CN1929861A - CTGF as target for the therapy of diabetic nephropathy - Google Patents

Abstract

The present invention relates to methods and compounds for treating specific early stage aspects and late stage aspects of diabetic nephropathy. Methods and compounds for treating various physiological features associated with early stage and with late stage diabetic nephropathy are also provided.

Description

CTGF is as the treatment target spot of diabetic nephropathy

The application requires the U.S. Provisional Application serial number 60/544,121 of submission on February 11st, 2004; The U.S. Provisional Application serial number 60/561,018 that on April 8th, 2004 submitted to; The U.S. Provisional Application serial number 60/578,401 that on June 9th, 2004 submitted to; With the priority of the U.S. Provisional Application serial number of submitting on October 20th, 2,004 60/620,802, each application is included this paper in as a reference in full.

Invention field

The present invention relates to treat the specific early symptom of diabetic nephropathy and late period symptom method and chemical compound.Also provide treatment with in early days and method and the chemical compound of the relevant various physiological features of diabetic nephropathy in late period.

Background of invention

Nephropathy is that the normal physiologic of kidney and any change of function cause.Nephropathy can be caused by various acute and chronic disease and incident, comprise physics, chemistry or biological damage, wound or wound, disease is as hypertension, diabetes, congestive heart failure, lupus, sicklemia, various inflammatory and autoimmune disease, HIV associated kidney disease etc.Nephropathy can cause renal function reduction, hypertension and renal failure, seriously reduces quality of life, needs dialysis sometimes, needs renal transplantation under some situation.

Diabetic nephropathy is the main long-term complications of diabetes, and is to dialyse and the main indications of renal transplantation in the U.S..(Marks and Raskin, 1998, Med Clin North Am, 82:877-907.) diabetic nephropathy can take place I type and the type 2 diabetes mellitus patient of 25-50%.Therefore, diabetic nephropathy is the common causes that cause end-stage renal disease and renal failure in western countries.

With 1 type or type 2 diabetes mellitus patient the relevant risks and assumptions that works of diabetic nephropathy (with other nephropathy) takes place and comprise that the glomerular hemodynamics of hyperglycemia, hypertension, change and various somatomedin increase or unusual expression, comprising transforming growth factor-beta (TGF β), insulin like growth factor (IGF)-I, VEGF-a (VEGF-A) and Connective Tissue Growth Factor (CTGF).(see, for example, Flyvbjerg (2000) Diabetologia 43:1205-23; Brosius (2003) Exp Diab Res 4:225-233; Gilbert etc., (2003) Diabetes Care 26:2632-2636; With international publication number WO 00/13706.)

Present therapeutic strategy is in all sorts of ways to slow down the progress of diabetic nephropathy, comprises best glycemic control (by regulating diet and/or insulinize) and hypertension control, the effect difference of these therapies.For example, angiotensin converting enzyme (ACE) inhibitor and angiotensin receptor blocker (ARB) all are used to reduce hypertension, and they show can postpone nephropathy and albuminuretic progress of DABAI or development.Some clinical trials are verified ACE inhibitor and ARB is to the effect of diabetics.Yet although ACE inhibitor shows that the kidney that can postpone the type 1 diabetes patient is weak, these medicaments are not clear to type i diabetes patient's kidney protection effect.(Raij(2003)Am JHypertens 16：46S-49S。)

In addition, because the therapy of blood sugar control and blood pressure significantly reduces the M ﹠ M of diabetic nephropathy by the progress that postpones related pathologies, this routine treatment can not fully stop the progress of disease, therefore can't provide therapeutic effect completely.In addition, present nursing standard is to give ACE inhibitor or ARB, this be not general effectively, just minimally postpones and does not get rid of the needs of renal transplantation.

Other treatment means concentrates on one or more somatomedin as the treatment target spot.After deliberation independent inhibition or unite the therapy that suppresses VEGF or TGF β with ACE inhibitor or ARB.(see, for example, De Vriese etc., (2001) J Am Soc Nephrol 12:993-1000; Flyvbjerg etc., (2002) Diabetes 51:3090-3094; Ziyadeh etc., (2000) Proc Natl Acad Sci 97:8015-8020; Chen etc., (2003) Biochem BiophysRes Commun 300:16-22; With Benigni etc., (2003) J Am Soc Nephrol 14:1816-1824.Yet), this Therapeutic Method can not improve all aspects (renal function and the structure that for example change and weaken) of the kidney pathology relevant with diabetic nephropathy.For example, suppress can not effectively alleviate the albuminuria of db/db mice as the TGF β of diabetic nephropathy treatment target spot, expansion has beneficial effect although this treatment is to glomerular matrix.(see Ziyadeh etc., the same.) in addition, for raising, the relevant kidney permeability of diabetes provides benefit although give diabetes db/db mice anti-VEGF antibodies, and observe to glomerular mesangium expansion and have only minimum benefit.(see Flyvbjerg etc., (2002), the same.) therefore, although separately or unite that to use this therapy be likely, for example diabetic nephropathy relevant early stage (for example glomerule excessively filters, glomerular filtration rate risings, microalbuminuria etc.) and late period, (for example glomerular filtration rate reduction, albuminuria, glomerular mesangium substrate overdistension etc.) greatly pathological characters did not all improve with chronic nephropathy.Therefore, this area need to improve in early days with late period symptom and with disease progression with make progress the complete therapy of treatment diabetic nephropathy of relevant pathology.

Except above-mentioned shortcoming, present diabetic nephropathy therapy is because shortage specificity so the suitability/effect are limited.Specifically, the therapy of targeting VEGF-or TGF β can comprise the useful activity of these somatomedin, as angiogenesis, tumor suppression and immune suitable growth.For example, because TGF β is relevant with the fibrosis development, it also is the important medium of immunity growth and tumor suppression, and prompting suppresses TGF β and has deleterious potential adverse side effect.Therefore, this area need have more the method for optionally treating diabetic nephropathy.

In a word, the especially Therapeutic Method of diabetic nephropathy of nephropathy need be treated in this area, and this method is all effective in each stage of disease progression and progress (for example early stage and late period diabetic nephropathy).Specifically, the complete therapy that needs diabetic nephropathy, this therapy can effectively treat diabetic nephropathy early sign and late period feature, described feature for example: excessively filter that (in early days), glomerule permeability raise (in early days), glomerular filtration rate raises (in early days), microalbuminuria (in early days), albuminuria (late period) and glomerular filtration rate reduction (late period) greatly.Need more fully at the development of diabetic nephropathy and other nephropathy and the Therapeutic Method of relevant various various process of making progress.Specifically, need be the therapy of target with non-fibrosis relevant (for example excessively filtering) and fibrosis (for example glomerular mesangium substrate expansion) process with diabetic nephropathy.In addition, the Therapeutic Method that the 26S Proteasome Structure and Function benefit can be provided that needs treatment nephropathy, especially diabetic nephropathy.

The present invention identified CTGF with the development of nephropathy such as diabetic nephropathy and the effect in relevant each process of making progress, and provide the method that suppresses and prevent these processes, satisfied these needs.The present invention also provides and can be used to treatment and prevention nephropathy, especially relevant nephropathy with diabetes, and the method and the medicament of the diabetic nephropathy of more specifically saying so have satisfied existing needs.

The accompanying drawing summary

Fig. 1 shows, resist-kidney weight that CTGF antibody has reduced diabetes db/db mice increases.

Fig. 2 shows, resist-CTGF antibody reduced the creatinine clearance of diabetes db/db mice.

Fig. 3 shows, resist-CTGF antibody reduced the urinary albumin excretion of diabetes db/db mice.

Fig. 4 has shown the dependency between interior CTGF of people's vitreous body and VEGF level.

Fig. 5 shows, resist-CTGF antibody reduced the urine volume of diabetes db/db mice.

Fig. 6 shows, resist-CTGF antibody reduced the kidney basement membrane thickened of diabetes db/db mice.

Fig. 7 shows, resist-CTGF antibody reduced the albuminuria of diabetic nephropathy rat model.

Fig. 8 shows, resist-CTGF antibody reduced the BUN level of diabetic nephropathy rat model.

Fig. 9 shows, resist-CTGF antibody improved the glomerular filtration rate of diabetic nephropathy rat model.

Summary of the invention

The present invention relates to treat or the specific early symptom of diabetes and nephropathy preventing and late period symptom method and chemical compound, and provide treatment or prevention with in early days and the method and the chemical compound of the relevant various physiological features of diabetic nephropathy in late period.

Specifically, in the preferred embodiment of following each side method, preferably to liking human subjects.

In one embodiment, the invention provides a kind of method of creatinine clearance that reduces diabetes or early diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose, thereby reduces the creatinine clearance of object.People's normal creatinine clearance rate level is about 97-137 ml/min (adult male) and 88-128 ml/min (adult female).Therefore, especially considered the creatinine clearance level is reduced to these levels or the about method of these levels.

The excessive filterable method of glomerule of the object that alleviates diabetes or early diabetic nephropathy patient or ill risk is arranged also is provided here, this method comprises the CTGF inhibitor that gives the patient treatment effective dose, and the method that alleviates the glomerule hyperperfusion also is provided.

On the other hand, the present invention includes and alleviate or prevent diabetes or diabetic nephropathy patient or the method that has the object kidney weight of ill risk to increase, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

The present invention also provides the method for the glomerular filtration rate normalization of the object that makes diabetes or diabetic nephropathy patient or ill risk is arranged, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.Described diabetic nephropathy can be, for example, and early stage, late period, initial stage or explicitly diabetic nephropathy.In early days or under the situation of initial stage diabetic nephropathy, normalization may reduce glomerular filtration rate, and late or under the situation of explicitly diabetic nephropathy, the normalization glomerular filtration rate that may raise.Adult's normal GFR is about 120 ml/min.When the GFR of object is higher than normal level, need reduce GFR, consider especially GFR is brought down below about 150 ml/min, is lower than about 140 ml/min, is lower than the method for about 130 ml/min and about 120 ml/min.Impaired or when being lower than normal level as the GFR of object, GFR need be risen to and be higher than about 15 ml/min, be higher than about 30 ml/min, be higher than about 60 ml/min, be higher than the method for about 90 ml/min, about 120 ml/min.

In another embodiment, the invention provides the method for the object glomerule hypertrophy that alleviates diabetes or diabetic nephropathy patient or ill risk is arranged, described diabetic nephropathy comprises in early days, late period, initial stage or explicitly diabetic nephropathy, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

The albuminuretic method that alleviates diabetes or diabetic nephropathy patient or the object of ill risk is arranged also is provided here, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.The present invention also comprises the method for the albuminuria of the object that alleviates diabetes or diabetic nephropathy patient or ill risk is arranged, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.The method that also comprises the microalbuminuria of the object that alleviates diabetes or diabetic nephropathy patient or ill risk is arranged, wherein said diabetic nephropathy is early stage or initial stage diabetic nephropathy, described method comprises the CTGF inhibitor that gives the patient treatment effective dose, and provide the albuminuretic method of DABAI of the object that alleviates diabetes or diabetic nephropathy patient or ill risk is arranged, wherein said diabetic nephropathy is late period or explicitly diabetic nephropathy, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

Adult's normal urine albumin excretion rate is about the 15-30 mg/day usually.When being about the 30-300 mg/day, the urinary albumin excretion rate of object is diagnosed as microalbuminuria usually.The albuminuretic typical characteristic of DABAI is that the urinary albumin excretion rate is greater than about 300 mg/day.Therefore, the present invention provides the method that reduces the urinary albumin excretion rate of object especially, and described method comprises the CTGF inhibitor of the object effective dose that gives urinary albumin excretion rate rising (for example the urinary albumin excretion rate is higher than normal level).Here considered especially the urinary albumin excretion rate is brought down below about 300 mg/day, is lower than about 200 mg/day, is lower than about 100 mg/day, is lower than about 50 mg/day, and most preferably be lower than the embodiment of about 30 mg/day.

In some aspects, the invention provides the method for the BUN level of the object that reduces diabetes or diabetic nephropathy patient or ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.Adult's normal BUN level is 7-20 milligram/decilitre.Therefore, provide and make the BUN level be brought down below 20 milligrams/deciliter method.The present invention also provides the method for the inulin clearance of the object that reduces diabetes or diabetic nephropathy patient or ill risk is arranged, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.In particular aspects, described diabetic nephropathy is a diabetic nephropathy or explicitly diabetic nephropathy in late period.

Again in another embodiment, the invention provides in the object that the diabetic complication risk is arranged this complication of prevention, reduce its risk or postpone the method for its outbreak, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.In different embodiments, described diabetic complication comprises at least a following complication that is selected from: creatinine clearance raises, glomerular filtration rate rising or reduction, glomerular basement membrane thickening, glomerule excessively filter, glomerule hyperperfusion, glomerule hypertrophy, the rising of urinary albumin excretion rate, microalbuminuria, big albuminuria, BUN level raise, inulin clearance raises, kidney weight increases and impaired renal function.

The present invention also comprises treatment initial stage diabetic nephropathy patient or the method for initial stage diabetic nephropathy of the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose, and comprise treatment early diabetic nephropathy patient or the method for initial stage diabetic nephropathy of the object of ill risk arranged that described method comprises the CTGF inhibitor that gives the patient treatment effective dose.Here also considered treatment explicitly diabetic nephropathy patient or the method for explicitly diabetic nephropathy of the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

The present invention has considered method of the present invention and other therapies coupling.In one embodiment, described method and other therapies coupling, for example, with the therapeutic effect of further increase to some pathology affair, or the like.For example, during the treatment time-histories or in progression of disease and the back two kinds of processing of alleviation, can simultaneously or give continuously.In another embodiment, described method is united use with another kind of Therapeutic Method with similar or different binding modes, for example, and ACE inhibitor, ARB, inhibin, advanced glycosylation end product (AGE) inhibitor etc.Therefore, in one embodiment, the invention provides the method for the diabetic nephropathy of the object for the treatment of diabetic nephropathy patient or ill risk being arranged, described method comprises with the angiotensin-convertion enzyme inhibitor of amount of suppression unites the CTGF inhibitor that gives the patient treatment effective dose.The present invention also provides the treatment diabetic nephropathy patient or the method for diabetic nephropathy of the object of ill risk has been arranged, and described method comprises with the angiotensin receptor blocker of amount of suppression unites the CTGF inhibitor that gives the patient treatment effective dose.

In one embodiment, provide the method for the carrying out property renal failure of treatment target, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.In another embodiment, the invention provides microalbuminuria risk that reduces object or the method that postpones the microalbuminuria development, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.In another embodiment, also provide the method that reduces the big albuminuria risk of object or postpone big albuminuria development, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

One particular aspects the present invention relates to following discovery, and promptly CTGF is accredited as the key factor of the early stage PD that comprises diabetic nephropathy complication and vitreoretinopathy here.Therefore, on the one hand, the present invention relates to the method for this disease early symptom of the object treating or prevent the PD patient or ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.On the other hand, described PD is relevant with the somatomedin outside the CTGF, one concrete aspect, described other somatomedin is VEGF.On the one hand, described PD is a nephropathy, and in a particular aspects, described PD is relevant with diabetes or diabetic complication, or diabetic nephropathy.

The present invention also comprises the method for the renal function of the object that improves the impaired renal function patient or ill risk is arranged, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

As mentioned above, the present invention relates to following discovery, the various physiological features of diabetic nephropathy in early stage and late period can effectively be treated or prevent to promptly anti--CTGF therapy.Therefore, the invention provides treatment or prevention and at least a method that is selected from the relevant nephropathy of following feature: creatinine clearance raises; Glomerular filtration increases or glomerule excessively filters; Albuminuria; Urinary albumin excretion increases; Glomerular volume increases; The glomerule hypertrophy; Kidney weight increases; Glomerular basement membrane thickening; Glomerular filtration rate reduces; The BUN level raises; Raise with inulin clearance.Under the various situations, described method comprises the CTGF inhibitor that the object of these needs effective dose is arranged.These methods all comprise and give progress or the development of object CTGF inhibitor to prevent above-mentioned arbitrary complication.

The present invention comprises that also the CTGF inhibitor is used for the application of the medicine of following purposes in manufacturing: reduce creatinine clearance; Alleviating glomerule excessively filters; Alleviate the glomerule hyperperfusion; Alleviate the glomerule hypertrophy; Reduce renal glomerular basement membrane thickness; Reduce the urinary albumin excretion rate; Alleviate albuminuria (proteinuria); Alleviate albuminuria (albuminuria); Alleviate microalbuminuria; Alleviate big albuminuria (macroalbuminuia); Reduce the BUN level; Make glomerular filtration rate normalization; Reduce inulin clearance; Alleviate or prevent kidney weight to increase; The prevent diabetes complication, reduce its risk or postpone its outbreak; Treatment or prevention initial stage diabetic nephropathy progress; Treatment or prevention early diabetic nephropathy progress; Treatment or prevention explicitly diabetic nephropathy progress; Treatment or carrying out property of prevention renal failure; The PD early symptom development of treatment or object of prevention; With improve diabetes or diabetic nephropathy patient or the renal function of the object of ill risk arranged.In different embodiments, described reagent is selected from antibody, micromolecular inhibitor, antisensenucleic acids and siRNA.

In the preferred embodiment of said method, described to liking human subjects.

In any said method, considered that especially the reagent of inhibition CTGF is polypeptide, polynucleotide or micromolecule; For example, in conjunction with the antibody of CTGF, antisense molecule, siRNA, micromolecular compound etc.Specifically, the present invention considers and can realize suppressing CTGF to regulate expression and the activity of CTGF by any method well known in the art.Suppress the expression of CTGF encoding gene, inhibition CTGF generation or suppress the active any method of CTGF although the present invention includes, preferred use resists-CTGF reagent, for example human monoclonal antibodies of anti-CTGF.For example, available micromolecular compound suppresses expression, generation or the activity of CTGF.Because CTGF expresses and suppressed by the ring nucleus thuja acid, this chemical compound can comprise, for example, and ring-type nucleotide analog or phosphodiesterase (PDE) inhibitor.(see, for example, Duncan etc., (1999) FASEB J 13:1774-1786.) in addition, comprise that the polynucleotide of small interference ribonucleic acid (siRNA), Microrna (miRNA), ribozyme and antisense sequences can be used for method of the present invention to suppress expression and/or the generation of CTGF.(see, for example, Kondo etc., (2000) Biochem Biophys Res Commun 278:119-124.) this technology is that the association area those of skill in the art know.The example that is used for the antibody of the inventive method is described in, and for example, among the international publication number WO 2004/108764, this application is included in this paper as a reference in full.

Invention is described

Should be understood that to the invention is not restricted to concrete grammar described herein, scheme, cell line, mensuration and reagent, these can change.Should also be understood that terminology used here is used for describing specific embodiments of the present invention, rather than will limit the scope of the invention by any way that scope of the present invention is limited by additional claim.

Essential note, in the text with additional claim in, unless offer some clarification in addition in the literary composition, singulative " ", " a kind of " and " this " comprise its plural number.Therefore, comprise a plurality of this fragments when for example, mentioning " fragment ", be meant one or more antibody and its equivalent known to those skilled in the art when mentioning one " antibody ", or the like.

Unless otherwise defined, used here all technology and scientific terminology and the technical field of the invention those skilled in the art's routine is understood and is had identical meanings.Although all can be used to practice or test the present invention with any method and the material of method described here and materials similar or equivalence, what describe now is preferable methods, device and material.For describe and openly can with reported method, reagent and instrument in the publication that the present invention is used in combination, all publications of quoting are here included in this paper as a reference in full.Here all publications of mentioning can not be interpreted as enough admitting that the present invention does not have qualification early than disclosure of an invention formerly.

Except as otherwise noted, practice of the present invention will be adopted chemistry, biochemistry, molecular biology, cytobiology, hereditism, immunology and the pharmacological method of routine known in the art.This technology has at length in the literature to be explained.See, for example, Gennaro, A.R. compiles, and 1990, " Lei Mingdun pharmaceutical science " (Remington ' s PharmaceuticalSciences), the 18th edition, Mack Publishing Co.; Colowick, volumes such as S., " Enzymology method " (MethodsIn Enzymology), Academic Press, Inc.; " experiment immunization is learned handbook (Handbook of ExperimentalImmunology), the I-IV volume (D.M.Weir and C.C.Blackwell compile, and 1986, Blackwell ScientificPublications); Maniatis, volumes such as T., 1989, " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), second edition, I-III volume, Cold Spring Harbor Laboratory Press; Ausubel, volumes such as F.M., 1999, " fine works molecular biology experiment guide " (Short Protocols in MolecularBiology), the 4th edition, John Wiley ﹠amp; Sons; Volumes such as Ream, 1998, " Protocols in Molecular Biology: experiment is smart to be wanted " (Molecular Biology Techniques:An Intensive Laboratory Course), AcademicPress); " PCR (biotechnology book series introduction) " (PCR (Introduction to Biotechniques Series)), second edition, Newton and Graham compile, and 1997, Springer Verlag.

The present invention partly relates to following discovery, be that Connective Tissue Growth Factor (CTGF) is brought into play pivotal role in the specific early symptom of nephropathy, described early symptom comprises, for example, glomerule excessively filters, the rising of glomerule permeability, glomerular filtration rate rising, microalbuminuria etc.Known CTGF is relevant with the renal tubules interstitial fibrosis with nephropathy symptom in specific late period such as glomerulosclerosis, but is not accredited as the crucial target spot of the various early stage kidney pathological characteristicses of influence.Considered that especially treatment or prevention for example comprise the method for the nephropathy of diabetic nephropathy and the method for treatment or prevention related diseases Neo-Confucianism.

The invention provides by suppressing the method and composition of the complication that the multiple different pathological process relevant with nephropathy such as diabetic nephropathy that CTGF alleviates or alleviates object be correlated with.In some embodiments, described to as if animal, more preferably mammal, optimum is chosen.

The present invention also provides the compositions that is used for described method.This compositions can comprise micromolecular compound; Peptide and protein comprise antibody or its functional activity fragment; And polynucleotide, comprise small interference ribonucleic acid (siRNA), Microrna (miRNA), ribozyme and antisense sequences.(see, for example, Zeng (2003) Proc Natl Acad Sci USA100:9779-9784; And Kurreck (2003) Eur J Biochem 270:1628-1644.)

The present invention's part is based on the unexpected benefit of having found to suppress CTGF when treating a plurality of particular aspects of nephropathy such as diabetic nephropathy.The invention provides to prove that inhibition CTGF had alleviated in the past and the data of each pathology aspect of nephropathy that CTGF is irrelevant.In some aspects, evidence provided by the invention shows, suppressing CTGF provides Therapeutic Method for treatment or the specific physiology of diabetes and nephropathy preventing aspect, and described specific physiology aspect is relevant with the pathology activity with the biologic activity of VEGF, as glomerule excessively filtration and hyperperfusion.

Diabetic nephropathy

Diabetes are main causes of whole world M ﹠ M, and about 40% diabetics develops into diabetic nephropathy, need kidney dialysis or renal transplantation.Diabetes are first causes of latter stage nephropathy, and therefore, any diabetics all has the risk that develops into diabetic nephropathy.

The process of diabetic nephropathy is a feature with foreseeable event schema.Usually, the time-histories of diabetic nephropathy development is as follows.Glomerule excessively filters and the first year generation of renal hypertrophy behind onset diabetes, shows as glomerular filtration rate rising (for example, Ren Lei normal glomerular filtration rate is that about 120 ml/min are to about 150 ml/min).In preceding 5 years of diabetes, can observe pathological changes such as glomerule hypertrophy, glomerular basement membrane thickening and glomerular mesangium volume expanded.It is normal that glomerular filtration rate recovers gradually.Behind the diabetes 5-10, albumin (microalbuminuria) is in a small amount discharged in individual beginning in urine.Microalbuminuria (diabetic individual that microalbuminuria occurs is the initial stage diabetic nephropathy patient hereinafter referred to as) is the important indicator of making progress into explicitly diabetic nephropathy (part is a feature with big albuminuria or explicitly albuminuria).Basement membrane thickened and the glomerular volume expansion seen in early days in disease can be accumulated in the diabetic nephropathy in late period, cause the capillary lumen obturation, finally cause glomerular sclerosis.In case the explicitly diabetic nephropathy occurs, glomerular filtration rate will be stablized decline, and only about half of patient develops into end-stage renal disease in 7-10.

The development and the progress stage of human diabetes nephropathy have well been described clinically.I phase diabetic nephropathy and kidney (being glomerule (glormerular)) filter to be increased (promptly excessively filter, cause owing to flow through the blood increase of kidney and glomerule), glomerular filtration rate raises, the glomerule hypertrophy is relevant with renomegaly.II phase diabetic nephropathy is and continues excessively to filter the clinical reticent stage relevant with renal hypertrophy.The expansion of glomerular basement membrane thickening and glomerular mesangium takes place.III phase diabetic nephropathy (being also referred to as the initial stage diabetic nephropathy) is relevant with microalbuminuria (micro proteinuria) with microalbuminuria.Microalbuminuria is defined in the urine of collecting in 24 hours 30-300 mg/day urinaryalbumin, 20-200 microgram/minute urinaryalbumin, perhaps in the collection (spot collection) 30-300 microgram/milligram kreatinin is arranged.Kidney is lost the ability of filtering refuse gradually, and the blood levels of kreatinin and blood urea nitrogen raises simultaneously.Glomerular basement membrane thickening and glomerular mesangium expansion are along with the state of an illness increases the weight of to continue to take place.To continue rising relevant for kreatinin and blood urea nitrogen (BUN) level in IV phase diabetic nephropathy (being also referred to as the explicitly diabetic nephropathy) and big albuminuria (being clinical albuminuria) and the blood.Big albuminuria is defined in the urine of collecting in 24 hours to be had greater than 300 mg/day urinaryalbumin, greater than 200 micrograms/minute urinaryalbumin, and perhaps checking and accepting to concentrate has greater than 300 micrograms/milligram kreatinin.In case the explicitly diabetic nephropathy occurs, glomerular filtration rate reduces in the several years gradually.V phase diabetic nephropathy takes place with latter stage nephropathy and renal failure.

Excessively filter and hyperperfusion

Early diabetic nephropathy is relevant with impaired renal function, and its Partial Feature is that glomerule excessively filters and hyperperfusion.It is that the glomerulonephritis unit relevant with hyperglycemia and diabetes runs off that glomerule excessively filters.Along with the loss of functional nephron material, remaining functional nephron is loose and bear the workload of increase, thereby the overall forfeiture of renal function is minimized.Glomerule consequently takes place excessively to be filtered and hyperperfusion.

Glomerule excessively filters and hyperperfusion is reflected as the glomerular filtration rate rising.Glomerular filtration rate is the cubing of the filtrate of kidney per minute generation.The glomerular filtration rate measurement of human subjects is healthy and the best down comprehensive index of renal function of morbid state.(Smith, " kidney and urinary disease " (Diseases of the kidney and urinarytract) select from " 26S Proteasome Structure and Function when health and disease " (Structure and Function in Health andDisease), New York; The Oxford University Press, 1951:836-887.) glomerular filtration rate can in all sorts of ways definite, as measure filtering the urine clearance rate of label such as polysaccharide, iothalamate or iohexol.Method more commonly used is, can estimate glomerular filtration rate by determining kreatinin (a kind of produce and be discharged into protein in the blood by muscle) clearance rate.Creatinine clearance (being typically expressed as ml/min) can be determined by the creatinine levels of relatively collecting in (for example 12 or 24 hours) the interior urine preset time and the creatinine levels in the blood.The typical creatinine clearance of adult male is about the 97-137 ml/min, and the adult female is about the 88-128 ml/min.

In the clinical practice, the most common is from serum creatinine concentration estimation creatinine clearance.Creatinine clearance is drained with the urine creatine acid anhydride and is directly proportional, and is inversely proportional to serum creatinine concentration.This area set up and standardization the formula of many estimation creatinine clearances, therefore can adopt parameter estimation glomerular filtration rates such as serum creatinine concentration, age, sex and body weight.(see, for example, Cockcroft and Gault (1976) Nephron 16:31-41; Levey etc., (1999) Annals of Internal Medicine 130:462-470; Rule etc., (2004) Ann Intern Med 141:929-937.)

Method of the present invention and chemical compound can reduce the creatinine clearance of diabetes animal model.(see that for example, embodiment 1.) therefore, the invention provides and be used for reducing creatinine clearance increase or rising or creatinine clearance rising method and the chemical compound above the creatinine clearance of the object of normal level.The present invention confirms, suppresses CTGF (for example, by giving CTGF antibody) and has reduced the creatinine clearance relevant with nephropathy especially diabetic nephropathy.The creatinine clearance that raises and excessively filtration of glomerule, hyperperfusion, hypertrophy are relevant with the glomerular filtration rate rising, and it is for example early stage change in renal function of diabetic nephropathy development or impaired indication of nephropathy.On the one hand, the invention provides by suppressing method and the chemical compound that CTGF reduces creatinine clearance.On the other hand, the invention provides by suppressing method and the chemical compound that CTGF reduces glomerule kreatinin permeability and recovers glomerule selectivity and function.On the other hand, provide by suppressing CTGF treats or prevention is relevant with hyperglycemia or diabetes glomerule hypertrophy, the excessively method and the chemical compound of filtration and hyperperfusion.Again on the other hand, provide by suppressing CTGF treats or prevention is relevant with nephropathy especially diabetic nephropathy glomerule hypertrophy, the excessively method and the chemical compound of filtration and hyperperfusion.On the one hand, described nephropathy is an early diabetic nephropathy.

On the other hand, the invention provides by suppressing method and the chemical compound that CTGF reduces the glomerular filtration rate of glomerular filtration rate rising object.On the one hand, the invention provides method and the chemical compound that reduces glomerular filtration rate by the object CTGF inhibitor that gives the impaired or object that raises of glomerular filtration rate or this risk is arranged.On the one hand, described glomerular filtration is impaired raises relevant with early nephropathy with glomerular filtration rate.

In some embodiment, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat to raise relevant or, thereby treat or prevent this disease as the method and the chemical compound of the nephropathy of feature with creatinine clearance.In other embodiment, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat to increase or glomerule excessively filters relevant or as the method and the chemical compound of the nephropathy of feature, thereby treat or prevent this disease with glomerular filtration.

Find that method of the present invention and chemical compound can improve the glomerular filtration rate of diabetic nephropathy animal model in late period.(see embodiment 3.) therefore, the invention provides by the glomerular filtration rate that suppresses that CTGF improves or the normalization glomerular filtration rate is lower than normal value and reduce or the method and the chemical compound of the glomerular filtration rate of impaired object.On the one hand, the invention provides by giving the impaired or object that reduces of glomerular filtration rate or this object CTGF inhibitor that this risk is arranged improves or the method and the chemical compound of normalization glomerular filtration rate.On the other hand, described glomerular filtration rate is impaired relevant with end-stage renal disease or explicitly diabetic nephropathy with the glomerular filtration rate reduction.

On the one hand, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat or prevention is impaired with glomerular filtration rate and glomerular filtration rate reduces the method and the chemical compound of relevant nephropathy, thus prevention or prevent this disease.On the other hand, described glomerular filtration rate is impaired reduces relevant with end-stage renal disease with glomerular filtration rate.

Consider that method of the present invention can be used for having the object of any nephropathy of accepting clinically or nephropathy measurement index standard or the object that develops into this nephropathy risk arranged to improve renal function, normalization glomerular filtration rate, alleviate excessive filtration of glomerule and hyperperfusion or to reduce creatinine clearance.In some embodiment, described object suffers from diabetic nephropathy.In different embodiments, described object suffers from I phase nephropathy, II phase nephropathy, III phase nephropathy, IV phase nephropathy or V phase nephropathy.

Method of the present invention is used to prevent, alleviate or postpone to occur the outbreak of this complication of object of the relevant renal complication risk of early nephropathy, or be used for making medicine, this medicine is used to have any disease relevant with early nephropathy described here or the object of feature, preferred human subjects.On the one hand, described object suffers from diabetes.Diabetes can define with those skilled in the art's acceptance and any measurement of adopting.But the blood sugar level of human subjects is diagnosed as diabetes when surpassing 200 milligrams/deciliter (determining with fasting glucose test, oral glucose tolerance test or random blood sugar test).Therefore, in some aspects, it is can be with the proper object of method provided by the invention or medicinal usage treatment that blood sugar level is higher than about 200 milligrams/deciliter human subjects.

Can there be glomerular filtration rate impaired with other proper object of method treatment of the present invention.In one embodiment, the glomerular filtration rate of human subjects is higher than normal glomerular filtration rate, for example, is higher than about 120 ml/min.Therefore, the glomerular filtration rate human subjects that is higher than about 120 ml/min, is higher than about 130 ml/min, is higher than about 140 ml/min or is higher than about 150 ml/min is can be with the proper object of method provided by the invention or medicinal usage treatment.In different embodiments, the glomerular filtration rate that the method that also consider to reduce object (for example, glomerule excessively filters and the object of the hyperperfusion) glomerular filtration rate that glomerular filtration rate raises can be used to reduce human subjects is to being lower than about 150 ml/min, being lower than about 140 ml/min, being lower than the level of about 130 ml/min or arriving the level of about 120 ml/min.

Method of the present invention can reduce the diabetes animal model kidney weight relevant with diabetes or early diabetic nephropathy with chemical compound and increase.(see embodiment 1.) therefore, the present invention has considered that treatment or prevention and kidney weight increase the method for relevant nephropathy, this method comprises the object CTGF inhibitor that gives the patient or ill risk is arranged, thus treatment or prevent this disease.The present invention has considered that also treatment or prevention and glomerular volume increase the method for relevant nephropathy, and this method comprises and give patient or suspected patient CTGF inhibitor, thus treatment or prevent this disease.

Method of the present invention and chemical compound also are used to prevent, alleviate or postpone to occur the outbreak of this complication of object of the relevant renal complication risk of end-stage renal disease, or be used for making medicine, this medicine is used to have any disease relevant with end-stage renal disease described here or the object of symptom, preferred human subjects.On the one hand, the glomerular filtration rate of described human subjects is lower than normal glomerular filtration rate, for example, is lower than about 120 ml/min.Therefore, the glomerular filtration rate human subjects that is lower than about 120 ml/min, is lower than about 90 ml/min, is lower than about 60 ml/min, is lower than about 30 ml/min or is lower than about 15 ml/min is considered to the proper object with method provided by the invention or medicinal usage treatment.

In different embodiments, consider also that the rising glomerular filtration rate reduces or the method for impaired human subjects (for example, explicitly diabetic nephropathy patient) glomerular filtration rate can be used to raise glomerular filtration rate to being higher than about 15 ml/min, being higher than about 30 ml/min, being higher than about 60 ml/min, being higher than the level of about 90 ml/min and the level that arrives about 120 ml/min.

In some embodiment, described nephropathy is relevant with 1 type or type 2 diabetes mellitus.In other embodiment, described nephropathy is a diabetic nephropathy.

Microalbuminuria

The early stage clinical evidence that comprises the nephropathy of diabetic nephropathy is that albumin level reduces unusually in the urine, and this symptom is called as microalbuminuria.The individuality of the microalbuminuria initial stage nephropathy of being thought suffering from is arranged, perhaps, if relevant with diabetes then suffer from the initial stage diabetic nephropathy.There is the diabetic individual of microalbuminuria to develop into the risk of explicitly diabetic nephropathy than the individual height 42% of normal albuminuria (normoalbuminuria) (Bruno etc., 2003, Diabetes Care 26:2150-2155).Therefore, diabetic individual occur with the development microalbuminuria with develop into explicitly diabetic nephropathy (being big albuminuria) and final latter stage nephropathy and the risk of renal failure and improve relevant greatly.(see, for example, Mogensen and Christensen (1984) N Engl J Med 311:89-93; Mogensen etc., (1983) Diabetes 32[supplementary issue 2]: 64-78; Viberti etc. (1982) Lancet 1:1430-1432.)

Microalbuminuria can in all sorts of ways definite, comprising: (1) is checked and accepted to concentrate in urine at random and is measured albumin: creatinine ratio; (2) twenty-four-hour urine liquid is collected and is measured kreatinin, measures creatinine clearance simultaneously; (3) regularly (for example, 4 hours or a night) collection.Human normal urine albumin excretion rate is less than 30 micrograms/milligram kreatinin (point is collected), less than 30 milligrams/24 hours (collecting in 24 hours), perhaps less than 20 micrograms/minute (regularly collecting).Microalbuminuria patient's urinary albumin excretion rate is 30-299 microgram/milligram kreatinin (point is collected), 30-299 milligram/24 hour (collecting in 24 hours), perhaps 20-199 microgram/minute (regularly collecting).Big albuminuria (for example, clinical albuminuria) patient's urinary albumin excretion rate is more than or equal to 300 micrograms/milligram kreatinin (point is collected), more than or equal to 300 milligrams/24 hours (collecting in 24 hours), perhaps more than or equal to 200 micrograms/minute (regularly collecting).

The very first time of the present invention confirms, suppresses CTGF (for example, by giving CTGF antibody) and can reduce the urinary albumin excretion rate relevant with nephropathy especially diabetic nephropathy.(see that for example, embodiment 1.) the urinary albumin excretion rate raises with the albumin permeability of glomerule and optionally changes relevantly, and is in early days or nephropathy change in renal function or impaired indication in developing.On the one hand, the invention provides by suppressing the method that CTGF reduces the urinary albumin excretion rate.On the other hand, the invention provides by suppressing CTGF reduction glomerule albumin permeability and recovering optionally method of glomerule.Again on the other hand, the invention provides by suppressing the method that CTGF alleviates microalbuminuria.Therefore, by reducing microalbuminuria and urinary albumin excretion rate, method of the present invention provides the method for treatment early nephropathy and initial stage nephropathy.

As mentioned above, in the nephropathy, the generation of microalbuminuria (being the initial stage nephropathy) increases relevant with the risk that big albuminuria, explicitly nephropathy, latter stage nephropathy and renal failure take place for development and diabetics in early days.Therefore, method and composition of the present invention also can be used for preventing, alleviating or postpone to occur the outbreak of these complication of object of the relevant renal complication risk of end-stage renal disease, and described complication comprises big albuminuria, explicitly nephropathy, latter stage nephropathy and renal failure.

The present invention confirms, suppresses CTGF (for example, by giving CTGF antibody) and can reduce and nephropathy proteins associated urine, BUN level and creatinine clearance.It is change in renal function or indication impaired and the nephropathy development that albuminuria, BUN level and creatinine clearance raise.On the one hand, the invention provides by suppressing CTGF and alleviate albuminuretic method and chemical compound.On the other hand, the invention provides by suppressing method and the chemical compound that CTGF reduces the BUN level.On the other hand, provide by suppressing method and the chemical compound that CTGF reduces creatinine clearance.

The present invention confirms, suppresses CTGF (for example, by giving CTGF antibody) and can improve renal function.When diabetic nephropathy proceeded to end-stage renal disease, for example reducing the glomerular filtration rate decline that records by inulin clearance was change in renal function or impaired indication.The present invention also confirms, suppresses CTGF (for example, by giving CTGF antibody) and has improved the impaired or reduction of the glomerular filtration rate relevant with end-stage renal disease.(see embodiment 3.) on the one hand, the invention provides by suppressing method and the chemical compound that CTGF improves glomerular filtration rate.On the other hand, the invention provides by suppressing method and the chemical compound that CTGF reduces inulin clearance.The method and the chemical compound of especially relevant with nephropathy such as diabetic nephropathy impaired renal function more on the other hand, are provided by suppressing CTGF treatment or prevention impaired renal function.On the other hand, described nephropathy is relevant with glomerular filtration rate reduction, big albuminuria or explicitly nephropathy.

Late period, diabetic nephropathy was relevant with the various pathology and the metamorphosis of kidney.This variation comprises that producing the increase glomerular mesangium relevant with the accumulation of glomerular mesangium extracellular matrix with substrate expands; The mesangial cell cell enlarges; The glomerular basement membrane thickening relevant in the late period diabetic nephropathy with glomerulosclerosis; And the development of renal tubules interstitial fibrosis.(Gilbert etc., (1999) Kidney Int 56:1627-1673.Thereby) glomerulosclerosis and renal tubules interstitial fibrosis be with renal insufficiency cause glomerular filtration rate to reduce and may cause latter stage nephropathy and renal failure late period diabetic nephropathy structural end-stage renal disease sign.

Before the present invention, CTGF is relevant with the feature in late period of kidney pathology, especially produces excessive extracellular matrix, glomerular mesangium substrate overextends and glomerulosclerosis (glomeruloscleorsis) and renal tubules interstitial fibrosis take place.(see, for example, international publication number WO 00/13706.) think that other factor such as VEGF causes the process relevant with early nephropathy, pathology and various feature, for example excessively filter and the increase of glomerule permeability.On the contrary, digital proof provided by the invention, the early stage of nephropathy and late period symptom development and progress in play a significant role, so be the desirable target spot for the treatment of diabetic nephropathy fully and effectively.

The invention provides the method for the various clinical and pathology methods of treatment and prevention diabetic nephropathy in early stage and late period.Specifically, method and composition of the present invention can be used for treatment or excessively filtration and the expansion of glomerular mesangium substrate of prevention glomerule.Therefore, the present invention has considered treatment nephropathy various aspects, comprises such as kidney and the loose and excessive method that filters early diabetic nephropathy features such as (being measured as creatinine clearance rising, the rising of urinary albumin excretion rate, glomerular filtration rate rising etc.) and diabetic nephropathy feature in late period (glomerular filtration rate reduction, the expansion of glomerular mesangium substrate, basement membrane thickened etc.) of glomerule.

The invention provides and be used for the treatment of with the method and composition of Connective Tissue Growth Factor (CTGF) as the disease or the symptom of mediated factor.Number of C TGF relevant disease has been described in the document; Yet all thought the main and fiber proliferative symptom of CTGF before the present invention, especially those symptoms relevant with TGF β are relevant.Manyly relate to the disease of fiber breeding and use although mentioned at providing or preventing that the active therapy of CTGF from treating these diseases, the present invention has enlarged this understanding, so the therapy of CTGF orientation is used for treating various non-fiber proliferative symptom and the complication relevant with diabetic nephropathy and nephropathy.

By for example measuring glomerular filtration rate, urinary albumin excretion rate, albuminuria and/or albuminuria as can be known, method of the present invention for example, suppresses CTGF, effectively reduces the excessive filtration of kidney and makes renal function normal or recover.Therefore, method and composition of the present invention can be used to treat the patient of diabetic nephropathy risk, comprises for example early diabetic nephropathy and initial stage diabetic nephropathy.This object comprises that diagnosis has the individuality of hyperglycemia, hypertension and/or diabetes.In addition, method of the present invention can be used to treat the patient who is diagnosed with nephropathy such as glomerulosclerosis, glomerulonephritis or diabetic nephropathy.

Method of the present invention for example, suppresses CTGF, has alleviated expansion of glomerular mesangium substrate and glomerular basement membrane thickening.Therefore, method of the present invention can be used to treat the patient of diabetic nephropathy danger with prevention albuminuria, glomerular filtration rate reduction etc.This object comprises that diagnosis has the individuality of hyperglycemia, hypertension and/or diabetes.In addition, method of the present invention can be used to treat the patient of explicitly diabetic nephropathy or other nephropathy such as glomerulosclerosis, glomerulonephritis etc.

Therefore, on the one hand, the present invention has considered that CTGF treats or the method for the process that prevention is relevant with early nephropathy or end-stage renal disease by suppressing.These pathological conditions comprise, for example, and excessively filtration, albuminuria, albuminuria, glomerule hypertrophy and glomerular mesangium volume expanded.Considered to treat or prevent the aspect of in the past relevant with TGF β early stage and end-stage renal disease especially with VEGF with method of the present invention.As mentioned above, this method can be used to treat the patient of diabetic nephropathy or related pathologies risk and the patient that treatment suffers from nephropathy such as glomerulosclerosis (glomeruloscerosis), glomerulonephritis, diabetic nephropathy.

The present invention considers method of the present invention and other therapies coupling.In one embodiment, described method and another kind of therapy coupling, for example, with the therapeutic effect of further enhancing to some pathology affair.For example, can be in the treatment time-histories or in progression of disease with alleviate the back simultaneously or give this two kinds of treatments in succession.In another embodiment, described method and another kind of Therapeutic Method coupling, for example ACE inhibitor, ARB, inhibin, advanced glycosylation end product (AGE) inhibitor etc. with similar or different binding modes.At present the therapeutic scheme of treatment diabetic nephropathy is that those skilled in the art are known, ACE inhibitor, angiotensin receptor blocker, inhibin, advanced glycosylation end product inhibitor, liver cell growth factor gene treatment, pyridoxamine, enalapril (Enapril), PPAR antagonist, sulfonylurea, matrix metallo-proteinase inhibitor, cox 2 inhibitor, pirfenidone are for example arranged, wishes Shu Luo, high dose thiamine and benfotiamine, calcium channel blocker etc.Consider purposes especially with any of these therapeutic agent of method coupling of the present invention.

The present invention provides the verified curative effect to two kinds of different nephropathy related pathologies aspects (for example the early sign of diabetic nephropathy and late period feature) first.Although anti--CTGF therapy here is to adopt the human monoclonal antibodies of anti-CTGF for example, the present invention includes any inhibition CTGF encoding gene and express, suppress the CTGF generation or suppress the active method of CTGF.For example, available micromolecular compound suppresses the CTGF expression, produces or activity.When suppressing CTGF and express with the ring nucleus thuja acid, this chemical compound can comprise, for example, and ring-type nucleotide analog or phosphodiesterase (PDE) inhibitor.(see, for example, Duncan etc., (1999) FASEB J 13:1774-1786.) in addition, comprise that the polynucleotide of small interference ribonucleic acid (siRNA), Microrna (miRNA), ribozyme and antisense sequences can be used for method of the present invention to suppress the CTGF expression and/or to produce.(see, for example, Kondo etc., (2000) BiochemBiophys Res Commun 278:119-124; With Shimo etc., the same.) this technology is that the association area those of skill in the art know.

The invention provides exemplary evidence proves, anti--CTGF monoclonal anti body method can improve creatinine clearance and glomerule hypertrophy, reduction kidney weight, inhibition TGF is beta induced and the glomerule fibrosis CTGF mediation and glomerular mesangium expansion in described here using in diabetes animal model.Therefore, method of the present invention has been improved two kinds of pathology of diabetic nephropathy, is glomerular mesangium expansion and glomerular filtration.

In some aspects, the invention provides by suppressing the method and composition that CTGF treats the TGF ss related diseases.On the other hand, the invention provides by suppressing the method and composition that CTGF treats the VEGF relevant disease.Again on the other hand, the invention provides by suppressing the method and composition that CTGF treats TGF β and VEGF relevant disease.Method and composition of the present invention also can be used to treatment and other somatomedin such as diseases associated such as IGF-1, endothelin.

On the one hand, the invention provides the method for treatment or the prevention nephropathy relevant with the creatinine clearance rising, this method comprises that the object that gives the patient or ill risk is arranged suppresses the reagent of CTGF (for example, suppressing or reduction CTGF expression or CTGF activity), thereby treats or prevent this nephropathy.On the other hand, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat or prevention and glomerular filtration increase and excessively filter the method for relevant nephropathy, thus treatment or prevent this disease.On the other hand, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat or the method for the nephropathy that prevention is relevant with basement membrane thickened, thus treatment or prevent this disease.On the other hand, the invention provides by giving the patient or have the object CTGF inhibitor of ill risk to treat or prevent and urinate volume and increase the method for relevant nephropathy, thus treatment or prevent this disease.Also provide by giving the patient or have the object CTGF inhibitor of ill risk to treat and the raise method of relevant nephropathy of urinary albumin excretion rate, thus treatment or prevent this nephropathy.

On the one hand, the invention provides the method for creatinine clearance that reduction needs the object of this treatment, described method comprises and gives patient CTGF inhibitor.The urinary albumin excretion rate that reduces also is provided in the object of this treatment of needs, has reduced glomerular filtration and excessively filter, alleviate the glomerular volume expansion or alleviate the method that kidney weight increases, described method comprises and gives patient CTGF inhibitor.In one embodiment, the invention provides the method for treatment or prevention and nephropathy proteins associated urine, this method comprises the object CTGF inhibitor that gives the nephrotic or ill risk is arranged.In the embodiment, described albuminuria is an albuminuria again.In each embodiment, described albuminuria is microalbuminuria or big albuminuria.In another embodiment, the invention provides the method for treatment or prevention kidney basement membrane thickened, this method comprises the patient who gives the kidney basement membrane thickened or the object CTGF inhibitor of ill risk is arranged.Again in another embodiment, the invention provides by urinating the method that patient that volume increases or the object CTGF inhibitor that ill risk is arranged alleviate or prevent the increase of urine volume.

Method of the present invention comprises the reagent (for example, reducing CTGF expression or active) of the inhibition CTGF of the object treatment effective dose that needs treatment.In some embodiment, described reagent is CTGF antibody.In preferred embodiments, described antibody is the monoclonal antibody of CTGF.In another preferred embodiment, described antibody is people or humanization CTGF antibody.In another embodiment, described reagent is micromolecule.In another embodiment, described reagent is antisense oligonucleotide.

Identified all ingredients that suppresses CTGF.U.S. Patent No. 5,408,040; International publication number WO99/07407; International publication number WO 99/33878; With the antibody of having described among the international publication number WO 00/35936 in conjunction with CTGF.A kind of exemplary antibodies that is used for the inventive method is described in international publication number WO 2004/108764, and the document is included this paper in as a reference in full.Can adopt the known any method of those skilled in the art to use this antibody or its fragment.For example, normally intravenous, intraperitoneal or hypodermic of antibody.

Suppressing CTGF expression and/or active micromolecule also described; For example, international publication number WO96/38172 with cAMP instrumentality such as cholera toxin and 8Br-cAMP as the CTGF expression inhibitor.Therefore, such as prostaglandin and/or prostacyclin analogs such as iloprost (see, for example, international publication number WO00/02450; Ricupero etc., (1999) Am J Physiol 277:L1165-1171; Also can be referring to Ertl etc., (1992) Am Rev Respir Dis 145:A19) and potential phosphodiesterase IV inhibitors (see, for example, Kohyama etc., (2002) Am J Respir Cell Mol Biol 26:694-701) etc. chemical compound can be used to regulate CTGF and express.Simultaneously, especially p38, the kinases that depends on cyclin such as CDK2 and glycogen synthase kinase (GSK)-3 also can reduce the expression of CTGF to the inhibitor of the mitogen-activated protein kinase of serine/threonine.(see, for example, Matsuoka etc., (2002) Am J Physiol Lung Cell Mol Physiol 283:L103-Ll 12; Yosimichi etc., (2001) Eur J Biochem 268:6058-6065; International publication number WO01/38532; With international publication number WO03/092584.) these reagent can be used to reduce thereby CTGF express to improve or the prevention arthrosis in the inductive pathological process of CTGF.This chemical compound can be prepared and use according to means known in the art.

Antisense technology comprises that small interference ribonucleic acid (siRNA), Microrna (miRNA), ribozyme and the antisense sequences of expressing at CTGF also can be used to treat arthrosis.(see, for example, Zeng (2003) Proc Natl Acad Sci USA100:9779-9784; And Kurreck (2003) Eur J Biochem 270:1628-1644.) the targeting CTGF antisense construct thing of expressing described and be used to reduce the expression of CTGF in the various cell types.(see, for example, international publication number WO 96/38172; International publication number WO 00/27868; International publication number WO 00/35936; International publication number WO 03/053340; Kothapalli etc., (1997) Cell Growth Differ 8 (1): 61-68; Shimo etc., (1998) J Biochem (Tokyo) 124 (1): 130-140; With Uchio etc., (2004) Wound Repair Regen12:60-66.Thereby) expression that can be used to reduce CTGF of this antisense construct thing alleviate or the prevention joint disease in the inductive pathological process of CTGF.Available suitable carriers and express instrumentality and design this construction with cell-or tissue-specific expressed and composing type or inducible expression.This genetic constructs is made and used to the method that available this area has been set up.

Pharmaceutical preparation and route of administration

Know as this field, compositions of the present invention can directly be transmitted or form transmission from the pharmaceutical composition that contains excipient.Therapeutic Method of the present invention can comprise suffering from diabetic nephropathy, especially excessively filters with glomerule for example and the object of diseases related such as glomerule hyperperfusion (hyperperfusion), microalbuminuria, initial stage diabetic nephropathy, big albuminuria, explicitly nephropathy or have the object of ill risk to treat the The compounds of this invention of effective dose.In a preferred embodiment, described to liking mammal, in the most preferred embodiment, described to liking the people.

Chemical compound or effective amount of drug as dosage, are not difficult to determine with routine test, can be used as effectively and route of administration and appropriate formulations easily.(see that for example, Gennaro compiles, (2000) " Lei Mingdun pharmaceutical science " is the same for existing various preparations in this field and drug delivery system; And Hardman, Limbird and Gilman compile, and (2001), " pharmacological basis of treatment " (The Pharmacological Basis of Therapeutics), the same).

Suitable route of administration can comprise, for example, and oral, rectum, part, nose, lung, eye, intestinal and intestines and stomach external administration.Main intestines and stomach external administration approach comprises intravenous, intramuscular and subcutaneous administration.Second kind of route of administration comprises in intraperitoneal, intra-arterial, intraarticular, intracardiac, the brain pond, in the Intradermal, intralesional, ophthalmic, pleura, in the sheath, administration in intrauterine and the ventricle.The indication that should preferably will treat is with physics, the chemistry and biology performance of medicine, regulation preparation type and used route of administration, and part or whole body is defeated passs whether.

The pharmaceutical dosage form of The compounds of this invention can promptly be released, controlled release, slow release or targeted drug delivery system provide.Dosage form commonly used for example comprises, solution, suspension, (little) Emulsion, ointment, gel and paster, liposome, tablet, dragee, soft or hard capsule, suppository, ovulum, implant, amorphous or crystallization shape powder, aerosol and lyophilized formulations.The route of administration that depends on use may need special device to apply or give medicine, for example, and syringe and syringe needle, inhaler, pump, injection pen, applicator or special-purpose bottle.Pharmaceutical dosage form often comprises medicine, excipient and container/sealing system.Can in chemical compound of the present invention, add one or more and be called the excipient of non-activity composition, with improve or help making, the administration and the safety of stability, medicine, a kind of instrument of realizing the required delivery mode of medicine can be provided.Therefore, the type that adds the excipient in this medicine depends on multiple factor, for example the physicochemical property of medicine, route of administration and manufacture method.This field of pharmaceutically acceptable excipient has, comprise listed those in the various pharmacopeia (see, for example, American Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP) and British Pharmacopoeia (BP); FDA's webpage (www.fda.gov), " inert fraction guide " (InactiveIngredient Guide) 1996 and " medicated premix handbook (Handbook of Pharmaceutical Additives), Ash volume; Synapse Information Resources Inc., 2002.)

Any method that available this field is known prepares the pharmaceutical dosage form of The compounds of this invention, as the mixing method of routine, sieve, dissolve, thawing, granulation, dragee preparation, film-making, suspension, extruding, spray drying, grinding, emulsifying, (Nano/micron) wrap up, hold back or lyophilizing processing.Compositions of the present invention as mentioned above can comprise one or more physiologically acceptable non-activity compositions, is used for medicinal usage to help that bioactive molecule is processed into preparation.

Appropriate formulations depends on required route of administration.For intravenous injection, for example, said composition can be mixed with aqueous solution, if desired, can adopt the physiological compatibility buffer agent, for example comprises, phosphoric acid, histidine or citric acid are regulated the pH of preparation and adopted tension regulator such as sodium chloride or glucose.For through mucous membrane or intranasal administration, preferred semisolid, liquid preparation or paster can contain penetration enhancers, and this class penetration enhancers is that this field is known usually.For oral administration, this chemical compound can be mixed with the liquid or solid dosage form and promptly release or controlled release/slow releasing preparation.Be fit to the oral dosage form of object and comprise tablet, pill, sugar pill, hard and soft shell capsule, liquid, gel, syrup, syrup, suspension and Emulsion.This chemical compound also can be formulated in the rectal compositions, in suppository or enema,retention, as contains conventional suppository bases such as cocoa butter or other glycerol.

Can adopt excipient to obtain solid oral dosage form, described excipient comprises filler, disintegrating agent, binding agent (do or wet), dissolving blocker, lubricant, fluidizer, antitack agent, cation exchange resin, wetting agent, antioxidant, antiseptic, coloring agent and aromatic.These excipient can be synthetic or natural origin.The example of this class excipient comprises cellulose derivative, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, lauric acid magnesium sulfate/sodium, mannitol, Polyethylene Glycol, polyvinylpyrrolidone, silicate, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid and salt thereof, sugar (being glucose, sucrose, lactose etc.), Pulvis Talci, tragacanth mucilage, vegetable oil (hydrogenant) and wax.The second alcohol and water can be used as the granulation auxiliary agent.In some cases, need wrap up tablet with the film of for example odour masking, anti-gastric acid film or delayed discharge film.Usually the natural and synthetic polymer of coupling and coloring agent, sugar, organic solvent and water wrap up tablet, produce sugar pill.When capsule during preferably with tablet, the hard or soft shell capsule that medicated powder, suspension or solution can be compatible is failed and is passed.

In one embodiment, can be local, for example, give chemical compound of the present invention as gel, (little) Emulsion, ointment, solution, (Nano/micron) suspension or foam by transdermal patches, semisolid or liquid preparation.For example can adopt penetration enhancer; Suitably select and combination lipotropy, hydrophilic and both sexes excipient, comprise water, organic solvent, wax, oil, synthetic and natural polymer, surfactant, emulsifying agent, by regulating pH, adopting chelating agent, regulate the infiltration of medicine to skin and its undertissue.Can adopt other technology,, regulate the infiltration of The compounds of this invention skin as ionotherapy.Preferred percutaneous or topical, for example local defeated drug delivery under the situation that needs the contact of bottom line whole body.

For inhalation or intranasal administration, chemical compound of the present invention can solution, the semi-solid aerosol form of suspension, Emulsion or pressurized package or adopt the aerosol apparatus of propellant usually, and the halocarbon that produces as methane and ethane, carbon dioxide or any other suitable gas transmits easily.For local aerosol, can adopt butane, iso-butane and pentane hydrocarbon.With regard to pressurized aerosol, can determine suitable dosage unit by a defeated aerocolloidal valve of definite amount of passing is provided.Can prepare the capsule and for example gelatin box that are used for inhaler or insufflator.These capsules or box contain the mixed-powder of chemical compound and the powder substrate that is fit to usually, as lactose and starch.

Usually with unit dosage forms,, provide the aseptic composite that is used for the outer injection of gastrointestinal tract of preparation as in ampoule, syringe, injection pen or multi-dose container (often containing antiseptic).This compositions can be taked suspension, solution or oiliness Emulsion or aqueous carrier form, can contain reagent preparation, as buffer agent, penetrating agent, viscosity-increasing agent, surfactant, suspension and dispersant, antioxidant, biocompatible polymer, chelating agen and antiseptic.Depend on the injection site, carrier can moisture, synthetic oil or vegetable oil and/or organic cosolvent.Under some situation, during as freeze-drying prods or concentrated product, can before administration, rebuild or dilute gastrointestinal tract external administration preparation.The durative action preparation of The compounds of this invention controlled release or slow release can be provided, can comprise Nano/micron grain or Nano/micron level or the crystalline injectable suspensions of non-littleization.Polymer, those that know except that this field, as poly-(lactic acid) poly-(glycolic), or its copolymer or as controlled release/sustained-release matrix.Can implant provide other long-acting delivery system with the pump form that needs cutting.

The carrier of well known suitable intravenous injection molecule of the present invention, comprise can form the ionizing chemical compound contain alkali such as sodium hydroxide, as the sucrose of penetrating agent or the group water solution of sodium chloride, for example, the buffer of phosphoric acid or histidine.Can add cosolvent, as Polyethylene Glycol.These water based systems can effectively be dissolved chemical compound of the present invention, produce hypotoxicity when the whole body administration.The component ratio of solution system is can be quite different and do not destroy dissolubility and toxic characteristic.In addition, the homogeneity of component can change.For example, can adopt the surfactant of low toxicity, as Polysorbate or poloxamer, can add Polyethylene Glycol or other cosolvent, biocompatible polymer such as polyvinylpyrrolidone, other sugar and polyhydric alcohol replace glucose.

For present Therapeutic Method compositions for use, originally available various technology well known in the art are estimated the treatment effective dose.The used initial dosage of zooscopy can be decided according to the valid density that cell culture test is determined.The dosage range that is fit to human can be determined with the data that zooscopy and cell culture obtained.

The treatment effective dose or the dosage of chemical compound of the present invention, medicament or medicine refer to that this chemical compound or medicine can cause the remission of object or amount or the dosage that existence prolongs.The toxicity of this quasi-molecule and therapeutic effect can be determined by the pharmacy program of carrying out standard in cell culture or experimental animal, for example, determine by measuring LD50 (dosage that causes the death of 50% colony) and ED50 (the effective dosage of 50% mass treatment).The dose ratio of toxicity and curative effect is a therapeutic index, and available LD50/ED50 compares value representation.The preferred preparation that shows high therapeutic index.

Effective dose or treatment effective dose are that tissue, system, the animal or human that researcher, veterinary, doctor or other clinical staff are sought brings out the described chemical compound of biology or medical response or the amount of pharmaceutical composition, for example, can reduce creatinine clearance, glomerule excessively filtration and hyperperfusion, urinary albumin excretion or microalbuminuria, treatment is early stage or late period diabetic nephropathy, or the like.

Preferred dosage should comprise ED50 and have little or do not have in the toxic circulation composition scope.Dosage can change according to used dosage form and/or route of administration in this scope.Should be according to means known in the art according to the accurate preparation of the feature selection of object situation, route of administration, dosage and spacing of doses.

Can adjust dosage individually and can fully obtain required effect to provide at interval, as regulating glucose metabolism, reducing blood sugar level etc., i.e. minimal effective concentration (MEC).The MEC of each chemical compound is different but can estimate from for example vitro data and animal experiment.The dosage that needs to obtain MEC depends on individual feature and route of administration.With regard to topical or selectivity absorption, effective local concentration of medicine may be irrelevant with plasma concentration.

Can be according to multiple factor, the preparation that gives or the amount of compositions are determined in the order of severity, administering mode and doctor's the judgement that comprises sex, age and body weight, the disease of object to be treated.

If desired, compositions of the present invention can be contained in the filling or dispersal device that contains one or more unit dosage forms (containing active component).For example, this filling or device can comprise metal or plastic foil, load in thing or glass and rubber closure such as the bottle as bubbling.This filling or dispersal device can be equipped with the administration description.Also can prepare the compositions that contains The compounds of this invention with the preparation of compatibility pharmaceutical carriers, place proper container, certain indicates the label of disease to stick treatment.

By reading this paper content, those of ordinary skills are not difficult to implement these and other embodiment of the present invention.

Embodiment

Referring to following examples is that the explanation embodiments of the invention will further be understood the present invention purely.Provide these embodiment just for claim of the present invention is described.The invention is not restricted to purpose is the described scope of one exemplary embodiment of explanation one aspect of the invention.Any method that equates on the function all within the scope of the invention.Those skilled in the art understand that as described above and accompanying drawing can make various modifications beyond described herein to the present invention.It is in the scope of additional claims that this class is revised.

Embodiment 1: the early sign of treatment diabetic nephropathy

Confirm wide spectrum effect to some aspect animal model of early diabetic nephropathy, following carrying out with method of the present invention.From Harlan, Indianapolis IN obtains disappearance leptin receptor (Ob-R; By the db gene code) function mutation 8 the week age mice.These db/db mices are as the type 2 diabetes mellitus fat animal model, especially the early stage aspect with diabetic nephropathy is the fat model of type 2 diabetes mellitus nephropathy of feature, described early stage aspect comprises, for example, and excessive filtration of kidney and albuminuria and with slight interstitial fibrosis.By slight interstitial fibrosis can prove this be early diabetic nephropathy rather than late period the diabetic nephropathy animal model.The db/db (diabetes) that isozygotys has hyperglycemia when 8 ages in week.Isozygoty db/db (diabetes) and heterozygosis db/+ (non-diabetic) animal handled (peritoneal injection) with anti--CTGF monoclonal antibody (α CTGF) (is the cell line preparation of PTA-6006 by the description preparation of international publication number WO 2004/108764 or with the accession number in preservation on the 20th May in 2004 that ATCC identifies) or contrast human IgG (cIgG).All animals are injected 300 microgram antibody, inject 100 micrograms then, 3 times weekly, totally 60 days.When the processing phase begins and period ground collect blood sample and measure body weight.Write down food consumption simultaneously.

Following table 1 has shown the 0th day and the db/+ mice of cIgG processing in the 60th day, the db/+ mice that α CTGF handles, the diabetes db/db mice of cIgG processing and average weight (BW), blood sugar level (BG) and the food consumption (FC) of the db/db mice that α CTGF handles.All data are expressed as meansigma methods ± SEM.The number of every group of mice (n) is 9-15.Non-diabetic (db/+) animal that polycystic kidney occurs is excluded outside analyzing.As shown in table 1, body weight, blood sugar level and the food consumption of diabetes (db/db) animal and non-diabetic (db/+) animal have marked difference.TBW increase, blood sugar level or the food consumption handled diabetes (db/db) or non-diabetic (db/+) animal with anti--CTGF antibody or cIgG have no significant effect.

Table 1

Group is handled (number of animals)	The 0th day			The 60th day
							BW (gram)	BG (mM)	FC (restraining/24 hours)	BW (gram)	BG (mM)	FC (restraining/24 hours)
	db/+，cIgG (n＝11)	20.6±0.3	6.3±0.2	4.8±0.1	22.4±0.4	6.0±0.2							5.0±0.1
db/+，αCTGF (n＝9)							20.2±0.4	6.1±0.2	4.7±0.2	21.6±0.3	6.2±0.1	5.1±0.2
	db/db，cIgG (n＝15)	39.4±0.6 *	16.7±1.0 *	10.0±0.2 *	47.1±1.0 *	22.1±0.8 *							10.8±0.2 *
db/db，αCTGF (n＝14)							38.0±1.0 *	15.8±0.7 *	10.2±0.1 *	47.7±1.2 *	21.7±0.8 *	10.5±0.1 *

Data are expressed as meansigma methods ± SEM. ^*Compare db/+ mice P＜0.01.

The progress that suppresses the early sign of diabetic nephropathy

Above-mentioned anti--obtain the various measurements of renal function and nephropathy after the CTGF antibody treatment phase, comprise kidney weight, creatinine clearance, urinary albumin excretion rate and urinate volume.Following table 2 has shown db/+ mice, the db/+ mice that α CTGF handles, the db/db mice of cIgG processing and the 60th day average kidney weight (KW) of db/db mice, creatinine clearance (CrCl) and the twenty-four-hour urine albumin excretion rate (UAE) that α CTGF handles that cIgG handles.All data are expressed as meansigma methods ± SEM.The number of every group of mice (n) is 9-15.As mentioned above, non-diabetic (db/+) animal that polycystic kidney occurs is excluded outside analyzing.

Table 2

Group is handled (number of animals)	KW (milligram)	CrCl (milliliter/hour)	UAE (microgram/24 hour)
				db/+，cIgG (n＝11)	133.8±5.1	2.17±0.29	0.30±0.02
db/+，αCTGF (n＝9)	141.0±4.3	2.37±0.19	0.23±0.04
				db/db，cIgG (n＝15)	207.8±3.9 **	5.39±0.36 **	2.52±0.20 **
db/db，αCTGF (n＝14)	177.4±4.5 *	2.76±0.31 △	0.98±0.09 □

Data are expressed as meansigma methods ± SEM. ^*Compare db/+ mice P＜0.01. ^*Compare db/+ mice P＜0.01 and compare db/db mice P＜0.05 that cIgG handles. ^△Compare db/db mice P＜0.01 that cIgG handles. Compare db/db mice p＜0.01 that db/+ mice and cIgG handle.

As shown in table 2, as kidney expansion (being that kidney weight increases) (Fig. 1), creatinine clearance raises (Fig. 2) and urinary albumin excretion rate rising (Fig. 3, * compare db/+ mice P＜0.01 that anti--CTGF handles) pointed, the kidney Presentation Function of db/db mice is hyperfunction.Comparing the diabetic animal of handling with cIgG with the diabetic animal of anti--CTGF antibody treatment demonstrates kidney weight and increases less.

The creatinine clearance of the db/db animal that cIgG handles is about the twice of observed value in the db/+ animal, illustrates that diabetic animal has impaired renal function, hypertrophy and excessively filtration.Compare observed urinary albumin excretion rate in non-diabetic db/+ animal, the urinary albumin excretion rate of diabetes db/db animal has also improved.Creatinine clearance and urinary albumin excretion rate level with the db/db animal of anti--CTGF antibody treatment significantly are lower than observed value in the db/db animal that cTgG handles.Specifically, the creatinine clearance level of the diabetic mice of anti--CTGF processing is than observed value in the diabetic mice of handling at cIgG low 82%.The urinary albumin excretion rate level of the diabetic mice that anti--CTGF handles is than observed value in the diabetic mice of handling at cIgG low 69%.These results confirm that the renal function of the mice of anti--CTGF antibody treatment has surprising improvement.Handle the non-diabetic animal with method of the present invention and show that kidney weight or function are not had ill effect.These data show, give diabetic animal anti--the CTGF antibody capable reduces kidney weight increase, creatinine clearance and urinary albumin excretion rate.

In addition, compare non-diabetic (db/+) mice, diabetes (db/db) mice demonstrates the urine volume of increase.Resist as mentioned above-CTGF antibody reduced the urine volume of diabetes (db/db) mices.(see Fig. 5, db/+ mice P＜0.01 of anti--CTGF processing that * compares.) these data point out, give diabetic animal anti--CTGF antibody reduced the urine volume.These results also point out, suppress CTGF and can reduce the urine volume increase relevant with diabetic nephropathy, and therefore the method for improving renal function is provided.

The variation of analyzing glomerular volume (for example, the minimizing of glomerular volume expansion) and basement membrane thickened has also confirmed to suppress the effect of CTGF for treatment and diabetes and nephropathy preventing development and progress.(db/+ of anti--CTGF processing that * is not different from) reduced basement membrane thickened with anti--CTGF antibody treatment diabetes (db/db) animal as shown in Figure 6.

In a word, these data show have alleviated renal hypertrophy (can be confirmed by the kidney weight that has reduced the diabetic animal that anti--CTGF handles) and have recovered renal function (can be confirmed by the creatinine clearance and the homaluria rate that have reduced the diabetic animal that anti--CTGF handles) with anti--CTGF antibody treatment diabetes (db/db) animal.These results also point out, suppress CTGF and can reduce glomerule permeability and excessively filtration, alleviate glomerular mesangium expansion and basement membrane thickened simultaneously.Therefore, suppress the early sign that CTGF can treat diabetic nephropathy.

Embodiment 2:CTGF participates in the early sign of carrying out property vitreoretinopathy

CTGF and the relation that comprises the oculopathy of retinal disorder have been set up before.(see, for example, international publication number WO 03/049773.) detected the eye concentration of CTGF and VEGF and the relation between neovascularization and the fibrosis here determining, if any, the relation between vitreous body CTGF and the vegf expression.Prompting CTGF and early stage with late period the oculopathy symptom relevant.The proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), macular pucker or the macular hole patient that carry out the ciliary ring vitrectomy from preparation obtain undiluted vitreous body sample (0.5-1 milliliter).The vitreous humor sample collection uses dry ice freezing in sterile tube immediately, and is stored into mensuration CTGF and VEGF in-80 ℃.

The neovascularization following classification relevant with all retinal disorders: 0 grade, no neovascularization; 1 grade, only there is not dabbling colloid pipe (gliotic vessel) in static neovascularization; With 2 grades, dynamically neovascularization has the preceding capillary tube of dabbling retina.(see Aiello etc., (1994) N Engl J Med 331:1480-1487.)

Level by CTGF and VEGF in the ELISA measurement vitreous body sample.In brief, with the vitreous body sample 14, centrifugal 15 minutes of 000rpm, and collect supernatants in 4 ℃.Two kinds of monoclonal antibodies of personnel selection CTGF are measured the CTGF level by sandwich ELISA, the zones of different of every kind of monoclonal antibody specific recognition CTGF N-end portion, as mentioned above.Anti--CTGF the monoclonal antibody (10 mcg/ml) of catching is added coating buffer (50mM sodium borate, pH 9.6), spend the night with its coating microtitration plate at 4 ℃.Flat board sealed 2 hours in room temperature with buffered 1% BSA of phosphate-buffered saline with 100 microlitres, used lavation buffer solution (phosphate-buffered saline that contains 0.05% Tween 20) washing then.Vitreous body sample analysis buffer (50mM TRIS, pH 7.7,0.1% BSA, 4mM MgCl ₂, 400mM ZnCl ₂, 0.05% NaN ₃, 50 mg/litre heparin sodiums and 0.1% Triton X-100) and dilute 5 times.The vitreous body sample and the biotinylated monoclonal anti-human CTGF of 50 microlitres that add the dilution of 50 microlitres in each hole detect antibody (being diluted in analysis buffer).Dull and stereotyped 37 ℃ of cultivations 2 hours, wash with lavation buffer solution, and cultivated 1 hour in room temperature with the link coupled alkali phosphatase of the mould avidin of 100 microlitres/pore chain (1 mcg/ml is diluted in analysis buffer) (Jackson Immunoresearch Laboratories).Wash dull and stereotyped with lavation buffer solution after current the cultivation and in each hole, add 100 microlitres and be diluted in diethanolamine buffer (1M diethanolamine, 0.5mM MgCl ₂, 0.02% NaN ₃, pH 9.8) substrate solution (1 mg/ml, p-nitrophenyl phosphoric acid, SigmaChemical Co.).On Bio-Rad microplate reader, read the absorbance of 405 nanometers.With the recombined human CTGF of purification as standard.With commercially available sandwich ELISA according to (the R ﹠amp of manufacturer; D Systems) the vitreous body level of VEGF-165 is measured in explanation.

Observe CTGF level and the obvious positive correlation of neovascularization degree (p=0.0153).As shown in table 3 below, the patient's of neovascularization degree the highest (2 grades) CTGF level is apparently higher than the patient of neovascularization degree lower (0 grade or 1 grade).Fig. 4 show CTGF and the horizontal positive correlation of VEGF in the vitreous body (r=0.544, p=0.001).Obviously, these results confirm first, and are directly related between CTGF that records from the same sample of disease same phase and the VEGF level.

Table 3

The neovascularization rank	The CTGF level	95％CI
			0 grade	8.7 nanograms/milliliter	6.9-11.0
1 grade	14.0 nanograms/milliliter	11.2-17.5
			2 grades	20.8 nanogram/ml	16.2-26.9

Above-mentioned experimental result shows, CTGF be present in people's vitreous body and the existence of its concentration and neovascularization and degree significantly and relevant consumingly, the while vitreous body level of CTGF is relevant strongly with the vitreous body level of VEGF.Though the development of known CTGF and eye fibrosis and retinopathy aspect in other and make progress relevant in late period, in conjunction with as can be seen, CTGF and VEGF are the key factors that comprises the PD early-stage development of diabetic nephropathy and various vitreoretinopathies with the result of embodiment 1 in this result and the literary composition and embodiment 3.Therefore, the invention provides the method in early stage (for example, neovascularization) and late period (for example, fibrosis) stages of retinopathy such as treatment PVR, PDR.

Embodiment 3: the feature in late period of treatment diabetic nephropathy

Detected the effect of anti--CTGF therapy late in the diabetic nephropathy animal model.As mentioned above, employing to one-sided renal ischaemia again the diabetes rat of influx height susceptible as animal model, one-sided renal ischaemia again perfusion can cause rapidly carrying out property nephropathy and latter stage renal failure, be badly damaged simultaneously with fibrosis development, renal atrophy and glomerular filtration rate.(see, for example, Melin etc., (1997) Kidney Int 52:985-991.) in this diabetes animal model, the ischemia grievous injury renal function of diabetes rat.In this animal model, renal function and hyperglycemia and the pathological kidney effect of ischemia are similar to observed effect in people's diabetic nephropathy in late period and latter stage nephropathy (ESRD).

In male Sprague Dawley rat, bring out diabetes by a streptozotocin of intravenous injection (STZ) (50 mg/kg).Clamp left renal artery and in a kidney, caused one-sided renal ischaemia to pour into (IR) again in 30 minutes, thereby prevent that blood from flowing to left kidney.Renal ischaemia pours into preceding 1 day (being 2 week of diabetes development back) again to begin with anti--CTGF monoclonal antibody (peritoneal injection 5 mg/kg) treatment, and treatment 3 times weekly continued for 10 weeks.Give not accept to resist-the control animal PBS (peritoneal injection 5 ml/kg) of CTGF antibody.Obtain blood sample from the tail vein.The 0th, 4,8 and 10 weeks by Quality Clinical Labs, Inc. (Mountain View, CA) analyzing blood clinical chemistry.Determine 24 hours total urinary proteins in the 5th and 9 weeks.Be placed on each rat in the metabolic cage and collect the twenty-four-hour urine sample.Measure volume of urine and analyze urine protein with BCA protein determination test kit (Pierce Chemical Co.).

Glomerular filtration rate (GFR) is the extended measurements of renal function.Inulin clearance is the measurement of glomerular filtration rate.In these embodiments, determined the glomerular filtration rate (for example, renal function) of individual kidney by measuring urine volume and inulin clearance.Collect urine and collect blood by ureter from femoral artery.By gravimetric analysis estimation urine volume.Determine inulin concentration with anthrone method.Determine indication-inulin clearance of GFR with following formula: (U concentration * U volume)/S concentration.The excision kidney carries out biochemistry and histopathology evaluation when off-test.

Data are expressed as mean+/-SEM.The data of experimental group when adopting one way analysis of variance (ANOVA) and Student-Newman-Keuls method (SIGMASTAT) to compare each time point.When only comparing two groups, adopt t-check (two samples hypothesis is equal to variance analysis instrument, Microsoft Excel).It is significant that the value of P＜0.05 is considered to.

Rise as can be known from blood sugar level, the animal that gives single dose STZ suffers from diabetes.Blood sugar level is lower than 200 milligrams/deciliter and rises to animal that STZ handles greater than 600 milligrams/deciliter level from contrast (non-STZ handle) animal, illustrates that these animals suffer from diabetes.The kidney IR of non-diabetic (being that non-STZ handles) animal rises above the level (data not shown) of control animal blood sugar level.In 10 weeks behind one-sided kidney IR, the blood sugar level of the animal that STZ handles still raises.(data not shown.)

Late protein urine

The microalbuminuria feature of early diabetic nephropathy is made progress into big albuminuria and late protein urine.In diabetic animal, observe 24 hours total urinary proteins and significantly raise (being late protein urine), illustrate that glomerule excessively filters increase and renal failure development.As shown in Figure 7, non-diabetic animal (pseudo-(the sham)+PBS that handles; IR+PBS) total urinary protein is about 100 milligrams/24 hours.(in Fig. 7, in corresponding week, * represents to be higher than non-diabetic (p＜0.001), and # represents to be lower than DM+IR+PBS (p＜0.05).Yet), the total urinary protein horizontal exceeding 350 milligrams/24 hours of the diabetic animal of kidney IR is arranged.Compare with untreated diabetic animal, have the diabetic animal of kidney IR anti--CTGF antibody causes 24 hours total urinary proteins in the 5th and 9 weeks significantly to be reduced to respectively about 225 milligrams/24 hours and 250 milligrams/24 hours.(see Fig. 7.) this data show, give the albuminuria that CTGF antibody has reduced diabetic animal.These presentation of results, suppressing CTGF is to reduce excessively filterable a kind of Therapeutic Method of kidney.These results confirm that first anti--CTGF therapy can be used to prevent the development and the progress of late protein urine.

Blood urea nitrogen (BUN)

It is indication with the relevant impaired renal function of diabetic nephropathy in late period that the BUN level raises.Observing the BUN level in these diabetic animals significantly raises.The 0th, 4 and 10 weeks contrast non-diabetic animal (pseudo-processing+PBS in research; IR+PBS) BUN level is lower than 20 milligrams/deciliter.In the diabetic animal that kidney IR is arranged, the BUN level is elevated to more than 40 milligrams/deciliter from about 22 milligrams/deciliter of the 0th week.(see Fig. 8, in the 4th week, * represents to be higher than pseudo-processing+PBS and IR+PBS (p＜0.01), and # represents to be lower than DM+IR+PBS (p＜0.01).) with resisting-diabetic animal of CTGF antibody in observed value compare, have the diabetic animal of kidney IR anti--the CTGF monoclonal antibody causes the BUN level in the 4th week and the 10th week to drop to about 30 milligrams/deciliter and 35 milligrams/deciliter respectively.(see Fig. 8.) these results show first, anti--CTGF therapy can be used to reduce the BUN level of diabetes (object), and prompting suppresses CTGF provides a kind of Therapeutic Method that improves renal function.

Glomerular filtration rate

Determine the glomerular filtration rate of individual kidney under the above-mentioned various different tests condition.Control animal (be non-diabetic, non--IR) in, GFR is greater than 0.3 ml/min/kidney/100 gram.There is the GFR of the non-diabetic animal of kidney IR to be about 0.28 ml/min/kidney/100 grams.The GFR of the diabetic animal of no kidney IR is about 0.17 ml/min/kidney/100 grams.(data not shown.)

The glomerular filtration rate of diabetic animal ischemia kidney is sharply being reduced to about 0.01 ml/min/kidney/100 grams the 10th week.(see Fig. 9.) resist-CTGF antibody makes and significantly improved to the level that is higher than 0.035 ml/min/kidney/100 grams by the glomerular filtration rate of the individual kidney of diabetic animal that kidney IR influences.This data show gives CTGF antibody and makes that to suffer from end-stage renal disease be that the glomerular filtration rate of diabetic animal raises.These results confirm that first anti--CTGF therapy can effectively improve the glomerular filtration rate of diabetic nephropathy in late period, and a kind of Therapeutic Method that improves the renal function of end-stage renal disease (object) therefore is provided.

Except shown in this paper and describe, those skilled in the art understand and can do various modifications to the present invention according to above-mentioned description.These are revised all in the scope of claim of the present invention.

It is for referencial use to fit into this paper in all lists of references that this paper quotes.

Claims (24)

1. method of creatinine clearance that reduces diabetes or early diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

2. excessive filterable method of glomerule that alleviates diabetes or early diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

3. method of glomerule hyperperfusion that alleviates diabetes or early diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

4. method of urinary albumin excretion rate that reduces diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

5. one kind alleviates or prevent diabetes or diabetic nephropathy patient or the method that has the object kidney weight of ill risk to increase, and described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

6. method of glomerular filtration rate normalization that makes diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

7. method that alleviates diabetes or diabetic nephropathy patient or the object glomerule hypertrophy of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

8. albuminuretic method that alleviates diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

9. method of albuminuria that alleviates diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

10. method of microalbuminuria that alleviates diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

11. the albuminuretic method of DABAI that alleviates diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

12. the method for BUN level that reduces diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

13. the method for inulin clearance that reduces diabetes or diabetic nephropathy patient or the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

14. a this complication of prevention in the object that the diabetic complication risk is arranged, reduce its risk or postpone the method for its outbreak, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

15. the method for initial stage diabetic nephropathy for the treatment of the initial stage diabetic nephropathy patient or the object of ill risk being arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

16. the method for initial stage diabetic nephropathy for the treatment of the early diabetic nephropathy patient or the object of ill risk being arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

17. the method for explicitly diabetic nephropathy for the treatment of the explicitly diabetic nephropathy patient or the object of ill risk being arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

18. the method for diabetic nephropathy for the treatment of diabetic nephropathy patient or the object of ill risk being arranged, described method comprises with the angiotensin-convertion enzyme inhibitor of amount of suppression unites the CTGF inhibitor that gives the patient treatment effective dose.

19. the method for diabetic nephropathy for the treatment of diabetic nephropathy patient or the object of ill risk being arranged, described method comprises with the vasotonia rope receptor blocking agent of amount of suppression unites the CTGF inhibitor that gives the patient treatment effective dose.

20. a treatment or prevention PD patient or the method for this disease early symptom of the object of ill risk is arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

21. the method for the carrying out property renal failure of a treatment target, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

22. the method for renal function of improving the impaired renal function patient or the object of ill risk being arranged, described method comprises the CTGF inhibitor that gives the patient treatment effective dose.

23.CTGF inhibitor is used for the application of the medicine of following purposes in manufacturing: reduce creatinine clearance; Alleviating glomerule excessively filters; Alleviate the glomerule hyperperfusion; Alleviate the glomerule hypertrophy; Reduce renal glomerular basement membrane thickness; Reduce the urinary albumin excretion rate; Alleviate albuminuria; Alleviate albuminuria; Alleviate microalbuminuria; Alleviate big albuminuria; Reduce the BUN level; Make glomerular filtration rate normalization; Reduce inulin clearance; Alleviate or prevent kidney weight to increase; The prevent diabetes complication, reduce its risk or postpone its outbreak; The progress of treatment or prevention initial stage diabetic nephropathy; The progress of treatment or prevention early diabetic nephropathy; The progress of treatment or prevention explicitly diabetic nephropathy; Treatment or carrying out property of prevention renal failure; The development of the PD early symptom of treatment or object of prevention; With improve diabetes or diabetic nephropathy patient or the renal function of the object of ill risk arranged.

24. application as claimed in claim 23 is characterized in that, described reagent is selected from antibody, micromolecular inhibitor, antisensenucleic acids and siRNA.

Images