-5676 mice are accepted described chemical compound by intravenous injection.Period 1 comprises 0-14 days and used Nab-5404 or Nab-5676 in per 3 days, uses 4 dosage, and then second round, it comprises 15-30 days and used Nab-5404 or Nab-5676 in per 3 days, uses 4 dosage.Per 3 days of irinotecan is used by intravenous injection with the dosage of 60mg/kg, uses 4 dosage.Each group is shown in the table 2, and wherein the period 1 referred to 0-14 days and referred to second round 15-30 days.

[0138] table 2

Handle	Period	1 dosage	The period 1 scheme	Second round dosage	Second round scheme
Handle	Period	1 dosage	The period 1 scheme	Second round dosage	Second round scheme	Control vector
Irinotecan	60mg/kg	q3dx4				Control vector
Irinotecan	60mg/kg	q3dx4			Nab-5404	3.4mg/kg	q3dx4	40mg/kg	q3dx4
Nab-5404	2.5mg/kg	q3dx4	30mg/kg	q3dx4	Nab-5404	3.4mg/kg	q3dx4	40mg/kg	q3dx4
Nab-5404	2.5mg/kg	q3dx4	30mg/kg	q3dx4	Nab-5404	1.7mg/kg	q3dx4	20mg/kg	q3dx4
Nab-5676	3.4mg/kg	q3dx4	40mg/kg	q3dx4	Nab-5404	1.7mg/kg	q3dx4	20mg/kg	q3dx4
Nab-5676	3.4mg/kg	q3dx4	40mg/kg	q3dx4	Nab-5676	2.5mg/kg	q3dx4	30mg/kg	q3dx4

Nab-5676

1.7mg/kg

q3dx4

20mg/kg

q3dx4

[0139] shown in Figure 10 and 11, at lower dosage, Nab-5404 and Nab-5676 suppress tumor growth significantly, wherein 3.4,2.5 and the p value of the Nab-5404 of 1.7mg/kg be 0.02,0.007,0.001, and 3.4,2.5 and the p value of the Nab-5676 of 1.7mg/kg be 0.04,0.003 and 0.0004.Nab-5404 and the Nab-5676 anti-tumor activity under low dosage shows that these chemical compounds have anti-angiogenesis activity.At higher dosage, compare with Nab-5676, after handling with Nab-5404 significantly bigger tumour regression is arranged.Based on the data that lose weight, in this model, the MTD with Nab-5404 of remarkable tumour regression is about 30mg/kg.

[0140] combretastatin 4-phosphate ester (CA4P) also is given the mice that suffers from the HT-29 tumor in routine tests.Reach 900mm in tumor ³The time, handle mice (n=10) with the report MTD of 100mg/kg based on administration every day 4 dosage with carrier or CA4P.Find CA4P non-activity in this tumor model, do not have difference (Figure 12) with control vector.

[0141] although foregoing invention, by setting forth and quite at length being described for example, is apparent that for those of ordinary skills that variation that some are little and change can be implemented for the clear purpose of understanding.Therefore, the scope that should not be construed as limiting the invention of described description and example.

Claims

1. suppress the method that individual medium vessels generates, comprise that giving described individuality comprises the dimeric compositions of carrier protein and colchicine or muscoril, wherein said compositions is in the amount of effective inhibition angiogenesis, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in described individuality.

2. the method for non-tumprigenicity angiogenesis-associated diseases in the treatment individuality, comprise that giving described individuality comprises the dimeric compositions of carrier protein and colchicine or muscoril, the amount of wherein said compositions is the described non-tumprigenicity angiogenesis-associated diseases of treatment effectively.

3. method as claimed in claim 2, wherein said non-tumprigenicity angiogenesis-associated diseases is an oculopathy.

4. method as claimed in claim 3, wherein said oculopathy are age-related macular degeneration, diabetic retinopathy or neovascular glaucoma.

5. method as claimed in claim 2, wherein said non-tumprigenicity angiogenesis-associated diseases is a cardiovascular disease.

6. method as claimed in claim 5, wherein said cardiovascular disease are restenosis or atherosclerosis.

7. method as claimed in claim 2, wherein said non-tumprigenicity angiogenesis-associated diseases is rheumatic arthritis or psoriasis.

8. method as claimed in claim 2, the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in described individuality.

9. method for cancer in the treatment individuality, comprise that giving described individuality comprises the dimeric compositions of carrier protein and colchicine or muscoril, wherein said compositions is the amount of effectively treating cancer, and the quantity not sufficient of wherein said compositions is to induce significant cytotoxicity in described individuality.

10. method as claimed in claim 9, the dimeric amount of colchicine or muscoril described in the wherein said compositions is the about below 15% of described colchicine or the dimeric corresponding MTD of muscoril.

11. method as claimed in claim 10, wherein said cancer are solid tumor.

12. method as claimed in claim 11, wherein said cancer are metastatic solid tumors.

13. as each described method among the claim 1-12, wherein said colchicine or muscoril dimer are the chemical compounds of formula (II):

Wherein:

B ₁Be methoxyl group or methyl mercapto;

B ₂Be methoxyl group or methyl mercapto;

N is from 0 to 8 integer;

Y is CH ₂Group, or when n was 1, Y also can be the group of formula NH.

14. method as claimed in claim 13, wherein Y is CH ₂

15. method as claimed in claim 13, wherein n be 1 and Y be NH.

16. method as claimed in claim 13, wherein B ₁And B ₂It all is methyl mercapto.

17. method as claimed in claim 13, wherein said chemical compound are IDN5404.

18. method as claimed in claim 13, wherein said chemical compound are IDN5676.

19. as each described method among the claim 1-12, the dimeric amount of colchicine or muscoril described in the wherein said compositions is at about 15mg/m ²Below.

20. as each described method among the claim 1-12, wherein said compositions is used once weekly.

21. method as claimed in claim 20, wherein said compositions is used about more than 3 months without interruption.

22. as each described method among the claim 1-12, wherein said compositions does not contain surfactant substantially.

23. as each described method among the claim 1-12, wherein said carrier protein is an albumin.

24. method as claimed in claim 23, the dimeric ratio of wherein said albumin and colchicine or muscoril are about below 18: 1.

25. as each described method among the claim 1-12, wherein said compositions comprises and contains described carrier protein and described colchicine or the dimeric nano-particle of muscoril.

26. method as claimed in claim 25, wherein said carrier protein are albumin.

27. method as claimed in claim 26, wherein said albumin and described colchicine or the dimeric ratio of muscoril are about below 18: 1.

28. method as claimed in claim 25, wherein said nano-particle have 200nm or littler average diameter.

29. method as claimed in claim 25, wherein said compositions do not contain surfactant substantially.

30. as each described method among the claim 1-12, wherein said compositions is Nab-5404.

31. as each described method among the claim 1-12, wherein said individuality is the people.