CN101048160B - Purine and pyrimidine cdk inhibitors and their use for the treatment of autoimmune diseases - Google Patents

Purine and pyrimidine cdk inhibitors and their use for the treatment of autoimmune diseases Download PDF

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CN101048160B
CN101048160B CN2005800370898A CN200580037089A CN101048160B CN 101048160 B CN101048160 B CN 101048160B CN 2005800370898 A CN2005800370898 A CN 2005800370898A CN 200580037089 A CN200580037089 A CN 200580037089A CN 101048160 B CN101048160 B CN 101048160B
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medicine
purine
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mice
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CN101048160A (en
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阿里拉·贝尼格尼
卡拉·佐加
朱斯普·雷马齐
阿索斯·詹内拉-博拉多里
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Cyclacel Ltd
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Abstract

The present invention relates to the use of an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a disease associated with antinuclear antibodies, wherein the inhibitor of CDK2 and/or CDK7 and/or CDK9 or pharmaceutically acceptable salt thereof is administered in an amount sufficient to down-regulate the levels of antinuclear antibodies. A further aspect of the invention relates to a combination comprising an inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, and methylprednisolone, and its use in the treatment of diseases associated with antinuclear antibodies, such as SLE.

Description

Purine and pyrimidine cdk inhibitors and be used for treating the purposes of the medicine of autoimmune disease in preparation
The present invention relates to the method for a kind of treatment and antinuclear antibody diseases associated.More clearly but exclusively non-, the present invention relates to treat the LADA rheumatism, human system's property lupus erythematosus (SLE) for example, method, and pharmaceutical composition and combination thereof.
Background of invention
Immune purpose is that the protection body is avoided potentially harmful substance (antigen), for example microorganism, toxin, cancerous cell and from the external blood of another one people or species or the harm of tissue.Through immunoreation antigen is destroyed, wherein immunoreation comprises the generation of antibody (combine with antigen and make it easier ruined molecule) and the activation of lymphocyte (discerning and destroy special antigenic special leukocyte).
Incorrect when immunoreation, too much or when lacking, just disease of immune system can appear.Autoimmune disease be meant any not normal with function of immune system be the disease of characteristic, it causes immune system to produce the antibody of anti-autologous tissue.This is caused by allergy, and wherein immune system is to common negligible material, and just normal " self " body tissue reacts.
Under the normal condition, immune system can be distinguished " self " and " non-self " tissue.It is responsive to " self " histiocyte that some immune system cells (lymphocyte) can become, but these wrong lymphocytes receive other lymphocytic control (inhibition) usually.When this normal control procedure is upset,, perhaps normal body tissue makes when it no longer is regarded as " self ", just autoimmune disease can take place if changing.
Autoimmune disease typically can cause the body tissue of one or more types to be destroyed, the organs abnormality growth, and perhaps organ dysfunction changes.This disease can only influence organ or tissue's type, perhaps can influence a plurality of organ or tissues.The common organ and the tissue that influenced by autoimmune disease comprise blood constituent (like erythrocyte), blood vessel, connective tissue, endocrine gland (like thyroid or pancreas), organ (like kidney or liver), muscle, joint and skin.
The instance of autoimmune or the disease relevant with autoimmune comprises: chronic lymphocytic thyroiditis, pernicious anemia, Addison's disease, type i diabetes, rheumatoid arthritis, systemic lupus erythematosus (sle), dermatomyositis, xerodermosteosis, DILE, multiple sclerosis, myasthenia gravis, Reiter syndrome and Ge Leishi disease.
The symptom of autoimmune disease has a great difference according to disease type.Yet autoimmune disease usually is attended by nonspecific symptom, for example tired, feel dizzy, uncomfortable (nonspecific uncomfortable sensation), fever and low grade fever (low-grade temperature elevations).
Special autoimmune disease can cause certain organ or tissue to be destroyed; Cause the function of organ or tissue to reduce (for example in diabetes; The islet cells of pancreas is destroyed), and/or the size of organ or tissue increases (for example in grave disease, thyroid increases).Symptom is along with specific disorders and affected organ or tissue and a great difference is arranged.
Autoimmune can be controlled through immune system being carried out balance suppress.Its objective is the immunoreation of minimizing, and keep complete immunoreation microorganism and abnormal structure to normal body tissue.The clinical treatment autoimmune disease comprises that typically use reduces immunoreactive corticosteroid and immunosuppressant (comprising cyclophosphamide or azathioprine).Yet many treatments that can obtain at present are all with serious adverse.
The present invention attempts to be provided for treating the alternative medicine of autoimmune disease, and it ideally can mitigation symptoms and the control self-immunprocess, keeps the ability of antagonism disease simultaneously.More particularly, the present invention relates to treatment and antinuclear antibody diseases associated, particularly autoimmune disease, for example human system's property lupus erythematosus.The present invention also attempts to provide drug regimen and the compositions that is suitable for treating these diseases.
Summary of the invention
First aspect of the present invention relates to uses inhibitor or the acceptable salt of its medicine of CDK2 and/or CDK7 and/or CDK9 to prepare medicine; Be used for treatment and antinuclear antibody diseases associated, wherein the dosage of CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine is enough to reduce the level of antinuclear antibody.
Second aspect of the present invention relates to the method for treating with the antinuclear antibody diseases associated; Said method comprises that its consumption is enough to reduce the level of antinuclear antibody to the CDK2 of object administration doses and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine.
The third aspect of the invention relates to through the horizontal stretcher of antinuclear antibody and the method for antinuclear antibody diseases associated in the downward modulation subject; Said method comprises that its consumption is enough to reduce the level of antinuclear antibody to the CDK2 of said object administration doses and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine.
Fourth aspect of the present invention relates to through the horizontal stretcher of antinuclear antibody and the method for antinuclear antibody diseases associated in the downward modulation subject; Said method comprises administration CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine, thereby treats said disease.
The 5th aspect of the present invention relates to the method for antinuclear antibody level in the downward modulation subject; Said method comprises that its consumption is enough to reduce the level of antinuclear antibody to the CDK2 of said object administration doses and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine.
The 6th aspect of the present invention relates to the method for antinuclear antibody level in the downward modulation cell; Said method comprises that the CDK2 that makes said cell and doses and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine contact, and its consumption is enough to reduce the level of antinuclear antibody in the said cell.
The 7th aspect of the present invention relates to the pharmaceutical composition that is used to treat with the antinuclear antibody diseases associated; Said compositions comprises CDK2 and/or CDK7 and/or CDK9 inhibitor; Its consumption is enough to reduce the level of antinuclear antibody, and mixes with medicine acceptable diluent, excipient or carrier.
Eight aspect of the present invention relates to the combination that comprises CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone (methylprednisolone).
The 9th aspect of the present invention relates to a kind of pharmaceutical composition, and it comprises according to combination of the present invention and medicine acceptable carrier, diluent or excipient.
The tenth aspect of the present invention relates to a kind of drug products, and it comprises CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine and the prednisolone of combined preparation, and they are simultaneously, in order or separately use in treatment.
The of the present invention the tenth relates in one aspect to a kind of pharmaceutical composition, and it comprises:
CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine; And prednisolone; Mix with medicine acceptable diluent, excipient or carrier.
The 12 aspect of the present invention relates to a kind of according to combination of the present invention, is used to prepare the medicine that is used to treat with the antinuclear antibody diseases associated.
The 13 aspect of the present invention relates to the medicine that uses CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable processed with salt of its medicine to be ready for use on treatment and antinuclear antibody diseases associated, and wherein this medicine is used for and the prednisolone combination is used.
The 14 aspect of the present invention relates to uses the prednisolone preparation to be used to treat the medicine with the antinuclear antibody diseases associated, and wherein this medicine is used for and CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt combination of its medicine are used.
The 15 aspect of the present invention relates to uses CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine and prednisolone to prepare to be used to the medicine of treating with the antinuclear antibody diseases associated.
The 16 aspect of the present invention relates to the medicine that uses CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable processed with salt of its medicine to be ready for use on treatment and antinuclear antibody diseases associated, and wherein said treatment comprises simultaneously, in order or dividually to object administration CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone.
The 17 aspect of the present invention relates in the treatment target method with the antinuclear antibody diseases associated, and said method comprises to the object drug treatment can acceptable dose:
CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine; And prednisolone.
For easy reference, now will with suitable division header discuss of the present invention these with further aspect.Yet the teachings of each part needn't only limit to each concrete part.
Detailed Description Of The Invention
CDK2 and/or CDK7 and/or CDK9 inhibitor
The present invention relates to use one or more CDK2 and/or CDK7 and/or CDK9 inhibitor.For fear of query occurring, this inhibitor can be each a inhibitor among CDK2, CDK7 or the CDK9, or the inhibitor of its combination in any.
In a preferred embodiment, this inhibitor is the inhibitor of CDK2.
In a further preferred embodiment, this inhibitor is the inhibitor of CDK7.
In a further preferred embodiment, this inhibitor is the inhibitor of CDK9.
In a further preferred embodiment, this inhibitor is the inhibitor of CDK7 and CDK9.
The method that is used to detect the CDK vigor is known to those skilled in the art.Further details is listed in the additional embodiment part.
Preferably, inhibitor is for the IC of one or more CDK2, CDK7 or CDK9 50Value is less than 1mM, and is preferred less than 100 μ M, more preferably less than 50 μ M, be more preferably less than 25 μ M, be more preferably less than 10 μ M, be more preferably again less than 1 μ M or 0.5 or 0.1 μ M.
The suitable example of CDK2 and/or CDK7 and/or CDK9 inhibitor comprises purine derivative; For example at EP 0874847B (CNRS); WO 03/002565 (Cyclacel Limited), disclosed among WO04/016613 (Cyclacel Limited) and the WO 04/016612 (Cyclacel Limited); And pyrimidine derivatives, for example at WO 01/72745, WO 02/079193, and WO 03/029248, and WO04/043953 (all with Cyclacel Limited name) is disclosed.
In a preferred embodiment of the invention, the inhibitor of CDK2 and/or CDK7 and/or CDK9 is selected from following material and the acceptable salt of medicine thereof:
Figure G05837089820070429D000051
In an especially preferred embodiment, CDK2 and/or CDK7 and/or CDK9 inhibitor are selected in [5]-[8], [11] and [12] from chemical compound [1]-[2].
In another particularly preferred embodiment, CDK2 and/or CDK7 and/or CDK9 inhibitor are selected in [9] and [10] from chemical compound [4].
In another particularly preferred embodiment, CDK2 and/or CDK7 and/or CDK9 inhibitor are chemical compound [7].
So far, still there is not hint to show that CDK2 and/or CDK7 and/or CDK9 inhibitor can reduce the level of antinuclear antibody effectively.Also have no technically to instruct or hint and show that these inhibitor can use with the prednisolone combined therapy, are used to treat autoimmune disease.
In a preferred embodiment, the inhibitor of CDK2 and/or CDK7 and/or CDK9 is selected from roscovitine, olomoucine and purvalanol A.
In the embodiment that the present invention is more preferably, CDK2 and/or CDK7 and/or CDK9 inhibitor are roscovitine.
Roscovitine or 2-[(1-ethyl-2 ethoxy) amino]-6-benzyl amino-9-isopropyl purine also is known as 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine.As used herein, term " roscovitine " comprises dissolved R and S enantiomer, its mixture and racemate thereof.
As used herein, term " CYC202 " is meant the R enantiomer of roscovitine, i.e. 2-(1-R-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine, and structure is as follows.
Figure G05837089820070429D000061
The external activity of roscovitine is following:
Kinases IC 50(μM)
The Cdk1/ cell periodic protein B 2.7
Cdk2/ cyclin A 0.7
Cdk2/ cyclin E 0.1
Cdk7/ cyclin H 0.5
Cdk9/ cyclin T1 0.8
The Cdk4/ cyclin D1 14.2
ERK-2 1.2
PKA ＞50
PKC ＞50
Although the existing in the art proof of using roscovitine treatment autoimmune disease up to now, does not claim that still it can reduce the proposal of antinuclear antibody level effectively.Also have no technically to instruct or hint and show that roscovitine can use with prednisolone, treats autoimmune disease in combined therapy.
For all embodiments of the present invention, the preferred form of roscovitine is the R enantiomer, i.e. 2-(1-R-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine, and hereinafter is called " CYC202 ".
Therapeutic activity
As stated; The present invention relates to use CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine to prepare medicine; Be used for treatment and antinuclear antibody diseases associated, wherein the dosage of CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine is enough to reduce the level of antinuclear antibody.
Antinuclear antibody (ANA) is uncommon antibody, in blood, can detect, and has and the bonded ability of endonuclear some structure.ANA can find immune system causes patient's body of inflammation easily to self body tissue in.Can guide the antibody of anti-autologous tissue to be known as autoantibody.The tendency of immune system antagonism self body is known as autoimmune.
The existence of antinuclear antibody is the sign of autoimmune disease.Antinuclear antibody comprises multiple antibody, and they are oriented to the maxicell complex that contains protein and nucleic acid compositions usually.The most recurrently be the composition of antinuclear antibody and DNA-albumen or RNA-albumen composition [the Van Venrooij W.J. etc. that react; Von Mulen C.A. etc.].Research shows that these autoantibodys are the IgG antibody of high-titer, high-affinity normally, and their generation depends on the T-cell, and receives host antigen to drive [Reichlin M. etc.].
Be defined as autoimmune disease for a kind of, disorganization must show and is because the adaptive immunity reaction of autoantigen caused.Autoimmune disease can receive the mediation of autoantibody and/or autoreactive T cell, and the reason of disorganization can be to the direct aggression with antigenic cell, the formation or the local inflammation of immune complex.The T cell can be directly related with inflammation or cytoclasis, and it is necessary that they still keep the autoantibody reaction.Similarly, the B cell possibly be the important antigen-presenting cell that keeps the reaction of autoantigen specific T-cells.
The bonded B cell of cd4 t cell selective activation and epitope, wherein this epitope and by the peptide physical connection of T cell recognition.Therefore, autoreactivity B cell and autoreactive T cell all are that the autoimmune relevant disease is necessary.
The generation of antinuclear antibody can only be studied in vivo, because these production of antibodies need the immune system of malfunction, comprises B and T cell and can not select and destroy the immunocyte of discerning self.
The external check of T cell function is to identify to have the suitable screening implement of under the complex situations of autoimmune disease, regulating the chemical compound of immunoreactive ability.A kind of check wherein is the check of T cell proliferation, and its further details provides in additional embodiment.In the autoimmune disease that relates to the antinuclear antibody generation, need normal T cell function, so that pass through the generation of B cytositimulation autoantibody.Therefore; The chemical compound (measurement of T cell function is the ability that the response immunologic stimulant is bred) that influences the T cell function can be through the ability of control T cell and B intercellular communication; And pass through to stop the T cell migration to the destructive position of autoimmune, and prevent the formation of autoantibody.
In this article, term " antinuclear antibody " comprises ADA, anti-RNA antibody simultaneously and directly is oriented to the antibody of anti-pyrenoids composition.Comprise LADA rheumatism and organ specificity autoimmune with the antinuclear antibody diseases associated.
Preferably, the LADA rheumatism is selected from drug-induced lupus, systemic lupus erythematosus (sle) (SLE) and rheumatic arthritis.
Rheumatic arthritis is a kind of chronic disease, and it comprises joint and/or other internal's film inflammation.Rheumatic arthritis is a kind of systemic disease, and it influences whole human body, is one of modal form of arthritis.It is characterized in that the synovium of joint inflammation causes pain, stiff, heating, rubescent and swelling.Skeleton and cartilage can invaded and destroy to inflamed joints film synovial membrane.Inflammatory cells discharges the enzyme that can digest skeleton and cartilage.The shape in related joint and aligning (alignment) can be lax, cause pain and LOM.Symptom typically comprises joint inflammation, swelling, dyskinesia and pain.Other symptom comprises that appetite decline, fever, energy descend and anemia.
Up to now, Therapeutic Method concentrates on ease the pain, reduce inflammation, stop or slowing down joint injury and raising patient's function and comfort level.Medicine can be divided into two types: the medicine (for example NSAIDS, aspirin, analgesic and corticosteroid) of (i) suiting the medicine to the illness, and it helps to reduce arthralgia, stiff and swelling; (ii) alleviate the antirheumatic of disease, for example the methotrexate of low dosage, leflunomide, Beracilline, sulfasalazine, aurotherapy, minocycline, imuran, hydroxychloroquine (with other antimalarial), ciclosporin and biological agent.
More preferably, the LADA rheumatism is systemic lupus erythematosus (sle) (SLE).
Systemic lupus erythematosus (sle)
In a preferred embodiment, the present invention relates to therapy system property lupus erythematosus and (be also referred to as lupus erythematosus disseminatus; SLE; Lupus; Lupus erythematosus), it is a kind of chronic inflammatory autoimmune disease, influences many tracts, comprises skin, joint and internal organs.Although suffer from the people of this disease multiple different symptom can be arranged, that more modal symptoms comprise is extremely tired, arthralgia or swelling (arthritis), unaccountable fever, erythra and kidney problems.The method of still not treating SLE at present.
This disease is 9 times of the man to Ms's influence, and seems more general in African crowd.It can take place at any age, but in the crowd of the 10-50 age bracket of being everlasting, occurred.SLE can also be caused by some drugs.When drug-induced, be called drug induced lupus erythematosus, it is reversible after medicine stops usually.
This sick process can be from slight sporadic disease to serious mortality disease.In a particular individual, along with the time, its symptom also has a great difference, it is characterized by the phase of taking a favorable turn and deterioration phase.The more modal symptoms of lupus comprise arthralgia or swelling (arthritis), unaccountable fever and extremely tired.Other symptom of lupus comprise mastalgia, alopecia, anemia (erythrocyte reduction), oral ulcer and cold and stress situation under finger and toe become ash or purpling.Some also can experience headache, feel dizzy, depressed, mental disorder or epilepsy.After initial diagnostic, may continue to occur new symptom for many years, and different symptoms can occur in different time.When beginning, have only an organ to be involved.Other organ also can be involved afterwards.
The characteristics of lupus are to have the numerous disease cycle, are called burst period and good phase, or the catabasis.Typically, in case diagnosis has SLE, the doctor will formulate a treatment plan according to patient's age, sex, health status, symptom and life style.In formulating treatment plan, the doctor has a plurality of purposes: prevent burst, when taking place, treat and organ is destroyed and complication minimizes.
Up to now, a large amount of different Therapeutic Method that are used for SLE patient are arranged.For people, often use the medicine that is known as NSAID (NSAID) to reduce inflammation with joint or mastalgia or fever.The common adverse effect of NSAID can comprise gastrointestinal upset, heartburn, diarrhoea and fluid retention.Some patients also produce liver, kidney, or even neural complication.
Antimalarial is the medicine that another kind is usually used in treating lupus.Although they generally are used for treatment fatigue, arthralgia, erythra and pneumonia, clinical research finds, continues to treat with antimalarial to prevent the generation again that happens suddenly.The eyes retina damage that the side effect of antimalarial can comprise stomach discomfort and seldom see.
The main Therapeutic Method of lupus comprises use corticosteroid hormone.Corticosteroid through rapid inflammation-inhibiting onset can per os, be applied to the emulsifiable paste of skin or pass through drug administration by injection.The short-term side-effects of corticosteroid comprises that swelling, appetite increase and weight increase.These side effect generally disappear after drug withdrawal.Yet, can frequent existence stop using the danger of corticosteroid suddenly like this, so after the long period, need let the patient break off using them.The long-term side-effects of corticosteroid comprises skin lineae ablicantes (stretchmark), skeleton is fragile or destroy (osteoporosis and osteonecrosis), hypertension, arterial injury, hyperglycemia (diabetes), infection and cataract.Typically, the dosage of taking is high more, the time is long more, and the danger of side effect and the order of severity are big more.
Receive the patient that lupus influences for kidney or central nervous system, can use immunosuppressant.Immunosuppressant suppresses the immune system of overacfivity through the generation of blocking immunity cell.That side effect can comprise is nauseating, the danger of vomiting, alopecia, bladder disease, fertility decline and cancer and infection increases.The danger of side effect increases along with the length of treatment.Identical with other method medicine that is used to treat lupus, after the immunosuppressant of stopping using, there is the danger of recurring.
The invention provides a kind of SLE of being used for and the rheumatismal alternative medicine of other LADAs.Especially, the object of the present invention is to provide the replacement therapy of immunosuppressant and steroid, it has excellent therapeutic effect in the acute stage of disease at least, and simultaneously main long-term side-effects is less.
Research in the body
In the mice body, study, investigate the curative effect of CYC202 and CYC202/ prednisolone combination.
The spontaneous generation of NZB/W F1 hybridize mice meeting shows the serious autoimmune disease of human system's property lupus erythematosus.The formation evidence of this disease is to form early stage antinuclear antibody, occurs developing into renal insufficiency with albuminuretic immune complex glomerulonephritis with after after a while, and this also can cause mice to die young.The most outstanding disease possibly be T and B cell dysfunction, and with the generation of the autoantibody of anti-nuclear and endogenous antigen (comprising nucleosome, DNA and histone complex).
In vivo, nucleosome produces through apoptosis, and as if in lupus patient body, apoptotic process is interfered.Under the inadequate situation of apoptotic cell clearance, nucleosome plays the effect of autoantigen, drives the immunoreation that the T cell relies on, and the key component of this reaction is the special antibody of nucleosome and nucleosome/IgG complex.These materials combine with the proper constituent of GBM, and promote inflammation.Kidney is inherent can to produce too much cytokine and chemoattractant with the cell that infiltrates, and amplifies and the injury perennity of immune complex mediation is gone down.
The histology is last, and the change of NZB/W mice body internal glomerulus comprises and the outer relevant too much disease of blood capillary inner cell of hypertrophy of focus property (focal) blood capillary; In mesangium and on the endothelium downside of GBM (GBM), can detect the immunologic pattern precipitate.Injury of renal tubular, interstitial inflammation and fibrosis are all more serious.
More evidence shows; Just (kinases that cyclin and cyclin rely on) and negative (inhibitors of kinases that cyclin relies on) cyclin is in regulating the reaction of cell to immunity and non-immune form injury; Comprise cell proliferation and apoptosis, have crucial effects.The propagation of intrinsic messangial cell (for example mesangial cell) is to immune-mediated glomerular injury, the for example characteristic of IgA nephropathy, lupus and membranoprolifer ative glomerulonephritis reaction.In the experimental membrano proliferative glomerulonephritis (Thy nephritis) that with mesangial cell propagation is characteristic, the expression of positive cyclin (cyclin D, E, A) and CDK2 protein level increase with active.In this model, suppress CDK2 and can reduce the propagation of mesangial cell and the accumulation and the raising kidney function of stromatin.
Research shows in the applicant's the body, significantly is longer than the mice (P＜0.05) with vehicle treated since 2 monthly ages with 200 with the time-to-live of the NZB/W F1 mice of the CYC202 processing of 100mg/kg dosage.When research finishes (8 months), have only 4/13 (31%) survival with the NZB/W mice of vehicle treated, then have 10/13 (77%) and 10/14 (71%) to survive with 200 respectively with the mice of 100mg/kg CYC202 processing.In the mice group of handling with CYC202 (100mg/kg) since 5 monthly ages, the mice of survival rate and vehicle treated does not have difference.
In a preferred embodiment of the invention, the consumption of CDK2 and/or CDK7 and/or CDK9 inhibitor is enough to postpone the time of origin of albuminuria and renal dysfunction.
In all experimental grouies, has the accumulative perception of the mice of severe albuminuria (＞4mg/ days) in the different phase assessment of disease.In vehicle group, the percentage ratio with albuminuretic mice is along with the time increases gradually.When research finished, the percentage ratio of albuminuria mice was 85%.Compare with carrier, 2 monthly ages begin to prevent administration CYC202 can with dose-dependent mode significantly postpone albuminuretic generation (% albuminuria mice, 8 months: 200mg/kg, 23%, compare P＜0.01 with carrier; 100mg/kg, 43%, compare P＜0.05 with carrier).When since 5 monthly ages during, can see comparing to have the trend that reduces albuminuria mice percentage ratio, but not reach the significant degree of statistics with carrier to lupus mice administration CYC202.
At 5 months and 8 months, estimate renal function through serum blood urea nitrogen (BUN).In the time of 5 months, the serum BUN level of all experimental grouies be in normal range (＜29mg/dl).In vehicle group, renal function was degenerated along with the time, in the time of 8 months, and the BUN level >=30mg/dl of 50% survival mice.Prevent administration CYC202 can make the renal function of lupus mice better since 2 monthly ages, but when since 5 monthly ages administrations, then do not have effect.
In a preferred embodiment of the invention, the consumption of CDK2 and/or CDK7 and/or CDK9 inhibitor is enough to reduce antinuclear antibody, especially the level of ADA.
The increase of ADA level is the characteristic of NZB/W F1 mice.The ADA level that gives the mice of carrier shows to be increased along with the prolongation of time.The mice of handling with CYC202 since 2 monthly ages, under two dosage, when 5 or 8 monthly ages, the level of its ADA significantly is lower than carrier.In accept the mice group of CYC202 since 5 monthly ages, in the time of 8 months, the concentration of ADA numerically is lower than vehicle group, but not remarkable.
In a preferred embodiment, the consumption of CDK2 and/or CDK7 and/or CDK9 inhibitor is enough to reduce the change of matter property between glomerule and renal tubules (tubointerstitial).
When research finished, the NZB/W mice that gives carrier showed glomerule and changes, and with the outer relevant too much disease of blood capillary inner cell of hypertrophy of focus property blood capillary.In mesangium He on the GBM endothelium downside, can detect immunoprecipitate.Can also observe injury of renal tubular and interstitial inflammation.Handle with CYC202 since 2 monthly ages and can significantly limit matter property damage between the too much disease of messangial cell, immunoprecipitate and renal tubules.When with the dosed administration CYC202 of 200mg/kg, these effects are more obvious.In the mice of trying administration CYC202 since 5 monthly ages, only observe the kidney form is had slight effect.
In a preferred embodiment, the consumption of CDK2 and/or CDK7 and/or CDK9 inhibitor is enough to suppress the T cell proliferation that brought out by PMA and ConA.
In a preferred embodiment, the consumption of CDK2 and/or CDK7 and/or the CDK9 inhibitor expression that is enough to reduce Mcl-1.
In a preferred embodiment, the consumption of CDK2 and/or CDK7 and/or CDK9 inhibitor is enough to reduce monocytic matter property accumulation.
Through immunohistochemistry technique the positive monocyte/macrophage of F4/80 of kidney is analyzed.Between the kidney of the NZB/W mice that gives carrier, there is tangible F4/80 positive cell accumulation in the matter.Carry out Prevention Processing since 2 monthly ages with 200mg/kg CYC202 and compare, can significantly reduce the number of the positive monocyte/macrophage of F4/80 with vehicle treated.The CYC202 of 100mg/kg dosage can limit mononuclear cell in a matter property accumulation, but does not reach the statistics significance degree.(since the processing of 5 monthly ages) can be observed the F4/80 positive cell and quantitatively decrease in treatment research.
Generally speaking, the result of this research clearly illustrates that, carries out prophylactic treatment with CYC202 (200 and 100mg/kg) since 2 monthly ages, compares with the animal that gives carrier, can stop the kidney performance of NZB/W mice lupus, and the significant prolongation life-span.Particularly, CYC202 can postpone the generation of albuminuria and renal function damage, and matter sexually revises between restriction glomerule and renal tubules, comprises monocytic matter property accumulation, the too much disease of renal tubular cell and immunoprecipitation.This effect is more remarkable under the dosage of 200mg/kg.One of this research find significantly to be that CYC202 has reduced the level of ADA, this possibly give the credit to CYC202 influences the T cell, itself so that influence the B cell.Through experiment in vitro, after handling, can be observed being the concentration dependent inhibition with inductive T hyperplasia of ConA and mixed lymphocyte reaction by PMA with CYC202.And; Evidence show and activatedly in SLE can offer help and (see summary Rekvig for the B cell differentiation becomes anti-DNA to generate plasma cell the special autoimmune t helper cell of histone and nucleosome; Arthritis & Rheumatism 48:300-312,2003).
CYC202 is also known influential to transcribing, and comprises downward modulation anti-apoptotic proteins mcl-1, therefore can change the balance of cells survival signal.Lacking immunoreactive T cell maybe be responsive especially to this exciting mechanism.
Compare with the carrier mice, can make percentage rate of albuminuria mice and kidney injury percentage rate that slight reduction is arranged since 5 monthly ages administration CYC202 (100mg/kg).Survival rate does not improve.This appropriateness effect possibly be because dosage is lower.
Combination
In an especially preferred embodiment, this medicine is used for carrying out combined therapy with prednisolone.
In this article; Term " combined therapy " is meant that prednisolone is with CDK2 and/or CDK7 and/or the administration of CDK9 inhibitor; They then are in identical time range if not the while administration, administration in order when the both can bring into play therapeutical effect.
Another aspect of the present invention relates to the combination that comprises CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone.
Preferably, this combination has synergism, and just, this combination is synergistic.
Prednisolone is the corticosteroid of a kind of synthetic (manual work).Chemical name is 11,17,21-trihydroxy-6-methyl-(6a, 11b)-pregnane-1,4-diene-3,20-diketone.Corticosteroid is the chemical agent of natural generation, is produced by the adrenal gland adjacent with kidney.Corticosteroid blocking-up inflammation, and be widely used in various inflammation diseases.A large amount of corticosteroid preparations has been arranged, comprised oral tablets, capsule, liquid, topical ointment and gel, inhalant, eye drop and injectable and intravenous fluid.Prednisolone is typically write out a prescription as oral tablets or liquid.
Prednisolone is used to promote the inhibition to inflammation.Can use the instance of the inflammation condition of prednisolone to comprise rheumatic arthritis, systemic lupus erythematosus (sle), acute gouty arthritis, psoriatic arthritis, ulcerative colitis and Crohn disease.The serious allergic symptom that conventional therapy is invalid also can use prednisolone.Instance comprises bronchial asthma, allergic rhinitis, drug-induced dermatitis and contact and atoipc dermatitis.The chronic dermatosis of available prednisolone treatment comprises dermatitis herpetiformis, pemphigus, severe psoriasis and severe seborrheic dermatitis.The chronic allergic of eyes tunica uvea, iris, conjunctiva and optic nerve and inflammation also can be treated with prednisolone.
Relate to a kind of pharmaceutical composition on the other hand, it comprises according to combination of the present invention and medicine acceptable carrier, diluent or excipient.
Another aspect of the present invention relates to a kind of drug products, and it comprises CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone, and they are used in simultaneously, in order or separately use of treatment as combination preparation.
CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone can be simultaneously, in combination, in order or separately (as the part of dosage regimen) administration.
In this article, the meaning of " simultaneously " is, two kinds of medicament administrations simultaneously, and the meaning of term " in combination " is meant if not the while administration, then " in order " administration when both all can bring into play curative effect in identical time range.Therefore, " in order " administration can allow the administration in 5 minutes, 10 minutes or several hours after another kind of medicament administration of a kind of medicament, and condition is that the circulating half-life of the medicament of first administration makes two kinds of medicaments to be on the dose therapeutically effective simultaneously.Time delay between each composition administration can be according to the definite character of composition, interaction between them with their separately half-life and different.
Different with " in combination " or " in order "; The meaning of " dividually " is meant among this paper; The dosing interval of a kind of medicament and another kind of medicament is significant; That is to say that when using second kind of medicament, the amount of the medicament of first administration in blood flow no longer is in effective dosage range in the treatment.
Of the present inventionly relate in one aspect to a kind of pharmaceutical composition in addition, it comprises:
(i) CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine; With
(ii) prednisolone;
Mix with medicine acceptable carrier, diluent or excipient.
Further the aspect relates to according to combination of the present invention, and its preparation is used to treat the medicine with the antinuclear antibody diseases associated.
The further aspect of the present invention relates to the medicine that uses CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable processed with salt of its medicine to be ready for use on treatment and antinuclear antibody diseases associated, wherein this medicine and prednisolone combination use.
The present invention relates on the other hand and uses the prednisolone preparation to be used to treat the medicine with the antinuclear antibody diseases associated, and wherein this medicine is used for and CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt combination of its medicine are used.
The further aspect of the present invention relates to uses CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone, preparation to be used to treat the medicine with the antinuclear antibody diseases associated.
Another aspect of the present invention relates to the medicine that uses CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable processed with salt of its medicine to be ready for use on treatment and antinuclear antibody diseases associated, and wherein said treatment comprises side by side, in order or dividually to object administration CDK2 and/or CDK7 and/or CDK9 inhibitor or acceptable salt of its medicine and prednisolone.
The further aspect of the present invention relates to the method for treating with the antinuclear antibody diseases associated, and said method comprises to the object drug treatment can acceptable dose:
(i) CDK2 and/or CDK7 and/or CDK9 inhibitor or the acceptable salt of its medicine; With
(ii) prednisolone.
For all above-mentioned embodiments, preferably, CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone are side by side or administration in order.
In a preferred embodiment, CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone administration simultaneously.
In an especially preferred embodiment, at first to object administration CDK2 and/or CDK7 and/or CDK9 inhibitor, afterwards to said object administration prednisolone in order or dividually.
Another aspect of the present invention relates to the method for treating proliferative disease, and it comprises CDK2 and/or CDK7 and/or CDK9 inhibitor, the prednisolone of administration dose therapeutically effective afterwards of administration dose therapeutically effective in order.
Another aspect of the present invention relates to the medicine that use CDK2 and/or CDK7 and/or the manufacturing of CDK9 inhibitor are used to treat proliferative disease; It comprises CDK2 and/or CDK7 and/or CDK9 inhibitor, the prednisolone of administration dose therapeutically effective subsequently of administration dose therapeutically effective in order.
In a selectable preferred embodiment, at first to object administration prednisolone, subsequently in order or dividually to said object administration CDK2 and/or CDK7 and/or CDK9 inhibitor.
In an especially preferred embodiment, CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone administration in order.
In a preferred embodiment of the invention, CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone basis composition separately is with the dose therapeutically effective administration.
In a preferred embodiment of the invention, CDK2 and/or CDK7 and/or CDK9 inhibitor and prednisolone basis composition separately is with inferior therapeutic dose administration.
That preferably, describes in CDK2, CDK7 or CDK9 inhibitor and the preceding text first aspect present invention is identical.
Pharmaceutical composition
As stated, various aspects of the present invention all relate to pharmaceutical composition.
Even chemical compound of the present invention (comprising the acceptable salt of its medicine, ester and medicine acceptable solvent thing) can be individually dosed, but usually all be and pharmaceutical carrier, excipient or mixing diluents administration, especially when being used for the human treatment.Pharmaceutical composition can be people or beastly consumption in human and beasts medicine.
The example that is used for the suitable vehicle of the various multi-form pharmaceutical compositions that this paper explains can find in " handbook of pharmaceutical excipients " second edition (1994) that Wade and PJ Weller write.
Treatment acceptable carrier or diluent are well-known at pharmaceutical field, and at for example Remington ' s Pharmaceutical Sciences, there is explanation in Mack publishing company in (A.R.Gennaro writes, 1985).
The suitable carriers instance comprises lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol etc.The instance of suitable diluent comprises ethanol, G & W.
The selection of pharmaceutical carrier, excipient or diluent can be selected according to expection route of administration and standard pharmaceutical practice.Pharmaceutical composition can be perhaps to comprise carrier, excipient or diluent, any suitable bonding agent, lubricant, suspending agent, coating materials, cosolvent.
Suitably the instance of bonding agent comprises starch, gelatin, natural sugar such as glucose, Lactis Anhydrous, free-flow (free-flow) lactose, beta lactose, corn sweetener, natural and paragutta; For example arabic gum, tragacanth or sodium alginate, carboxymethyl cellulose and Polyethylene Glycol.
The instance of proper lubrication agent comprises enuatrol, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride etc.
Antiseptic, stabilizing agent, dyestuff and even fumet can be provided in pharmaceutical composition.The instance of antiseptic comprises sodium benzoate, sorbic acid and p-Hydroxybenzoate.Can also use antioxidant and suspending agent.
Salt/ester
The chemical compound that the present invention uses can salt or the acceptable salt of form, particularly medicine of ester or the form existence of ester.
The acceptable salt of the medicine of The compounds of this invention comprises that it adds the salt of acid or alkali.Summary about suitable pharmaceutical salts can be at Berge etc., J Pharm Sci, and 66,1-19 finds in (1977).The formation of salt can be used for example strong inorganic acid, and mineral acid for example is like sulphuric acid, phosphoric acid or hydrochloric acid; Use strong organic carboxyl acid, for example have the hydrocarbon carboxylic acids of 1-4 non-replacement or replacement (for example halo) carbon atom, like acetic acid; Use saturated or unsaturation dicarboxylic acids, for example oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, four benzoic acids (tetraphthalic); Use hydroxy carboxylic acid, for example ascorbic acid, glycolic, lactic acid, malic acid, tartaric acid or citric acid; Use aminoacid, for example aspartic acid or glutamic acid; Use benzoic acid; Or using the acid of organic sulfo group, (the C1-C4)-hydroxyl of for example non-replacement or replacement (for example by halogen)-or aryl-sulfonic acid is like methyl-or p-tolyl sulfonic acid.
The formation of ester can be passed through organic acid or alcohol/hydroxide, depends on esterified functional group.Organic acid comprises carboxylic acid, for example has the alkanoic acid of 1-12 carbon atom of replacement or non-replacement (for example by halogen), like acetic acid; Saturated or unsaturation dicarboxylic acids, for example oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, four phthalic acids (tetraphthalic); Hydroxy carboxylic acid, for example ascorbic acid, glycolic, lactic acid, malic acid, tartaric acid or citric acid; Aminoacid, for example aspartic acid or glutamic acid; Benzoic acid; Or organic sulfonic acid, (the C of for example non-replacement or replacement (for example by halogen) 1-C 4)-alkyl-sulfonic acid or aryl-sulfonic acid are like methanesulfonic acid or p-methylphenyl sulfonic acid.Suitable hydroxide comprises inorganic hydroxide, for example sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.Alcohol comprises the alkanol of 1-12 carbon atom with non-replacement or replacement (for example by halogen).
Enantiomer/isomer
In all aspects of the present invention of discussing in front, the present invention includes use, if suitable, the whole enantiomer and the isomer of related chemical compound.Those skilled in the art can identify the chemical compound of have optical property (one or more chiral carbon atom) or isomer characteristic.Corresponding enantiomer and/or isomer can separate through methods known in the art/prepare.
Solid and geometric isomer
The used chemical compound of the present invention can exist stereoisomer and/or geometric isomer, and for example they can have one or more asymmetric and/or geometric centers, therefore can have two or more solids and/or geometric format.The present invention expects all independent stereoisomers and the geometric isomer that uses these medicaments, and composition thereof.The term that uses in the claim comprises these forms, and prerequisite is that said form keeps suitable functional activity (although needn't have identical degree).
The present invention also comprises makes all suitable isotope types of with medicament or the acceptable salt of its medicine.A kind of isotope type or the acceptable salt of its medicine of medicament of the present invention are defined as, and have at least an atom to be had the same atoms number but the atomic weight atom replacement different with the atomic weight of in nature, finding usually.The isotopic example that can introduce medicament and the acceptable salt of its medicine comprises the isotope of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F with 36Cl.Some isotope type of medicament and the acceptable salt of medicine thereof, for example comprise have radiosiotope as 3H or 14The type of C, useful in medicine and/or the research of substrate tissue distribution.Tritium, just 3H, and carbon-14, just 14C, isotope are preferred especially, because their preparation and detections easily.Further, (promptly with isotope such as deuterium 2H) replacement can provide some treatment advantage, because they have bigger metabolic stability, for example increasing interior half-life of body or minimizing needs dosage, is preferred in some cases therefore.The appropriate isotope type of the enough suitable agent of the general ability of the isotope type of medicament of the present invention and the acceptable salt of medicine thereof prepares through the program of routine.
Solvate
The present invention also comprises the solvate form thereof of using The compounds of this invention.The term that uses in the claim comprises these forms.
Polymorph
The invention further relates to various crystal forms, polymorph form and moisture (not moisture) form of using The compounds of this invention.In pharmaceutical industries, confirm preferably, can isolate any form of chemical compound, perhaps from the solvent that uses when synthetic this chemical compound of preparation, isolate its any form through slight change purification process.
Prodrug
The present invention further comprises the prodrug forms that uses The compounds of this invention.This prodrug generally is the adorned chemical compounds of wherein one or more suitable groups, and this being modified at when people or mammal administration can be reversed.This reverse is normally realized by naturally occurring enzyme in the subject, although also might be with second kind of medicine of this prodrug administration so that carry out this reverse in vivo.The instance of this modification comprises ester (for example front explanation any), wherein reverses and can carry out through esterase etc.Other similar system is well-known for a person skilled in the art.
Administration
Ingredient of the present invention applicable to per os, per rectum, transvaginal, outside intestinal, in the intramuscular, intraperitoneal, intra-arterial, sheath, in the bronchus, subcutaneous, intradermal, intravenous, per nasal, trans-oral or Sublingual administration.
For oral administration, use flat tablet, pill, tablet, gellules, drop and capsule especially.Preferably, the every dosage of these compositionss contains 1mg-5000mg, more preferably, contains the 10mg-3000mg active component.
Other form of medication comprises can be in intravenous, intra-arterial, sheath, subcutaneous, intradermal, intraperitoneal or intramuscular injection and with solution or Emulsion aseptic or that but germicidal solution prepares.Pharmaceutical composition of the present invention can be suppository, vaginal suppository, suspension, Emulsion, washing liquid, ointment, paste, gel, spray, solution or form of powder.
The another kind of approach of percutaneous dosing is to use patch.For example, active component can be incorporated in a kind of paste of being made up of Polyethylene Glycol aqueous emulsion or liquid paraffin.Active component can also 1-10% percentage by weight be incorporated into a kind of by in the basis set ointment that becomes of white beeswax or paraffinum molle alba with the stabilizing agent of necessity and antiseptic.
The every dosage of injectable form can contain 10-3000mg, preferably the 10-1000mg active component.
Compositions can be formulated for unit dosage form, just contains the discrete form of UD or many units or subunit dosage.
Dosage
Those of ordinary skill in the art can easily confirm the appropriate dose to one of object administration present compsn under not tested situation.Typically; The doctor will be according to various factors; Comprise activity, metabolic stability and the length of compound effects time, age, body weight, general health degree, sex, diet, mode of administration and time, drainage rate, the drug regimen of used particular compound, the order of severity and the individual human therapy experience of special circumstances, confirm to be suitable for most certain patient's actual dose.The dosage that this paper announces is the instance of average case.Independent case also can give higher or lower dosage certainly, and this also within the scope of the invention.
As required, dosage can be the 0.01-30mg/kg body weight, for example 0.1-10mg/kg body weight, more preferably 0.1-1mg/kg body weight.
In exemplary, use 10-3500mg/ days potion or multi-agent medicine to the patient.
The present invention is further explained with embodiment below with reference to accompanying drawing, wherein:
Fig. 1 has shown since 2 monthly ages or 5 monthly ages with 100 percentage survival NZB/W F1 mice handled with CYC202 with the dosage of 200mg/kg with the variation of handling the persistent period (moon).
Fig. 2 has shown that the albuminuria mice percentage ratio of NZB/W F1 mice being handled with CYC202 with the dosage of 100mg/kg and 200mg/kg is with the situation of change of handling the persistent period (moon).
Fig. 3 shown with the ADA (U/ml) in the 100 NZB/W F1 mice serums handled with CYC202 with the dosage of 200mg/kg (with the control vector group relatively).
Fig. 4 shown with and without the DMSO vehicle treated, do not stimulate and the BrdU of irriate PBMC and control cells mix (on) and survival ability (descending).
Fig. 5 has shown that reuse DMSO (upper left) or chemical compound are with 4 * IC after stimulating 48 hours with PHA or PMA/I 50, 3 * IC 50, 2 * IC 50And IC 50Concentration handle the PBMC cell, its before stimulating 2 hours (2h), stimulate in (0h) with stimulate the 2 hours afterwards (difference that+2h) BrdU mixes.
Fig. 6 has shown that reuse DMSO (upper left) or chemical compound are with 4 * IC after stimulating 48 hours with PHA or PMA/I 50, 3 * IC 50, 2 * IC 50And IC 50Concentration handle the PBMC cell, its before stimulating 2 hours (2h), stimulate in (0h) with stimulate 2 hours afterwards (+2h) survival ability poor.
Fig. 7 has shown the titration of concanavalin A, shows that maximal stimulus is 10 μ g/ml (upper left), and survival ability descends (upper right) under higher concentration, compares with stimulate (left side down) with PMA/I with PHA, and BrdU mixes increase.
Fig. 8 shown with and handled 48 and 72 hours without DMSO, with and the BradU of the PBMC cell that stimulates without PHA and ConA mix (on) and survival ability (descending).
Fig. 9 has shown with 4 * IC 50, 3 * IC 50, 2 * IC 50, IC 50, 0.5 * IC 50With 0.25 * IC 50Chemical compound and DMSO control treatment 48 and after 72 hours, receive mixing of BrdU in the PBMC that PHA (left side) and ConA (right side) stimulate.
Figure 10 has shown with 4 * IC 50, 3 * IC 50, 2 * IC 50, IC 50, 0.5 * IC 50With 0.25 * IC 50Chemical compound and DMSO control treatment 48 and after 72 hours, receive the survival ability of the PBMC that PHA (left side) and ConA (right side) stimulate.
Embodiment
CDK2 and/or CDK7 and/or CDK9 inhibitor
The preparation of various inhibitor compounds is that basis is at EP 0874847B (CNRS); And WO03/002565, WO 04/016613, and WO 04/016612, and WO 01/72745, and WO 02/079193, and WO 03/029248, the method for explanation among the WO 04/043953 (all with Cyclacel Limited name).
The kinases check
The check of kinase activity is through measuring radiophosphorus gets into suitable peptide substrate from ATP amount.Recombiant protein kinases and kinase complex are preparation or commercial the acquisitions.Use 96 orifice plates and suitable test buffer (25mM β-phosphoglycerol typically, 20mM MOPS, 5mM EGTA, 1mM DTT, 1mM Na 3VO 3, pH7.4) test, wherein in buffer, add 2-4 μ g organized enzyme and suitable substrate.Through adding Mg/ATP mixture (15mM MgCl 2+ 100 μ M ATP and 30-50kBq/ hole [γ- 32P]-ATP) initial action, and cultivate these mixture at 30 ℃ on request.In cessation reaction on ice, (Whatman Polyfiltronics, Kent UK) filter through p81 filter plate or GF/C filter plate subsequently.After the water-soluble orthophosphoric acid flushing of 75mM 3 times, plate is carried out drying, add scintillator, and in scintillation counter (TopCount, Packard Instruments, Pangbourne, Berks, UK) the middle radioactivity of mixing of measuring.The compound that will be used for the kinases check becomes the storage of 10mM at DMSO, in the check buffer, is diluted to 10%DMSO.Data analysis uses curve fitting software (GraphPad Prismversion 3.00 for windows, GraphPad Software, San Diego California USA) to confirm IC 50Value (inhibitory enzyme activity reaches the concentration of 50% test compound).
CDK7 and 9 checks
In the presence of not commensurability test compound, the cumulative volume in 96 hole titer plate is 20mM MOPS pH 7.2,25mM β-phosphoglycerol, 5mM EGTA, 1mM DTT, 1mM sodium vanadate, the 15mM MgCl of 25 μ l 2(contain trace with 100 μ M ATP 32P γ ATP) in, CTD peptide substrates (biotinyl-Ahx-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) 4-NH 21-2mg/ml) cultivated 45 minutes at 30 ℃ with recombined human CDK7/ cyclin H, CDK9/ cyclin T1 or CDK9/ cyclin K (0.5-2 μ g).Plate is placed on 2 minutes cessation reactions on ice.In each hole, add Avidin (50 μ g), plate was at room temperature cultivated 30 minutes.Sample transfer in 96 hole P81 screen plates, is washed (4 * 200 μ l/ hole) with 75mM phosphoric acid.Add Microscint 40 scintillation solutions (50 μ l) to each hole, with each sample of Packard Topcount microplate scintillation counter measurement 32The P incorporation.
CYC202
Study, whether YC202 can effectively slow down the nephropathy development that NZB/WF1 is prone to suffer from the lupus mice with assessment administration CDK2 inhibitor C.
Use when beginning experiment be 2 monthly ages the female Mus of NZBxNZW F1 (Harlan Italy s.r.l., Milano, Italy).The care of animal carries out according to Guide Book with handling; This Guide Book meets (Decreto Legislativo n.116, Gazzetta Ufficiale suppl 40,18 febbraio 1992 of country; Circolare n.8; Gazzetta Ufficiale 14 luglio 1994) and international regulations and policy (EECCouncil Directive 86/609, OJL358-1, in December, 1987; Management of laboratory animal and instruction, U.S.National Research Council, 1996).Animal feeding is in thermostatic chamber, and dark/12 hour illumination circulation in 12 hours gives standard diet.
Embodiment 1
NZBxNZW F1 mice is divided into following group at random:
The group 1 (n=18): every day the tube feed carrier mice (HCl 50mM);
The group 2 (n=17): every day tube feed CYC202 mice (200mg/kg);
The group 3 (n=19): every day tube feed CYC202 mice (100mg/kg);
Processing begins (Prevention Research) when 2 monthly ages, lasted till for 8 monthly ages; When 5 monthly ages, put to death 4-5 animal for every group, with the level of assessment serum BUN and circulation ADA.
The another one group is organized 4 (n=14 mices) since 5 tube feed CYC202 every day at monthly age (100mg/kg), and was lasted till for 8 monthly ages, wherein the actual immune complex deposition (treatment is studied) that takes place when 5 monthly ages.(Charles River Italia, Calco is Italy) as contrast for 5 normal CD-1 mices.
Following parameter is assessed:
Urinary protein excretion: measured once in every month, until 5 monthly ages, per then 2 weeks measure once.
During execution:
ADA in the serum;
Serum BUN;
Serum transaminase (AST, ALT);
Nephridial tissue;
Monocyte/macrophage is the accumulation in the matter between kidney.
Embodiment 2
The lupus mice is divided into following each group at random:
The group 1 (n=10): every day the tube feed carrier mice (HCl 50mM);
The group 2 (n=15): every day tube feed CYC202 mice, dosage is 200mg/kg;
The group 3 (n=12): every day the peritoneal injection prednisolone (MPS, Urbason, Hoechst s.p.a, Milano, mice Italy), dosage is 1.5mg/kg;
The mice of group 4 (n=16): administration every day CYC202 (200mg/kg) and MPS (1.5mg/kg) combination.
Processing is when 5 monthly ages, and actual generation of immune complex deposition this moment continued to for 12 monthly ages, accepted last animal dead of vehicle treated this moment.(CharlesRiver Italia, Calco is Italy) as contrast for 5 normal CD-1 mices.
Following parameter is assessed:
Survival rate;
Urinary protein excretion: measured once in every month, until 5 monthly ages, per then 2 weeks measure once.
Serum BUN: when 5 monthly ages, measure (before the processing), and measured once in every month, finish until research;
Nephridial tissue: be critically ill mice with the survival to 12 the monthly age mice biopsy in assess;
The accumulation in the matter between kidney of the positive monocyte/macrophage of F4/80 (with top identical biopsy in assess).
Embodiment 1 and 2: material and method
Albuminuria and renal function
Confirming of albuminuria concentration is to combine experiment through Coomassie brilliant blue G dyestuff, is standard with the bovine serum albumin.Renal function is through Reflotron test (Roche Diagnostics corporation, Indianapolis, USA) BUN in the evaluation heparinization blood.The BUN level that surpasses 30mg/dl is thought abnormal (this laboratory thinks that be 14-29mg/dl the normal range of mice).
ADA
(Milano Italy) assessed the level of the autoantibody of anti-dsDNA for Diastat anti-dsDNA test kit, Bouty Laboratory through the former enzyme immunoassay of describing (Kidney Int, 53:726-734,1998) in the serum.
Serum transaminase
(Fullerton CA) measures the serum levels of AST and ALT for CX5, Beckman Instruments Inc. with automatic analyzer.
Renomorphology
Optical microscope: the renal cortex fragment is fixing in Dubosq-Brazil, in ethanol, dewaters, and is embedded in the paraffin.Section (3 μ m) is dyeed with hematoxylin and eosin, Masson trichromic stain and PAS Albert'stain Albert reagent (PAS dyeing).The assessment of the too much disease of glomerular capillary inner cell is carried out with semiquantitative mode, and (0=does not have hypercellularity disease to the marking system of use 0 to 3+; 1+=is slight; 2+=is medium; 3+=is serious).Also a single mark is made a call in other change according to the percentage ratio of the whole damaged glomerule that involved.Blood capillary is outer outgrowthly to be classified as 0 to 3+ (0=not to have hypercellularity; 1+=is less than 25% glomerule and is involved; The glomerule of 2+=25%-50% is involved; 3+=is involved greater than 50% glomerule).Glomerule is sedimental to be classified as 0 to 3+ (0=not to have deposit; 1+=is less than 25% glomerule and is involved; The glomerule of 2+=25%-50% is involved; 3+=is involved greater than 50% glomerule).A renal tubules (atrophy, bending and expansion) and a matter sexually revise (fibrosis and inflammation) and are classified as 0 to 3+ (0=does not change; The 1+=variable effect is less than 25% of sample; The 2+=variable effect is the 25%-50% of sample; The 3+=variable effect is greater than 50% of sample).100 glomerule are checked in each biopsy at least.Each sample is checked 10 visual fields at least under low power (10x), be used for marking to stroma.All renal biopsy is analyzed with single blind mode by same pathologist.
Immunohistochemical analysis
Use anti-be present in the antigenic rat monoclonal antibody of kytoplasm in mouse monokaryon cell and the macrophage (F4/80,4 μ g/ml, Caltag Laboratories, Burlingame, CA), through immunoperoxidase procedure technology for detection infiltration cell.Section is with the 0.3%H in the methanol 2O 2Hatched 30 minutes, and made endogenous peroxide enzyme deactivation.Be organized in then among the 0.1%Triton X-100 that is dissolved in 0.01mol/L PBS (pH7.2) and permeated 30 minutes, use normal lowlenthal serum (Vector Laboratories) to hatch then 30 minutes.First antibody uses SA (the Mus IgG of the biotinylated goat Chinese People's Anti-Japanese Military and Political College, Vector Laboratories) and Avidin-biotin-peroxidase complex (ABC) solution to hatch 4 ℃ of incubated overnight afterwards, develops with DAB at last.Section is dyed with the Harris hematoxylin is counter.Negative control obtains through omitting first antibody.At least in 10 high power microscope visual fields of selecting at random (400x), count the cell of F40/80 labelling for each animal.
Statistical analysis
Data are represented with meansigma methods ± standard deviation (SE).Survival curve is analyzed through sequence check.The albuminuria data are analyzed with the Fisher Precision Test.Every other parameter is analyzed with Kruskall Wallis check.Statistically significant is defined as P＜0.05.
Embodiment 1: the result
Body weight and diet and drinking-water
As shown in table 1, lupus mice weight increase during studying.Body weight between each group of experiment is not observed difference.From the 2-5 month, per two weeks are measured the intake of food (table 2) and water (table 3), and between the mice of carrier and CYC202 processing, compare.
Lupus mice survival rate
Since 2 monthly ages with the 200 NZB/W F1 mices of handling with the CYC202 of 100mg/kg dosage, its time-to-live significantly (P＜0.05) be longer than carrier mice (seeing table 4 and Fig. 1).In fact, when research finishes (8 monthly age), four (31%) survival is only arranged, and 13 have only ten (77%) and 14 to have only ten (71%) to survive respectively with the mice of 100mg/kg CYC202 processing with 200 with 13 of the NZB/W mices of vehicle treated.Since the mice group of 5 monthly ages administration CYC202 (100mg/kg) (treatment administration), survival rate and vehicle treated mice do not have difference.
Albuminuria and renal function
The accumulative perception of all experimental group assessments being suffered from the mice of serious albuminuria (＞4mg/ days) in the different phase of disease.As shown in table 5, there is the percentage ratio of albuminuretic mice to increase (Fig. 2) in time gradually in the vehicle group.When research finished, the percentage ratio of albuminuria mice was 85%.Compare with carrier, prophylactic treatment administration CYC202 can with dose-dependent mode significantly postpone albuminuretic generation (% albuminuria mice, 8 monthly ages: 200mg/kg compares P＜0.01 with carrier; 100mg/kg compares P＜0.05 with carrier).When since 5 monthly ages during to lupus mice administration CYC202, compare with carrier, can observe the trend that albuminuria mice percentage ratio reduces, but it is remarkable not reach statistics.
Assess renal function at 5 and 8 monthly ages through serum BUN.When 5 monthly ages, the serum BUN level of all experimental grouies all in normal range (＜29mg/dl).In vehicle group, renal function worsens along with time lengthening, when 8 monthly ages, and the BUN level >=30mg/dl (table 6) of 50% survival mice.Prophylactic treatment administration CYC202 can make the renal function of lupus mice better, if administration does not afterwards then have effect.
ADA
The increase of ADA level is the characteristic of NZB/W F1 mice.As shown in table 7, the level that gives the mouse anti dna antibody of carrier increased along with the time.Since the mice that 2 monthly ages handled with CYC202,5 or 8 monthly ages showed all that the ADA level significantly was lower than carrier under two dosage.During at 8 monthly ages, ADA concentration numerically is lower than carrier in the mice group of accepting CYC202 since 5 monthly ages, but not significantly (Fig. 3).
The serum transaminase level
Measurement is since 2 monthly ages administration 200mg/kg CYC202 or since intravital Serum ALT of NZB/W F1 mice and the AST level of 5 monthly ages administration 100mg/kgCYC202.The serum transaminase level is handled and is changed, numerical value similar with control group mice (table 8).
Renomorphology
As shown in table 9, when research finished, the NZB/W mice that gives carrier showed the too much change of glomerular capillary inner cell, and with the outer hypertrophy of focus property blood capillary.Endothelium downside at mesangium and GBM can detect the immunologic pattern precipitate.Can also observe injury of renal tubular and interstitial inflammation.Handle with CYC202 since 2 monthly ages and can significantly limit matter property damage between the too much disease of messangial cell, immunoprecipitation and renal tubules.When the dosage of CYC202 was 200mg/kg, these effects were more remarkable.Giving since 5 monthly ages in the mice of CYC202, can be observed the kidney form is had only slight influence.
Matter property accumulation between Monocytes
Analyzed the positive Monocytes of F4/80 of kidney through immunohistochemistry technology.The F4/80 positive cell significantly accumulates (table 10) in the matter between the kidney of the NZB/W mice that gives carrier.Compare the number that can significantly reduce the positive Monocytes of F4/80 with carrier with 200mg/kgCYC202 prevention administration processing.Under the CYC202 of 100mg/kg dosage,, can limit monocytic matter property accumulation although not remarkable on the statistics.In treatment research, can observe the F4/80 positive cell and quantitatively decrease.
The result of this problem shows, prevents administration CYC202 (200 with 100mg/kg) to compare with the animal that gives carrier since 2 monthly ages, can postpone the generation of NZB/W mice lupus, and the significant prolongation life-span.Say that clearly CYC202 postpones the generation of albuminuria and renal dysfunction, matter sexually revises between restriction glomerule and renal tubules, comprise monocytic matter property accumulation, and effect is more obvious under the dosage of 200mg/kg.A great discovery of the present invention is, CYC202 can reduce the level of ADA, and this possibly be that it further possibly influence the B cell because CYC202 influences the T cell.Through experiment in vitro, can observe and can concentration dependent ground suppress by PMA and ConA is inductive and T cell proliferation in mixed lymphocyte reaction.On the other hand, obviously in SLE, histone that is activated or nucleosome specificity autoimmune T cell can help the B cell differentiation to become anti-DNA to generate plasma cell (summarizing referring to Rekvig Arthritis& Rheumatism 48:300-312,2003).
Compare with the carrier mice since 5 monthly ages administration CYC202 (100mg/kg), can slightly reduce the percentage ratio of albuminuria mice and injury of kidney mice.Survival rate is not seen improvement.
Embodiment 2: the result
Body weight
NZB/W F1 mice weight increase during studying.Body weight between each experimental group is not observed difference.
Survival rate
Since the NZB/W F1 mice of 5 monthly ages with CYC202 and prednisolone (MPS) combined treatment, its time-to-live, carrier mice (seeing table 11) was longer than in remarkable (P＜0.0001).It should be noted that when 12 monthly ages, and to have only ten (62%) survival with the mice 16 of combined therapy when all death of all drug administration carrier mices.Survival curve and the vehicle group of accepting the mice of single therapy do not have difference.
Albuminuria
Table 12 has shown the accumulative perception albuminuria＞4mg/ days mice of the different phase assessment of disease.CYC202 compares with vehicle group with the MPS drug combination can significantly postpone albuminuretic generation.In the interval at 7-10 monthly age, the ratio of albuminuria mice significantly is lower than vehicle group (6.2-43.8% is to 40-90%) in the combined therapy group.CYC202 or MPS compare with carrier as single therapy agent administration can only the albuminuretic generation of some effects, but when observing in 7.5 months, the CYC202 group can be seen albuminuria mice percentage ratio and significantly reduce.
Renal function
During 5 (before the processing)-Decembers, measure serum BUN in every month, assess renal function.Table 13 has shown the cumulative percentage of the mice of BUN level >=30mg/dl.When 5 monthly ages, the serum BUN level of all experimental grouies is in normal range (promptly 14 to 29mg/dl) all.In vehicle group, renal function worsens along with time lengthening.Therefore, when August and 12 monthly ages, the animal of BUN level >=30mg/dl is respectively 80% and 100%.Contrastively, the mice of combined therapy group has only 27% and 53% mice impaired renal function when these time points.The kidney protection effect of individually dosed CYC202 or MPS is weaker than composite reagent.
Renomorphology
The kidney sample that mice sacrificed is obtained during to the mice of being critically ill from different time or December carries out morphological analysis.Data are as shown in table 14.The NZB/W mice that gives carrier demonstrates the too much glomerule of blood capillary inner cell and changes, and with the outer hypertrophy of focus property blood capillary.Endothelium downside at mesangium and GBM can detect the immunologic pattern precipitate.Can also observe injury of renal tubular and interstitial inflammation.Adding MPS with CYC202 handles and can significantly limit matter property damage between the too much disease of messangial cell, immunoprecipitation and renal tubules.In the mice of individually dosed CYC202 or MPS, the kidney form had only minimal effect.
Matter property accumulation between Monocytes
Analyzed the positive Monocytes of F4/80 of kidney through immunohistochemistry technology.The F4/80 positive cell obviously accumulates (table 15) in the matter between the kidney of the NZB/W mice that gives carrier.CYC202 compares the decreased number 65% (p＜0.01) that can make Monocytes with the MPS combination medicine-feeding with carrier.Individually dosed CYC202 can make a matter property infiltrate reduce 33%.In the mice group of handling with MPS separately, monocytic matter property accumulation is similar with vehicle group.
In a word, the result shows the life-span that CYC202 and low dosage prednisolone composite reagent can significant prolongation lupus mices.It should be noted that this processing forms 5 monthly ages just in stage, actual generation of immune complex deposition this moment in disease.Combined therapy can postpone albuminuretic generation, and matter sexually revises between development, immunoprecipitation and the renal tubules of restriction renal dysfunction and the too much disease of messangial cell.Also demonstrate antiphlogistic effects simultaneously, reduce because mononuclear cell gets into the cumulant of matter between kidney.Individually dosed CYC202 or prednisolone have only faint kidney protection effect.
In a word, combination CYC202 and low dosage prednisolone have the therapeutic effect in the life-span of improving performance of lupus kidney and the ill lupus mice of prolongation, for the novel method of treatment of lupus nephritis provides the foundation.
Embodiment 3 and 4: material and method
Select T hyperplasia check as alternative check, and tested selected CDK2,7 and 9 inhibitor (transcription inhibitor), chemical compound [1]-[12] the antinuclear antibody effect.
As stated, the generation of antinuclear antibody is merely able to study in vivo, because these production of antibodies need the immune system of malfunction, comprises B and T cell simultaneously, and needs to select and to destroy the immunocyte of identification self.Yet external test T cell function (for example, T cell proliferating determining) is to identify the suitable screening implement that can under the complex environment of autoimmune disease, regulate immunoreactive chemical compound.Therefore, the T cell proliferating determining can be for providing measuring method to the antinuclear antibody compounds effective.
The preparation of PMBC
Embodiment 3 and embodiment 4 have carried out two independent checks.Obtained buffy coat blood from Scottish National BloodTransfusion Serices, and at room temperature, used BD Vacutainer from three or four healthy donors (being respectively instance 3 and instance 4) TM(4ml absorption amount contains sodium citrate to CPT cell preparation pipe, REF362781) separates in centrifugal 30 minutes with 300g.The recovery of PMBC (PBMC) layer is through drawing and collecting in the 50ml centrifuge tube, then at the Hank ' of 3 times of volumes s buffer salt solution (w/o CaCl 2And MgCl 2, Gibco#14175-053) in through 300g centrifugal 15 minutes (room temperature) washing 2 times.PBMC is resuspended in the RPMI1640 culture medium of having added 10% hyclone, and confirms the number of living cells through Trypan Blue exclusive method.
Cytositimulation and processing
With PBMC with 1 * 10 5Cells/well is seeded in 96 orifice plates, every hole 50 μ l culture medium.As the check contrast; CCRF-CEM leukemia and LP-1 boniness myeloma cell are seeded in 100 μ l/ hole RPMI, 1640 tissue culture medium (TCM)s/10% hyclone (FCS) with 4000 and 5000 cells/well respectively, and experimental session do not add stimulate or the situation of compound treatment under grow.All stimulants and chemical compound are all processed the ultimate density in the 4x tissue culture medium (TCM), in the hole, add every kind of medicine and stimulant 25 μ l then, and final volume is 100 μ l/ holes.For non-stimulation contrast, Xiang Kongzhong adds 25 μ l tissue culture medium (TCM)s, but not stimulant for non-processing cell, adds the tissue culture medium (TCM) that equal-volume DMSO (final 0.1%) is contained in 25 μ l/ holes.Under each stimulation/treatment conditions, handle 3 adjacent holes, and calculate meansigma methods analysis.
For embodiment 3; Make cell with 50 μ g/ml PHA (phytohemagglutinin PHA-P; Sigma L9132) or 50/250ng/ml PMA/I (phorbol 12-myristate 13-acetate/Ionomycin, Sigma P1839/I0634) stimulate before sedimentation 2 hours.In addition, using concentration is that the ConA (concanavalin A, Sigma C5275) of 0.5 μ g/ml-100 μ g/ml stimulates PBMC to identify its effect of stimulation and optium concentration.For instance 4, PBMC stimulates with 50 μ g/ml or 10 μ g/ml ConA after inoculation immediately.
For embodiment 3, irriate or unprovoked PBMC use concentration to be IC 50, 2 * IC 50, 3 * IC 50With 4 * IC 50Chemical compound [1]-[10] handle.Chemical compound is when stimulation 2 hours (during inoculation), stimulation before and stimulate interpolation in 2 hours afterwards, and cell is at 37 ℃, 5%CO then 2Cultivated 50,48 and 46 hours with chemical compound respectively down, perhaps until stimulating back 48 hours.For embodiment 4, with previous concentration, other is with 0.5 * IC with above-claimed cpd, chemical compound [11]+[12], all test compounds for the PBMC of irriate 50With 0.25 * IC 50Concentration handle.Chemical compound is interpolation immediately after stimulating, and cell culture 48 and 72 hours.According to 72 hours IC that on the tumor cell plate, record through indoor cytotoxicity check 50Meansigma methods obtains IC 50Value (table 16), but do not comprise chemical compound [1] and [3], its IC 50Acquisition be according to disclosed IC during before (in-house) used in laboratory 50, because do not obtain IC in the laboratory 50Data.
BrdU ELISA and Alamar Blue check
After stimulation and compound treatment, the proliferation activity of PBMC is through using cell proliferation ELISA, and BrdU (colorimetry) test kit (Roche#1 647 229) is measured mixing of BrdU and confirmed.This test kit is to be used for 3A kind of on-radiation alternative method that the H thymidine mixes.Before the collecting cell 2 hours, use the BrdU labeled cell.Then with 300g (room temperature) with centrifugal 10 minutes of plate, from the hole, remove supernatant through drawing, cell was 60 ℃ of dryings 1 hour.In plate, add FixDenat solution (100 μ l/ hole), cultivated 30 minutes, flick then and remove, in plate, add anti-BrdU-POD solution (100 μ l/ hole), and cultivated 90 minutes.With washing liquid flushing 3 times (200 μ l/ holes) afterwards, plate was cultivated 30 minutes with substrate solution (100 μ l/ hole), added 1M H then 2SO 4(25 μ l/ hole) reads out in the optical density of 450nm on the AscentFluoroskan plate reader.
Vigor with Alamar Blue reagent (Biosource#DAL1100) assessment PBMC.Prepared fresh 20%Alamar Blue in culture medium, every hole adds 100 μ l, and final concentration is 10%.Before acquisition time (48 or 72 hours), plate with Alamar Blue at 37 ℃, 5%CO 2The middle cultivation 3 hours read fluorescence at Tecan Ultra plate reader with 535nm excitation wavelength and 595nm emission wavelength then.
Embodiment 3: the result
Stimulate with irritation cell not with PHA and to compare, the BrdU incorporation is brought up to about 7 times.Do not influence that BrdU mixes or PHA stimulates or the vigor of stimulated cells (Fig. 4) not with the DMSO vehicle treated.As shown in Figure 5, handle the PHA stimulated cells with all test compounds and can reduce mixing of BrdU, be reduced to the not level of irritation cell in many cases.Except that chemical compound [9] and [10], be low to moderate IC 50Concentration under can significantly reduce the incorporation of BrdU, the incorporation of BrdU does not also have dependency with compound concentration, comprehensively shows, chemical compound is at IC 50The time reached its maximum proliferation inhibiting effect.For chemical compound [9] and [10], its effect is dose dependent significantly, at IC 50The time, many 3-4 BrdU incorporation doubly than other chemical compound and under higher concentration, just be reduced to the not level of irritation cell.
To most compounds, with respect to stimulation time, the mix level of the processing time of chemical compound to BrdU do not have very big influence, is likely because this stimulation is inoculated in back 4 hours at PBMC and also there is not the generation effect.And chemical compound [2], [3] and [4] are exceptions, and be wherein astonishing, and (BrdU 2h) mixes level and compound treatment and stimulates simultaneously or handle and after stimulation, compare minimizing less (Fig. 5) with the chemical compound pretreatment.
Similar with the PHA processing, when not having chemical compound, PMA/I stimulates makes not irritation cell high about 7 times (Fig. 5) of BrdU incorporation ratio.With other all compound treatment except that chemical compound [4], [9] and [10], under all concentration, the BrdU incorporation is reduced, even be superior to the situation in the PHA irritation cell, general all can reaching is lower than the not level of irritation cell.As stated, in Alamar Blue check, only show low-down vigor with the PMA/I stimulation and with the PBMC that DMSO handles, and the BrdU incorporation increases (Fig. 4).Similarly, after handling with the PMA/I stimulated cells with chemical compound [4], [9] and [10], the BrdU incorporation still is higher than the PHA stimulated cells, and just Alamar Blue value is for negative.
The concanavalin A titration shows that ConA stimulates PBMC with the mode that concentration relies on, and reaches maximum efficiency when 10 μ g/ml.The incorporation of BrdU is reduced to the not level of irritation cell (under the higher concentration) then, and consistent with vigor assessment and fractographic result, it shows that PBMC can not survive under higher ConA test concentrations.When comparing with PHA and PMA/I, can make the BrdU incorporation be increased to quite high level with 10 μ g/mlConA stimulation, perhaps than high about 6 times of the PBMC that does not stimulate, and do not reduce vigor (Fig. 7).
Embodiment 4: the result
The conclusion that instance 3 draws is that all test compounds are being low to moderate IC 50Concentration under all can influence the proliferation activity of irriate PBMC cell.Experiment (instance 4) research of carrying out subsequently is lower than IC at drug level 50The time, the concentration dependent of this effect.Repeat the processing of front, all add lower concentration with [12] for the chemical compounds and the chemical compound [11] of all previous tests, 0.5 * and 0.25 * IC 50And DMSO contrast.Because compound treatment is not the core that suppresses proliferation activity with respect to the time that stimulates, so after inoculation, carrying out PBMC immediately simultaneously stimulates and compound treatment.The stimulation of cell is perhaps handled with PHA as the front, perhaps carries out with 10 μ g/ml ConA.
Compare with irritation cell not, at 48 hours, PHA stimulated the BrdU incorporation that makes DMSO handle cell to increase to surpass 20 times, and ConA stimulates to increase and surpasses 10 times, further increases at 72 hours, and the DMSO carrier mixes BrdU or vigor does not all influence (Fig. 8).As shown in Figure 9, when stimulating with PHA, all chemical compounds all reduce the BrdU incorporation with the mode that concentration relies on, at IC 50Or 2 * IC 50Reach maximum efficiency during concentration.This has confirmed previous observation, surpasses IC in concentration 50The time, do not detect concentration dependent, because reached maximum efficiency.In general, stimulate PBMC to see identical effect with ConA, although maximum efficiency is to be issued in lower slightly concentration.This possibly be because the BrdU signal has been reduced to the result of background level when maximum efficiency, because for the ConA stimulated cells, its signal generally has only half height of PHA irritation cell, so detection sensitivity is slightly less than the former.
As before, the behavior of chemical compound [4], [9] and [10] is slightly different with all the other chemical compounds.Consistent with previous experiment, surpass IC in concentration 50The time, still can be clear that the concentration dependent of chemical compound [9] and [10], and extend to lower concentration.For the ConA stimulated cells, at 4 * IC 50The time, BrdU mixes still and to reduce, and signal is near background level, but is slightly higher than the PHA stimulated cells.Be not to hope to be associated with theory, but that this shows is the same with other chemical compounds, they can suppress proliferative activity fully, but reach the higher concentration of these purpose needs.
For the cell of handling with chemical compound [4], under two kinds of stimulations, the BrdU signal all is not reduced to background level, even at 4 * IC 50Also be like this.And, under higher concentration, BrdU mix with compound concentration between few of related, this is consistent with our previous result.Be not to hope to be associated with theory, but show chemical compound [4] to the influence of proliferation activity at about 2 * IC 50The time maximum, the same with other most chemical compounds, but the inhibition degree is lower.
Showing that through the active assessment of Alamar Blue check the activity of PBMC can reduce slightly when irriate, is roughly suitable but handle and be untreated between the PBMC at compound treatment, DMSO generally.And along with the increase of compound concentration, Alamar Blue value of calculation does not reduce, and proves that the reduction of BrdU incorporation does not cause owing to the chemical compound cytotoxicity, and has just represented the inhibition of proliferation activity merely.
In a word, experiment shows, handles with the transcription inhibitor of any series all to change PHA or the ConA effect to the T LS, and suppresses proliferation activity fully with dose-dependent mode.In a word, can see identical trend with the ConA stimulated cells, seem to be lower than the PHA stimulated cells although in the ConA stimulated cells, reach the required drug level of inhibition propagation fully for PHA.This possibly be owing to stimulate the illusion that causes less than PHA fully by what ConA obtained.Most compounds has identical pattern, wherein is being low to moderate 0.25 * IC at first 50Or 0.5 * IC 50Concentration under can see some inhibitory action to proliferation activity, then at about 2 * IC 50In time, reaches fully and to suppress, and some differences of existence are the increase along with compound concentration, and the speed that proliferation activity reduces is different.An instance that merits attention is chemical compound [7], and it is being low to moderate 0.25 * IC 50Concentration under promptly reach fully and to suppress.Yet the comparison between different chemical compound certain concentration must be carried out in discretion, because IC 50Value is based on the meansigma methods of different cell plates.
Chemical compound [9] and [10] can be seen and above-mentioned pattern condition of different; They need the concentration higher than other chemical compound just can reach identical effect; And for chemical compound [4]; It follows the concentration dependent identical with other chemical compound, but under higher concentration, can not realize suppressing fully proliferation activity.
Experiment shows that the PBMC activity reduces slightly, but not remarkable when irriate, further reduce relevant with compound treatment.The more important thing is that activity does not obviously rely on compound concentration, the true inhibition of proliferation activity has been represented in the minimizing that therefore in BrdU mixes, measures, rather than the cytotoxicity of chemical compound.
For a person skilled in the art, under the prerequisite that does not deviate from the scope of the invention and spirit, various modifications of the present invention and change are conspicuous.Special preferred embodiment is explained although the present invention is contact, should be appreciated that the present invention of application right is not limited in these special embodiment.In fact, the various modifications that said pattern of the present invention is carried out in obvious being used to for various equivalent modifications are also within coverage of the present invention.
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Table 1: body weight
? 2 3 4 5 6 7 August
Carrier 27.69± 0.61 27.85± 0.59 28.31± 0.56 28.08± 0.79 29.91± 0.65 31.60± 0.75 31.75± 1.11
The preventative research of CYC202 (200mg/kg) 27.85± 0.72 28.00± 0.68 28.54± 00.45 28.92± 0.35 31.58± 0.43 30.82± 0.35 31.60± 0.52
The preventative research of CYC202 (100mg/kg) 27.93± 0.62 27.86± 0.52 28.07± 0.57 28.64± 0.57 30.23± 0.67 31.50± 0.87 30.78± 0.91
CYC202 (100mg/kg) therapeutic studies 26.71± 0.74 28.14± 0.69 29.43± 1.01 29.71± 0.82 31.45± 0.96 30.80± 1.69 29.80± 1.46
Numerical value is represented with meansigma methods ± SE
Table 2: food intake
? 2 2.5 3 3.5 4 4.5 May
Carrier 3.77± 0.28 4.31± 0.17 4.15± 0.22 3.62± 0.35 3.54± 0.35 3.31± 0.35 3.50± 0.15
The preventative research of CYC202 (200mg/kg) 3.54± 0.29 4.08± 0.08 4.00± 0.11 4.08± 0.21 4.00± 0.16 4.00± 0.18 3.92± 0.29
The preventative research of CYC202 (100mg/kg) 3.69± 0.33 3.77± 0.23 3.92± 0.18 4.08± 0.18 3.92± 0.26 3.85± 0.30 3.85± 0.22
Numerical value is represented with meansigma methods ± SE
Table 3: water intake
? 2 2.5 3 3.5 4 4.5 May
Carrier 4.62± 0.33 5.00± 0.30 5.00± 0.30 4.54± 0.39 5.15± 0.15 5.00± 0.23 5.08± 0.23
The preventative research of CYC202 (200mg/kg) 5.31± 0.26 4.77± 0.20 4.92± 0.18 5.08± 0.21 4.85± 0.27 4.38± 0.43 4.23± 0.28
CYC202 (the preventative research of 100mg/ kg 6.64± 0.29 5.14± 0.40 4.21± 0.28 5.00± 0.28 5.57± 0.20 4.43± 0.20 3.93± 0.22
Numerical value is represented with meansigma methods ± SE
Table 4: survival rate %
? 3 4 5 5.5 6 6.5 7 7.5 August
Carrier
100 100 92 85 85 62 38 31 31
The preventative research of CYC202 (200mg/kg) 100 100 100 92 92 92 85 77 77
The preventative research of CYC202 (100mg/kg) 100 100 100 100 93 71 71 71 71
CYC202 (100mg/kg) therapeutic studies 100 100 100 93 71 64 36 36 36
Table 5: the cumulative percentage rate of albuminuria mice
? 3 4 5 5.5 6 6.5 7 7.5
Carrier 0 0 7.7 46.0 69.0 77.0 77.0 85
CYC2002(200mg/kg) 0 0 0 23.1 23.1 * 23.1 ** 23.1 ** 23.1 **
CYC202(100mg/kg) 0 0 0 0 ** 21.4 * 21.4 ** 42.9 42.9 *
CYC202(100mg/kg) 0 0 7.1 21.4 28.5 35.7 50.0 57
*P＜0.05, *P＜0.01 is compared with carrier
Table 6: with serum BUN assessment renal function
? August
Carrier
50％ (43-169mg/dl)
The preventative research of CYC202 (200mg/kg) 30％ (36-88mg/dl)
The preventative research of CYC202 (100mg/kg) 33％ (31-43mg/dl)
CYC202 (100mg/kg) therapeutic studies 40％ (30-111mg/dl)
Table 7: serum anti dna antibody (U/ml)
Figure G05837089820070429D000361
Numerical value is represented with meansigma methods ± SE
*P＜0.05, *P＜0.01 was compared with carrier in the corresponding time
Table 8: serum transaminase (IU/L)
? AST (IU/L) ALT (IU/L)
The preventative research of CYC202 (200mg.kg) 62.89±4.70 36.44±4.04
CYC202 (100mg/kg) therapeutic studies 74.40±1.72 36.00±2.45
Contrast (CD-1 mice) 78.00±2.28 36.40±2.64
Numerical value is represented with meansigma methods ± SE
Table 9: nephridial tissue
? The blood capillary inner cell is too much The outer hypertrophy of blood capillary The glomerule deposit Matter property damage between renal tubules
Carrier 2.00±0.44 1.00±0.38 1.14±0.34 0.71±0.23
The preventative research of CYC202 (200mg/kg) 0.36±0.15 **○○ 0.18±0.18 0.27±0.19 * 0.25±0.16 *○
The preventative research of CYC202 (100mg/kg) 0.92±0.23 * 0.67±0.26 0.92±0.23 0.46±0.17
CYC202 (100mg/kg) therapeutic studies 1.29±0.18 0.57±0.20 1.14±0.26 0.61±013
Numerical value is represented with meansigma methods ± SE. *P＜0.05, *P＜0.05, 00P＜0.01 couple CYC202 (100mg/kg) was from 5 months
The positive monocyte/macrophage of F4/80 between table 10 kidney in the matter
? August
Carrier 29.68±5.97#
The preventative research of CYC202 (200mg/kg) 8.56±1.94 ○*#
CYC202 (the preventative research of 100mg/kg () 18.26±4.89#
CYC202 (100mg/kg) therapeutic studies 21.87±5.13#
Contrast 0.25±0.25
Table 11: survival rate %
Figure G05837089820070429D000381
Processing is since 5 monthly ages.Relative carrier, CYC202+MPS (P＜0.0001) significantly prolong life.
Table 12: the cumulative percentage rate of albuminuria＞4mg/ world mice
Figure G05837089820070429D000382
Each point has reacted albuminuria level current in the survival mice and last measurement in the decline mice.0.05, *P＜0.01 pair carrier.
The cumulative percentage of the mice of table 13: renal function-BUN＞30mg/dl
? 5 6 7 8 9 10 11 December
Carrier
0 10％ 40％ 80％ 90％ 90％ 90％ 100％
CYC202 (200mg/kg) 0％ 7％ 15％ 43％ 57％ 64％ 64％ 64％
MPS (1.5mg/kg) 0％ 0％ 17％ 42％ 50％ 58％ 58％ 67％
CYC202+MPS 0％ 0％ 0％ 27％ 40％ 40％ 47％ 53％
BUN level＞30mg/dl thinks undesired (normal range: 14-29mg/dl)
Table 14: nephridial tissue
? ? Glomerule ? Matter property damage between renal tubules
? The blood capillary inner cell is too much The outer hypertrophy of blood capillary The immunity deposit ?
Carrier 1.60±0.24 1.00±0.00 2.20±0.37 1.70±0.09
CYC202 (200mg/kg) 1.14±0.26 0.86±0.14 1.43±0.30 1.29±0.23
MPS (1.5mg/kg) 1.40±0.40 0.80±0.20 1.80±0.20 1.20±0.14
CYC202 +MPS 0.70±0.30 0.30±0.15 * 0.90±0.23 *○ 0.45±0.21 **
Numerical value is represented with meansigma methods ± SE. *P＜0.05, *P＜0.01 pair carrier; ZeroP＜0.05 couple MPS.
Table 15: the F4/80 monocyte/macrophage between kidney in the matter (cell/HPF)
Carrier 61.5±4.6
CYC202(200mg/kg) 41.4±8.6
MPS(1.5mg/kg) 57.2±10.5
CYC202+MPS 21.8±4.8 **○
Numerical value is represented with meansigma methods ± SE.The HPF=high power field. *P＜0.01 pair carrier, ZeroP＜0.05 pair＜PS
Contrast CD-1 mice scope: 0-4 cell/HPF
Table 16: 72 hours IC of indoor cell plates 50Meansigma methods (μ M) and
The IC that uses 50About numerical value is so that calculate
Chemical compound IC 50Meansigma methods Cell line quantity IC 50Use value
1 27 * 1 * 25 *
2 16.45 62 16
3 56 * 1 * 50 *
4 1.04 47 1
5 1.43 13 1.5
6 10.4 13 10
7 5.31 18 5
8 0.876 35 1
9 0.250 6 0.25
10 0.753 11 1
11 0.291 7 0.5
12 0.353 7 0.5
*Do not have indoor cell toxicant data, the IC50 of HCT116 cell is disclosed in people such as Rayanud, Clin Cancer Res 11 (13): 4875-87,2005.
Table 17: the IC of the antagonism CDK/ cyclin of indoor kinase assay and the chemical compound of Aurora enzyme 50Meansigma methods (μ M)
Chemical compound CDK1B CDK2A CDK2E CDK4D1 CDK6D3 CDK7H CDK9T1 AurA AurB
1 ＞100 8.84 0.946 45.7 n/a 0.603 2.03 n/a n/a
2 17.5 2.80 0.208 27.5 55.5 0.438 1.04 ＞100 n/a
3 ＞100 83.08 7.67 ＞100 n/a ＞20 2.72 n/a n/a
4 7.46 1.05 0.505 2.82 1.04 8.57 1.88 0.038 0.030
5 15.9 1.24 0.931 2.75 1.94 27.0 2.71 0.051 0.023
6 7.60 0.373 0.126 19.3 9.19 1.33 0.338 ＞10 ＞10
7 4.95 0.223 0.071 7.63 6.27 0.983 0.195 ＞10 ＞10
8 1.99 2.00 0.354 0.089 0.168 0.053 0.034 0.652 0.399
9 2.00 1.67 0.204 0.118 0.110 0.147 0.097 0.094 0.021
10 1.49 0.115 0.013 5.10 6.40 0.455 0.081 ＞10 ＞100
11 0.667 0.304 0.054 0.066 n/a 0.266 0.029 0.028 0.008
12 1.03 0.076 0.025 1.02 n/a 2.38 0.215 0.161 0.025

Claims (23)

1. be selected from following purine
Figure FSB00000552537000011
Or the acceptable salt of its medicine is used for the purposes of the medicine of lupus, systemic lupus erythematosus (sle) or rheumatoid arthritis that medicine brings out in preparation.
2. according to the purposes of claim 1, wherein said medicine is used for therapy system property lupus erythematosus.
3. according to the purposes of claim 1 or 2, wherein said medicine further comprises prednisolone.
4. according to the purposes of claim 1 or 2, wherein said purine is selected from 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine and olomoucine.
5. according to the purposes of claim 1 or 2, wherein said purine is 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine or acceptable salt of its medicine.
6. according to the purposes of claim 3, wherein said purine is selected from 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine and olomoucine.
7. according to the purposes of claim 3, wherein said purine is 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine or acceptable salt of its medicine.
8. combination comprises being selected from following purine
Figure FSB00000552537000021
Or acceptable salt of its medicine and prednisolone.
According to Claim 8 be combined in the purposes that preparation is used for the medicine of lupus, systemic lupus erythematosus (sle) or rheumatoid arthritis that medicine brings out.
10. according to the purposes of claim 9, wherein this medicine is used for therapy system property lupus erythematosus.
11. according to the purposes of claim 9 or 10, wherein said purine is selected from 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine and olomoucine.
12. according to the purposes of claim 9 or 10, wherein said purine is 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine or acceptable salt of its medicine.
13. a pharmaceutical composition, it comprises according to Claim 8 combination and medicine acceptable carrier, diluent or excipient.
14. according to the pharmaceutical composition of claim 13, wherein this purine is selected from 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine and olomoucine, and the acceptable salt of medicine.
15. according to the pharmaceutical composition of claim 13, wherein this purine is 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine, or the acceptable salt of its medicine.
16. a drug products, it comprises and is selected from following purine
Figure FSB00000552537000031
Figure FSB00000552537000041
Or acceptable salt of its medicine and prednisolone, as combination preparation simultaneously, in order or separately use in treatment.
17. according to the drug products of claim 16, wherein this purine is selected from 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine and olomoucine, and the acceptable salt of medicine.
18. according to the drug products of claim 16, wherein this purine is 2-(1-D, L-hydroxyl first third amino)-6-benzyl amino-9-isopropyl purine, or the acceptable salt of its medicine.
19. according to the drug products of claim 16, the administration simultaneously of wherein said purine or the acceptable salt of its medicine and prednisolone.
20. according to the drug products of claim 16, it further comprises medicine acceptable carrier, diluent or excipient.
21., be used to treat LADA rheumatism or organ specificity autoimmune disease according to the drug products of claim 16.
22. according to the drug products of claim 21, wherein the LADA rheumatism is selected from drug-induced lupus, systemic lupus erythematosus (sle) or rheumatoid arthritis.
23. according to the drug products of claim 22, wherein this LADA rheumatism is a systemic lupus erythematosus (sle).
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