WO2005123070A1 - Dual blockade of renin-angiotensin system reduces connective tissue growth factor levels in diabetic nephropathy - Google Patents

Abstract

Methods are provided for reducing connective tissue growth factor (CTGF) levels in a subject suffering from diabetic nephropathy by administering an angiotensin converting enzyme inhibitor and an angiotensin II receptor blocker to the subject. Also provided are methods for determining the effectiveness of a course of treatment of a nephropathy patient by monitoring urinary CTGT levels.

Description

DUAL BLOCKADE OF RENIN-A GIOTENSHV SYSTEM REDUCES CONNECTIVE TISSUE GROWTH FACTOR LEVELS IN DIABETIC NEPHROPATHY

BACKGROUND INFORMATION CROSS REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Application Serial No. 60/578,566, filed June 9, 2004. The disclosure of the prior application is considered part of and is incorporated by reference in the disclosure of this application.

FIELD OF THE INVENTION [0002] The invention relates generally to methods of treating, and of monitoring the effectiveness of treatment of, a nephropathy patient, and more specifically to methods of ameliorating glomerulosclerosis in a subject by reducing urinary connective tissue growth factor (CTGF) levels in the subject, and to methods of monitoring the responsiveness of a nephropathy patient to treatment by measuring urinary CTGF levels during the therapeutic regimen.

BACKGROUND OF THE INVENTION [0003] Approximately 30% to 40% of patients with insulin dependent diabetes mellitus develop renal failure, as do about 20% of patients with non-insulin dependent diabetes mellitus. Diabetic nephropathy accounts for about one-third of all new cases of end-stage renal disease. Although some diabetic patients with nephropathy die from uremia, the majority ultimately succumb to cardiovascular disease, which presents a 40-fold greater risk to diabetics with nephropathy than that of the general population. Kidney disease due to diabetes is the most common reason for renal transplantation in adults (Rubin and Farber, "Pathology" 3d ed. Lippincott-Raven, publ., 1999; page 1216).

[0004] Diabetic nephropathy can be treated by blocking the renin-angiotensin system (RAS). Traditionally, treatment of diabetic nephropathy used either angiotensin converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARB). Such treatment can slow, but does not arrest, the progression to end-stage renal disease. However, recent studies indicate that dual blockade of the RAS using combination therapy with both an ACE inhibitor and an ARB can provide substantially greater renoprotection in type II diabetics, and may substantially improve quality of life and survival (Abraham et al., Diabetes Care 26:2268, 2003). [0005] While methods for treating diabetes in general, and diabetic nephropathy in particular, have improved, such improvements rely on trial and error methods due to a lack of understanding of the basic mechanisms the lead to nephropathy. Thus, a need exists for identifying the cellular factors involved in the initiation and progression of diabetic nephropathy so that specific treatment protocols can be developed.

SUMMARY OF THE INVENTION

[0006] The present invention is based, in part, on the discovery that changes in the level of urinary connective tissue growth factor (CTGF) correlate with the effectiveness of treatment of diabetic nephropathy patients and, further, that a synergistic (greater than additive) decrease in urinary CTGF levels are obtained following dual blockade therapy as compared to the decrease that would be expected using an angiotensin converting enzyme (ACE) inhibitor, alone, and an angiotensin II receptor blocker (ARB), alone. This result indicates that CTGF levels (e.g., urinary CTGF levels or plasma CTGF levels) can be used to monitor the course of a disorder associated with abnormal CTGF levels, for example, increased urinary CTGF levels as can occur in diabetic nephropathy or other glomerulosclerosis, in a patient, as well as the effectiveness of a treatment regimen directed at ameliorating the disorder. Further, when considered in view of recent evidence that CTGF regulates intracellular signaling pathways in cells such as kidney cells, the present results indicate that manipulation of CTGF levels in such patients can provide a means to ameliorate diabetic nephropathy.

[0007] Accordingly, the present invention relates to a method of reducing urinary CTGF levels in a subject suffering from a disorder associated with elevated urinary CTGF levels. In one embodiment, the method is directed to reducing urinary CTGF levels in a subject having a fibrotic disorder of the kidney. Such disorders include, for example, diabetic nephropathy or other disorder associated with glomerulosclerosis. In one aspect of this embodiment, the method is directed to using a combined modality therapy, wherein urinary CTGF levels are reduced to a greater amount than would be expected from adding together the reduction in urinary CTGF levels expected to be effected by each of the modalities of the combined modality when used alone.

[0008] By way of example, the invention provides a method of using dual blockade therapy to reduce urinary CTGF levels of a subject having diabetic nephropathy, which is associated with increased urinary CTGF. Such a method can be performed, for example, by administering an ACE inhibitor and an ARB to the subject, whereby the urinary CTGF level due to dual blockade therapy is reduced by a greater amount than would be expected from adding together the decrease in urinary CTGF level resulting from treatment with the ACE inhibitor, alone, and with the ARB, alone. The ACE inhibitor can be any ACE inhibitor typically used to treat a subject with diabetic nephropathy, including, for example, lisinopril/enalapril or captopril. Similarly, the ARB can be any ARB typically used to treat a subject with diabetic nephropathy, including, for example candesartan or losartan.

[0009] The present invention also relates to a method for monitoring a disorder associated with an elevated urinary CTGF level in a subject, for example, a CTGF-associated disorder such as a kidney fibrotic disorder (e.g., glomerulosclerosis), or a cardiovascular disorder. The monitoring (diagnostic) method can be performed, for example, by detecting (measuring) a first urinary CTGF level, for example, when the subject first presents with signs and symptoms of the disorder, and, thereafter, detecting (measuring) a second urinary CTGF level, for example, upon a return clinical visit. By comparing the first urinary CTGF level and the second urinary CTGF level, information can be obtained as to the status of the disease, i.e., whether the disease has become more severe (progressed), become less severe (resolved), or has stayed substantially the same. In particular, the detection of an increase in the second level of urinary CTGF as compared to the first level is indicative of progression of the CTGF-associated disorder; the detection of a decrease in the second level of urinary CTGF as compared to the first level is indicative of resolution of the fibrotic disorder; and the detection of no significant change in the second level as compared to the first level indicates that the fibrotic disorder has not substantially progressed or resolved.

[0010] In one embodiment, the diagnostic method of the invention is useful for confirming that the disorder associated with elevated urinary CTGF levels is progressing. As such, the method can be used to provide information to a clinician indicating that treatment of the patient should be initiated. In another embodiment, the method can be used for monitoring the effectiveness of treatment designed to ameliorate the disorder, wherein a first level of urinary CTGF is determined prior to initiating treatment (e.g., the second level as determined above can be used as the "first level" in the present embodiment), then the treatment is initiated, then a second (or more) level of urinary CTGF is determined. According to this embodiment, a decrease in the second level as compared to the first level is indicative that the fibrotic disorder is effectively being treated. By way of example, the fibrotic disorder can comprise diabetic nephropathy, and the treatment can comprise dual blockade therapy using an ACE inhibitor and an ARB, wherein a decrease in the second level of urinary CTGF as compared to the first level indicates that the nephropathy is being effectively treated.

[0011] The present invention further relates to a method of identifying an agent that reduce urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF. Such a method can be performed, for example, using an animal model of the disorder, wherein at least one agent (e.g., 1, 2, 3, 4, 5, etc.) that can or is suspected of being able to treat the disorder, is administered to the animal, and urinary CTGF levels are monitored (e.g., prior to and after administration of the agent, wherein a decrease in urinary CTGF levels following administration of the test agent identify the test agent as an agent that can reduce urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF levels.

[0012] A test agent examined according to a method of the invention can be any type of molecule, including, for example, a peptide, polynucleotide, peptidomimetic, or small organic molecule. In one embodiment, the method is performed using a combination of agents and/or test agents, including one or more agents known to be effective in treating the disorder and/or one or more test agents. In one aspect of this embodiment, combinations of agents that synergistically reduce urinary CTGF levels are identified. In respect to the present methods, it should be recognized that a first level of urinary CTGF need not necessarily be measured prior to initiating therapy where, for example, the population of animals comprising the animal model system typically exhibit a range of known, elevated urinary CTGF levels (i.e., the first level of urinary CTGF can be known based on a statistical analysis of a population being examined).

[0013] The present invention also relates to an agent identified by such screening assays, for example, a peptide, a polynucleotide, a small organic molecule, or a peptidomimetic agent. Where the agent is to be used for a therapeutic method, it can be formulated in a form suitable for administration to a subject, for example, as a pill or a liquid, and can be administered, for example, orally, by injection, or via inhalation. Accordingly, methods are provided for reducing urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF by administering an agent identified by a screening assay of the invention to the subject. Once disease is established and a treatment protocol is initiated, monitoring assays of the invention can be used to follow the course of the disease and effectiveness of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a graph showing the distribution of U-CTGF concentration for 20 subjects receiving either the placebo (top panel) or candesarten (bottom panel).

[0015] Figure 2 is a log-transformation graph showing distribution of U-CTGF concentration for 20 subjects receiving either the placebo (top panel) or candesarten (bottom panel).

[0016] Figure 3 is a graph showing the distribution of levels of 24 hr U-CTGF 24 hr excretion for 20 subjects receiving either the placebo (top panel) or candesartan (bottom panel).

[0017] Figure 4 is a log-transformation graph showing the distribution of levels of 24 hr U- CTGF 24 hr excretion for 20 subjects receiving either the placebo (top panel) or candesartan (bottom panel).

[0018] Figure 5 is a graph showing individual changes in the U-CTGF concentrations for 20 subjects receiving either the placebo (left) or candesartan (right).

[0019J Figure 6 is a graph showing U-CTGF excetion rates for 20 subjects receiving either the placebo (left) or candesartan (right).

[0020] Figure 7 is a graph showing the correlation between concentrations of U-CTGF and that of albuminuria ).

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is based, in part, on the discovery that changes in the level of urinary connective tissue growth factor (CTGF) correlate with the effectiveness of treatment of diabetic nephropathy patients and, further, that a synergistic (greater than additive) decrease in urinary CTGF levels are obtained following dual blockade therapy as compared to the decrease that would be expected using an angiotensin converting enzyme (ACE) inhibitor, alone, and an angiotensin II receptor blocker (ARB), alone. As such, the present invention provides methods of monitoring the course of a disorder such as diabetic nephropathy, which is associated with increased urinary CTGF levels, in a subject, as well as the effectiveness of a treatment regimen directed at ameliorating the disorder. Methods for reducing urinary CTGF levels to treat a disorder associated with elevated urinary CTGF levels also are provided, including, for example, methods of reducing urinary CTGF levels in a patient suffering from diabetic nephropathy by using dual blockade therapy.

[0022] The present invention is not limited to the particular methodology, protocols, cell lines, vectors, reagents, and the like, described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof (e.g., antigen binding antibody fragments) known to those skilled in the art, and so forth.

[0023] CTGF is a member of a family of growth regulators that include, for example, mouse (fisp-12) and human CTGF, Cyr61 (mouse), CeflO (chicken), and Nov (chicken). Based on sequence comparisons, it has been suggested that the members of this family have a modular structure consisting typically of at least one of the following: (1) an insulin-like growth factor domain responsible for binding; (2) a von Willebrand factor domain responsible for complex formation; (3) a thro bospondin type I repeat, possibly responsible for binding matrix molecules; and (4) a C-terminal module found in matrix proteins, postulated to be responsible for receptor binding. The sequence of the cDNA for human CTGF contains an open reading frame of 1047 nucleotides, with an initiation site at about nucleotide 130 and a TGA termination site at about nucleotide 1 177, and encodes a peptide of 349 amino acids (see U.S. Patent No. 5,408,040, which is incorporated herein by reference).

|0024] The synthesis and secretion of CTGF can be selectively induced by TGF-β and BMP-2, as well as potentially by other members of the TGF-β superfamily of proteins. Although TGF-β can stimulate the growth of normal fibroblasts in soft agar, CTGF alone cannot induce this property in fibroblasts. However, the synthesis and action of CTGF are essential for the TGF-β to stimulate anchorage independent fibroblast growth (see, e.g., Kothapalli et al., 1997, Cell Growth & Differentiation 8(l):61-68; Boes et al., 1999, Endocrinology 140(4): 1575-1580; Schiffer, et al., J. Clin. Investigation, 108:807-816 (2001); and U.S. Patent No. 6,150,101 , each of which is incorporated herein by reference).

[0025] CTGF is a cysteine rich monomeric peptide, having a molecular weight of about 38 kDa. CTGF has both mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells and is believed to be active upon interaction with a specific cell receptor. Polynucleotides encoding CTGF, fragments of CTGF, and CTGF regulatory sequences are disclosed in U.S. Patent Nos. 5,585,270; 6,069,006; 6,190,884; and 6,492,129; each of which is incorporated herein by reference.

[0026] As used herein, the term "disorder associated with elevated urinary CTGF'^' refers to any condition in which the level of urinary CTGF is significantly greater than the level of urinary CTGF characteristic of a corresponding normal population to which a subject belongs. Such a corresponding normal population is characterized, in part, in that the subjects of the population do not have the disorder for which a patient is being examined, monitored or treated (e.g., diabetes, particularly diabetic nephropathy), and can be, but need not be, matched based on sex, race, age, and the like as is known in the art.

[0027] Disorders associated with elevated urinary CTGF levels are exemplified herein by disorders associated with kidney fibrosis (e.g., diabetic nephropathy and glomerulosclerosis), and can further include, for example, cardiovascular disorders, immunological disorders, angiogenesis-related disorders, skin fibrotic disorders, excessive scarring resulting from acute or repetitive traumas, including surgery or radiation therapy, fibrosis of organs such as the kidney, lungs, liver, eyes, heart, and skin, including scleroderma, keloids, and hypertrophic scarring. Additional exemplary disorders associated with elevated urinary CTGF levels can include, diseases caused by vascular endothelial cell proliferation or migration (e.g., cancer, including dermatofibromas), conditions related to abnormal endothelial cell expression, breast carcinoma desmosplasia, angiolipoma, and angioleiomyoma. Other related conditions include atherosclerosis and systemic sclerosis, including atherosclerotic plaques, inflammatory bowel disease, Crohn's disease, angiogenesis, and other proliferative processes which play central roles in atherosclerosis, arthritis, cancer, and other disease states; neovascularization involved in glaucoma, inflammation due to disease or injury, including joint inflammation, tumor growth metastasis, interstitial disease; dermatological diseases; asthma; hepatitis; systemic lupus eryfhematosis; acquired immune deficiency syndrome; multiple sclerosis; psoriasis; meningitis; neurodegeneration; cachexia; euthyroid sick syndrome; glomerulonephritis; arthritis, including chronic rheumatoid arthritis, arteriosclerosis; diabetes, including diabetic nephropathy and retinopathy, hypertension, and other kidney disorders; ischemia/reperfusion injury; and fibrosis resulting from chemotherapy, radiation treatment, dialysis, and allograft and transplant rejection. (See, e.g., Baldwin, J. Clin. Invest. 107:3-6 (2001); Seaman, Dynamic Chiropractic, 21 (2003);

[0028] Fibrotic disorders include, but are not limited to, the diseases and disorders listed above, for example, scleroderma, pulmonary fibrosis, arthritis, hypertropic scarring, and atherosclerosis, diabetic retinopathy, hypertension, other angiogenesis-related disorders, including but not limited to blood vessels associated with tumor formation, and other proliferative processes that have a central role in atherosclerosis, arthritis, and other disease states, and, for example, in skin, cardiac, and pulmonary and renal fibrosis. In general, severe fibrosis involving kidney, liver, lung, and the cardiovascular system are included herein. There are numerous examples of fibrosis, including the formation of scar tissue following a heart attack, which impairs the ability of the heart to pump. Diabetes frequently causes damage/scarring in the kidneys which leads to a progressive loss of kidney function. Even after surgery, scar tissue can form between internal organs causing contracture, pain, and, in some cases, infertility. Major organs such as the heart, kidney, liver, lung, eye, and skin are prone to chronic scarring, commonly associated with other diseases. Hypertrophic scars (non-malignant tissue bulk) are a common form of fibrosis caused by burns and other trauma. In addition, there are a number of other fibroproliferative disorders such as scleroderma, keloids, and atherosclerosis which are associated respectively with general tissue scarring, tumor-like growths in the skin, or a sustained scarring of blood vessels which impairs blood carrying ability.

[0029] The present invention provides screening assays useful for identifying agents that can reduce urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF. As used herein, the term "test agent" means any compound that is being examined for the ability to reduce such elevated urinary CTGF levels. A test agent (and an agent identified by a method of the invention) can be any type of molecule, including, for example a peptide, a polynucleotide, an antibody, a glycoprotein, a carbohydrate, a small organic molecule, or a peptidomimetic.

[0030] Polynucleotides are known to specifically interact with proteins and, therefore, can be useful as test agents to be screened for the ability to reduce urinary CTGF levels. The term "polynucleotide" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the term "polynucleotide" includes RNA and DNA, which can be a synthetic RNA or DNA sequence, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the term "polynucleotide" as used herein includes naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). In various embodiments, a polynucleotide useful as a test agent can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond. In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. However, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994; Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15:68-73, 1997, each of which is incorporated herein by reference).

[0031] The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al., Nucl. Acids Res. 22:977-986, 1994; Ecker and Crooke, BioTechnology 13:351360, 1995, each of which is incorporated herein by reference). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.

[0032] A polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al., supra, 1995).

[0033] Peptides also can be useful as test agents. The term "peptide" is used broadly herein to refer to a molecule containing two or more amino acids or amino acid analogs (or modified forms thereof) linked by peptide bonds. As such, peptide test agents (or agents) can contain one or more D-amino acids and/or L-amino acids; and/or one or more amino acid analogs, for example, an amino acid that has been derivatized or otherwise modified at its reactive side chain. In addition, one or more peptide bonds in the peptide can be modified, and a reactive group at the amino terminus or the carboxy terminus or both can be modified. Peptides containing D-amino acids, or L-amino acid analogs, or the like, can have improved stability to a protease, an oxidizing agent or other reactive material the peptide may encounter in a biological environment. Further, the stability of a peptide agent (or test agent) can be improved by generating (or linking) a fusion protein comprising the peptide and a second polypeptide (e.g., an Fc domain of an antibody) that increases the half-life of the peptide agent in vivo. Peptides also can be modified to have decreased stability in a biological environment, if desired, such that the period of time the peptide is active in the environment is reduced.

[0034] Antibodies provide an example of peptides useful as test agents in a screening assay of the invention. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. Antibodies are characterized, in part, in that they specifically bind to an antigen, particularly to one or more epitopes of an antigen. The term "binds specifically" or "specific binding activity" or the like, when used in reference to an antibody, means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1 x 10^"6 M, generally at least about 1 x 10^"7 M, usually at least about 1 x 10^"8 M, and particularly at least about 1 x 10^"9 M or 1 x 10^"10 or less. As such, Fab, F(ab')₂, Fd and Fv fragments of an antibody that retain specific binding activity are included within the definition of an antibody.

[0035] The term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246:1275-1281 , 1989, which is incorporated herein by reference). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341 :544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference). In addition, modified or derivatized antibodies, or antigen binding fragments of antibodies, such as pegylated (polyethylene glycol modified) antibodies, can be useful for the present methods.

[0036] Antibodies can be tested for anti-target polypeptide activity using a variety of methods well-known in the art. Various techniques may be used for screening to identify antibodies having the desired specificity, including various immunoassays, such as enzyme- linked immunosorbent assays (ELISAs), including direct and ligand-capture ELISAs, radioimmunoassays (RIAs), immunoblotting, and fluorescent activated cell sorting (FACS). Numerous protocols for competitive binding or immunoradiometric assays, using either polyclonal or monoclonal antibodies with established specificities, are well known in the art. See, e.g., Harlow and Lane. Such immunoassays typically involve the measurement of complex formation between the target polypeptide and a specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on the target polypeptide is preferred, but other assays, such as a competitive binding assay, may also be employed. See, e.g., Maddox et al, 1983, J. Exp. Med. 158:121 1.

[0037] An antibody useful in the methods of the invention can be an intact antibody or antigen binding fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding the epitopic determinant. The antibodies used in the method can be polyclonal or, more preferably, monoclonal antibodies. Monoclonal antibodies with different epitopic specificities are made from antigen containing fragments of the protein by methods well known in the art.

[0038] An antibody also can be "human'^' or "humanized" antibody. Humanized antibodies are antibodies, or antibody fragments, that have the same binding specificity as a parent antibody, (i.e., typically of mouse origin) and increased human characteristics. Humanized antibodies may be obtained, for example, by chain shuffling or by using phage display technology. Techniques for generating humanized antibodies are well known (see, e.g., U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761 ; and 5,693,762), as are techniques for producing human antibodies in transgenic mice (see, e.g., U.S. Pat. Nos. 5,545,806 and 5,569,825).

[0039] A test agent also can be an antisense molecule, which can be DNA, RNA, or any nucleic acid mimic or analog. Antisense RNA or DNA molecules bind specifically with a targeted gene's RNA message, interrupting the expression of that gene's protein product. The antisense binds to the messenger RNA forming a double stranded molecule which cannot be translated by the cell. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can be attached to an antisense oligonucleotide, causing cleavage of the RNA at the site of hybridization. These and other uses of antisense methods to inhibit the translation of genes are well known in the art. See, for example, Marcu-Sakura, 1988, Anal. Biochem 177:278.

|0040] As used herein "hybridization" refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. In an /; vitro situation, suitably stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature. For example, hybridization under high stringency conditions could occur in about 50% formamide at about 37°C to 42°C. Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C. In particular, hybridization could occur under high stringency conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and 200 mg/ml sheared and denatured salmon sperm DNA. Hybridization could occur under reduced stringency conditions as described above, but in 35% formamide at a reduced temperature of 35°C. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.

[0041] The sequence of an antisense polynucleotide useful for inhibiting a target molecule, can be obtained, for example, by comparing the sequences of orthologous genes (sequences that are conserved between species), or the transcripts of orthologous genes, and identifying highly conserved regions within such sequences. Similarity in nucleic acid sequences may be determined by procedures and algorithms which are well-known in the art. Such procedures and algorithms include, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AM AS (Analysis of Multiple Aligned Sequences), and AMPS (Protein Multiple Sequence Alignment).

[0042] In selecting the preferred length for a given polynucleotide, various factors should be considered to achieve the most favorable characteristics. In one aspect, polynucleotides of the present invention are at least 15 base pairs (bp) in length and preferably about 15 to about 100 bp in length. More preferably, the polynucleotides are about 15 bp to about 80 bp in length and even more preferably, the polynucleotides of the present invention are about 15 to about 60 bp in length. Shorter polynucleotides, such as 10 to under 15-mers, while offering higher cell penetration, have lower gene specificity. In contrast, longer polynucleotides of 20 to about 30 bp offer better specificity, and show decreased uptake kinetics into cells. See, Stein et al., "Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression," Cohen, ed., McMillan Press, London (1988). Accessibility to transcript RNA target sequences also is of importance loop-forming regions and orthologous sequences in targeted RNAs thus offer promising targets. In this disclosure, the term "polynucleotide" encompasses both oligomeric nucleic acid moieties of the type found in nature, such as deoxyribonucleotide and ribonucleotide structures of DNA and RNA, and man-made analogues which are capable of binding to nucleic acid found in nature. The methods of the present invention can be based upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothionate or other bonds. They may also comprise monomer moieties which have altered base structures or other modifications, but which still retain the ability to bind to naturally occurring transcript RNA structures. Such polynucleotides may be prepared by methods well-known in the art, for example, by using commercially available machines and reagents such as those available from Perkin- Elmer/Applied Biosystems (Foster City, CA). For example, polynucleotides specific to a targeted transcript are synthesized according to standard methodology. Phosphorothionate modified DNA polynucleotides typically are synthesized on automated DNA synthesized on automated DNA synthesizers available from a variety of manufacturers. These instruments are capable of synthesizing nanomole amounts of polynucleotides as long as 100 nucleotides. Shorter polynucleotides synthesized by modem instruments are often suitable for use without further purification. If necessary, polynucleotides may be purified by polyacrylamide gel electrophoresis or reverse phase chromatography. See, Sambrook, et al., Molecular Cloning: A Laboratotγ Manual, Vol. 2, Chapter 1 1 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0043] An appropriate carrier for administration of a polynucleotide to a subject can include, for example, vectors, antibodies, pharmacologic compositions, binding or homing proteins, or viral delivery systems to enrich for the sequence into the target cell or tissue. In the methods of the present invention, a polynucleotide can be coupled to, for example, a binding protein which recognizes endothelial cells or tumor cells. As used herein, a "binding protein" is a protein in which peptide sequences from different proteins are covalently linked together. Following administration, a polynucleotide can be targeted to a recipient cell or tissue such that enhanced expression of, for example, cytokines, transcription factors, G- protein coupled receptors, tumor suppressor proteins, and apoptosis initiation proteins can occur.

[0044] Delivery of antisense molecules and the like can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia or preferably an RNA virus such as a retro vims. A number of the known retroviruses can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polynucleotide sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polynucleotide.

[0045] Another targeted delivery system for antisense molecules is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery systems in vivo and in vitro. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. In order for a liposome to be an effective gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.

[0046] A functional equivalent of an agent that reduces urinary CTGF levels can contain modifications depending on the necessity of such modifications for the performance of a specific function. The term "functional equivalent" is intended to include fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule. A molecule is considered to be a chemical derivative of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed, for example, in Gennaro, A.R., ed., 1990, Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton PA. Procedures for coupling such moieties to a molecule are well known in the art. [0047] In order to express a biologically active CTGF fragment, for example, the nucleotide sequence coding for the full length protein, or a functional equivalent as described above, the CTGF fragment is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods well known in the art can be used to construct expression vectors containing the CTGF or CTGF fragment sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See e.g., the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.

[0048] In one embodiment, test agents and agents identified according to the present methods are molecules other than CTGF polypeptides, CTGF polynucleotides (e.g., antisense molecules), or anti-CTGF antibodies. For example, the agents identified according to the present screening assays can be small molecules that reduce urinary CTGF levels in a subject suffering from a disorder associated with elevated CTGF levels.

[0049] The screening methods of the invention can be used to screen combinatorial libraries of test agents in order to identify those agents that can reduce elevated urinary (or plasma) CTGF levels. Methods for preparing a combinatorial library of molecules that can be tested for a desired activity are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Patent No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al., Gene 109:13 19, 1991; each of which is incorporated herein by reference); a peptide library (U.S. Patent No. 5,264,563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83 92, 1995; a nucleic acid library (O'Connell et al., Proc. Natl. Acad. Sci., USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold et al., Ann. Rev. Biochem. 64:763- 797, 1995; each of which is incorporated herein by reference); an oligosaccharide library (York et al., Carb. Res., 285:99 128, 1996; Liang et al., Science, 274:1520 1522, 1996; Ding et al., Adv. Expt. Med. Biol. 376:261 269, 1995; each of which is incorporated herein by reference); a lipoprotein library (de Kruif et al., FEBS Lett. 399:232 236, 1996, which is incorporated herein by reference); a glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol. 130:567 577, 1995, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem. 37:1385-1401 , 1994; Ecker and Crooke, BioTechnology 13:351-360, 1995; each of which is incorporated herein by reference). Polynucleotides can be particularly useful as agents that can modulate a specific interaction of molecules because nucleic acid molecules having binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having such specificity can be readily prepared and identified (see, for example, U.S. Patent No. 5,750,342, which is incorporated herein by reference).

[0050] In some instances, the identity of the agent can be determined by a nucleic acid tag, using the polymerase chain reaction for amplification of the tag. See, for example, WO93/20242. In this instance, the agents which are active may be determined by taking the lysate and introducing the lysate into a polymerase chain reaction medium comprising primers specific for the nucleic acid tag. Upon expansion, one can sequence the nucleic acid tag or determine its sequence by other means, which will indicate the synthetic procedure used to prepare the agent.

[0051] Alternatively, one may have tagged particles where the tags are releasable from the particle and provide a binary code which describes the synthetic procedure for the compounds bound to the particle. See, for example, Ohlmeyer, et al., Proc. Natl. Acad. Sci., USA 90: 10922, 1993. These tags can conveniently be a homologous series of alkylene compounds, which can be detected by gas chromatography-electron capture. Depending upon the nature of the linking group, one may provide for partial release from the particles, so that the particles may be used 2 or 3 times before identifying the particular compound.

[0052] While for the most part libraries have been discussed, any large group of compounds can be screened analogously, so long as the CTGF epitope can be joined to each of the compounds. Thus, compounds from different sources, both natural and synthetic, including macrolides, oligopeptides, ribonucleic acids, dendrimers, etc., may also be screened in an analogous manner.

[0053] For administration to a subject, an agent that reduce urinary CTGF levels is administered by a route and under conditions that facilitate contact of the agent with the target cell and, if appropriate, entry into the cell. Thus, the agent can be administered to the site of the cells to be treated, or can be administered by any method that provides the target cells with the agent. Furthermore, the agent generally is formulated in a composition (e.g., a pharmaceutical composition) suitable for administration to the subject. As such, the invention provides pharmaceutical compositions containing an agent that reduce urinary CTF levels in a pharmaceutically acceptable carrier. As such, the agents are useful as medicaments for treating a subject suffering from a disorder associated with elevated urinary CTGF (e.g., diabetic nephropathy). Further, such a composition can include one or more other compounds that, alone or in combination with the agent reduce urinary CTGF levels, particularly therapeutic agents that act synergistically, as well as other agents that provide a therapeutic advantage to the subject, for example, an antibiotic if the subject is susceptible to a bacterial infection, one or more additional antiviral agents known to be useful for treating the particular disease or disorder, a nutrient or vitamin or the like, a diagnostic reagent, toxin, a therapeutic agent such as a cancer chemotherapeutic agent, or any other compound as desired, provided the additional compound(s) does not adversely affect the activity of the agent that reduces urinary CTGF levels or, if the compound does affect the activity of the agent, does so in a manner that is predictable and can be accounted for in formulating the agent.

[0054] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the agent. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the agent that alters protein-protein interactions that affect hearing and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art.

[0055] An agent that reduces elevated urinary CTGF levels can be incorporated within an encapsulating material such as into an oil-in-water emulsion, a microemulsion, micelle, mixed micelle, liposome, microsphere or other polymer matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, FL 1984); Fraley et al., Trends Biochem. Sci. 6:77, 1981, each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos. 5,882,679; 5,395,619; and 5,225,212, each of which is incorporated herein by reference) are an example of such encapsulating materials particularly useful for preparing a composition useful for practicing a method of the invention, and other "masked" liposomes similarly can be used, such liposomes extending the time that the therapeutic agent remain in the circulation. Cationic liposomes, for example, also can be modified with specific receptors or ligands (Morishita et al., J. Clin. Invest. 91 :2580-2585, 1993, which is incorporated herein by reference). In addition, a polynucleotide agent can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al., J. Biol. Chem. 268:6866-6869, 1993, which is incorporated herein by reference).

[0056] The route of administration of a pharmaceutical composition containing an agent as disclosed herein will depend, in part, on the chemical structure of the molecule. Polypeptides and polynucleotides, for example, are not particularly useful when administered orally because they can be degraded in the digestive tract. However, methods for chemically modifying polypeptides, for example, to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., supra, 1995; Ecker and Crook, supra , 1995). In addition, a peptide agent can be prepared using D-amino acids, or can contain one or more domains based on peptidomimetics, which are organic molecules that mimic the structure of peptide domain; or based on a peptoid such as a vinylogous peptoid.

[0057] A pharmaceutical composition as disclosed herein can be administered to an individual by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant. A pharmaceutical composition also can be administered to the site of a pathologic condition, for example, intravenously or intra-arterially into a blood vessel supplying a tissue or organ comprising retrovirus infected cells.

[0058] The pharmaceutical composition also can be formulated for oral formulation, such as a tablet, or a solution or suspension foπn; or can comprise an admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications, and can be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, or other form suitable for use. The carriers, in addition to those disclosed above, can include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening or coloring agents and perfumes can be used, for example a stabilizing dry agent such as triulose (see, for example, U.S. Patent No. 5,314,695).

[0059] The total amount of an agent that reduces elevated urinary CTGF levels to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. An advantage of using a fractionated method is that, upon normal division of a retrovirus infected cell, replication of the retrovirus can be reduced or inhibited due to the presence of the agent. One skilled in the art would know that the amount of the composition to treat a retrovirus infection in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary. In general, the formulation of the pharmaceutical composition and the routes and frequency of administration for treatment of human subjects are determined, initially, using Phase I and Phase II clinical trials. Once disease is established and a treatment protocol is initiated, the monitoring (diagnostic ) methods of the invention can be repeated as desired to evaluate efficacy of the treatment.

[0060] The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1

URINARY CTGF LEVELS ARE REDUCED FOLLOWING DUAL BLOCKADE OF THE RENIN-ANGIOTENSIN SYSTEM

[0061] CTGF is a prosclerotic cytokine that is implicated in the pathogenesis of diabetic glomerulopafhy. Because urinary CTGF (U-CTGF) is significantly increased in patients with diabetic nephropathy, short-term changes in U-CTGF were monitored following dual blockade of the renin-angiotensin-system (RAS), wherein an angiotensin II receptor blocker (ARB) was added to treatment with maximal recommended doses of ACE-inhibitor (ACEI) in patients with type 2 diabetes (T2D) and nephropathy.

[0062J Twenty T2D patients with hypertension and nephropathy were enrolled in this double-blinded randomized two-period crossover trial (see Abraham et al., supra, 2003). Patients received eight weeks therapy with the ARB, candesartan, (16 mg daily) or placebo, added in random order to existing treatment with lisinopril/enalapril (40 mg) or captopril (150 mg daily). At the end of each treatment period, U-CTGF (ELISA; Fibrogen Corp., South San Francisco CA), albuminuria (turbidimetry), 24 hr ambulatory blood pressure measurement (ABPM; Takeda-TM2420 device), and glomerular filtration rate (GFR; ^5,Cr- EDTA plasma-clearance technique) were monitored.

[0063] During mono blockade of the RAS by ACEI treatment alone, U-CTGF was 5248 (3496 to 7878) ng/24 hr (geometric mean (95%CI), albuminuria was 706 (349 to 1219) mg/24 hr, 24 hr ABPM (mean (SEM)) was 138(3) / 72(2) mm Hg, and GFR was 77(6) ml/min/1.73 m². During dual blockade of the RAS by addition of candesartan, there was an overall mean reduction (95% Cl) in U-CTGF of 18% (0 to 33%), as compared to ACEI alone (p=.05). Albuminuria was reduced by 28% (17 to 38%; p<0.001), and there was a modest reduction in systolic/diastolic 24 hr ABPM of 3(-2 to 8) / 2(-2 to 5) mm Hg (NS) and in GFR of 4(-l to 9) ml/min/1.73 m² (not significant).

[0064] Remarkably, a significant carry-over effect by dual blockade on U-CTGF was observed, as reflected by a 36% (17 to 51%; p<0.001) reduction in the 10 patients who received ACEI, alone, in the first period and dual blockade in the second period, whereas an insignificant change in U-CTGF of -5% (-38 to 20%; p=0.71) was observed in patients who received dual blockade in the first period and mono blockade with ACEI in the second period. No significant carry-over effect was observed with respect to albuminuria or GFR.

[0065] Figure 1 shows the distribution of U-CTGF-concentration (ng/ml) following further characterization of all 20 patients included in the study during placebo (top panel) and candesartan (bottom panel) treatment; all available measurements of U-CTGF were used. Further, FIG. 2 is a log transformation of FIG. 1 due to the skewed distribution.

[0066] Figure 3 shows the distribution of 24 hr U-CTGF 24-h excretion (ng/24 hours) for all 20 patients included in the study with placebo (top panel) and candesartan (bottom panel) treatment. Figure 4 is a log transformation of FIG. 3 due to the skewed distribution.

[0067] Figure 5 shows individual changes in U-CTGF concentration, placebo (top panel) vs. candesartan (bottom panel). The y-axis in FIG. 5 shows log transformed U-CTGF concentration (logl 0(ng/ml). Again, the number of patients included in these measurements is 20 (n=20). Figure 6 shows that the day to day U-CTGF excretion coefficient of variation for U-CTGF 24 hr was higher, about 82%, versus that of U-CTGF , which was about 65%.

[0068] Additionally, levels of U-CTGF in urine samples were analyzed (see Table I) for changes in U-CTGF and 24h U-CTGF.

Table I: Total changes in U-CTGF and 24 hr U-CTGF excretion rate upon treatment Placebo (P) Candesartan (C) Relative reduction p-value (95%CI) %

*U-CTGF cone, 2.01 1.64 18 (1 to 33) 0.04 (ng/ml) (1.43 to 2.83) (1.22 to 2.23)

*U-CTGF 5248 3968 18 (0 to 33) 0.0504 excretion rate (3496 to 7878) (2954 to 5328) (ng/24-hours) U-CTGF/U-Crea 3.36 2.57 16 (-1 to 31) 0.07 (ng/mg) (2.27 to 4.95) (1.89 to 3.49)

Albuminuria 706 508 28 <0.001 (mg/24-hours) (350 to 1219) (228 to 999) (17 to 38)

*Geometric mean (95%CI for mean)

[0069] The treatment effect shown in Table I was evaluated in a random effect model, comparing a simple t-test for paired data, making it possible to include repeated measurements (several urine samples during each treatment visit). This analysis also revealed a significant carry-over effect with respect to 24-h U-CTGF excretion. This implies that patients treated with placebo in the first period (and candesartan in the second period) had a greater decline in 24-h U-CTGF than patients treated with candesartan in the first period (and placebo in the second period) as shown below in Table II.

Table II: Treatment response according to treatment sequence: Placebo first period and Candesartan second period Period 1 Period 2 Relative p-value Placebo (P) Candesartan (C) reduction % (95%CI) U-CTGF cone. 3.02 1.94 36 <0.001 (ng/ml)* (2.08 to 4.39) (1.35 to 2.79) (18 to 50) U-CTGF excretion rate 8009 5124 36 O.001 (ng/24-hours)* (5235 to (3369 to 7791) (17 to 51) 12253) U-CTGF/U-CREA 5.08 3.33 34 <0.001 (ng/mg)* (3.35 to 7.70) (2.15 to 5.17) (15 to 50) Albuminuria 888 546 39 <0,001 (mg/24-hours) (649 to 1215) (356 to 838) (27 to 49) Table II: Treatment response according to treatment sequence: Candesartan first period, placebo second period Period 1 Period 2 Relative p-value Candesartan (C) Placebo (P) reduction % (95%CI) U-CTGF cone. 1.39 1.35 -3 0.81 (ng/ml)* (1.15 to 1.69) (1.0 tol.82) (-37 to 22) U-CTGF excretion 3336 3167 -5 0,71 rate (2610 to 4264) (2553 to 3928) (-38 to 20) (ng/24-hours)* U-CTGF/U-Crea 2.16 2.02 -7 0.60 (ng/mg)* (1.77 to 2.64) (1.65 to 2.49) (-37 to 17) Albuminuria 473 561 16 0.12 (mg/24-hours) (334 to 670) (433 to 726) (-5 to 32)

* geometric mean (95%CI),

[0070] Figure 7 shows that the changes in U-CTGF correlated with changes in albuminuria. Table III shows the standardized and unstandardized coefficients as between the placebo and candesartan.

Table III: Standardizeds and unstandardized coeeficients

a Dependent Variable: Change in U-CTGF (Placebo - Candesartan - logl0(ng/ml))

[0071] These results demonstrate that dual blockade of the RAS induces a significant reduction in U-CTGF and albuminuria, as compared to monoblockade of the RAS by ACEI, in maximally recommended doses. Further, the significant carry-over effect on U-CTGF levels indicate that U-CTGF levels can be predictive of a prolonged effect of candesartan.

[0072] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method of reducing urinary connective tissue growth factor (CTGF) levels of a subject having a kidney fibrotic disorder associated with increased urinary CTGF, comprising administering to the subject an angiotensin converting enzyme (ACE) inhibitor and an angiotensin II receptor blocker (ARB), whereby the urinary CTGF level is reduced by a greater amount than the level of urinary CTGF reduction due to the ACE inhibitor, alone, and the level of urinary CTGF reduction due to the ARB, alone.

2. The method of claim 1 , wherein the kidney fibrotic disorder is diabetic nephropathy or other glomerulosclerosis.

3. The method of claim 1 , wherein the ACE inhibitor is lisinopril, enalapril or captopril.

4. The method of claim 1 , wherein the ARB inhibitor is candesartan or losartan.

5. The method of claim 1, wherein the ACE inhibitor is lisinopril, enalapril or captopril.

6. The method of claim 1 , wherein the ARB inhibitor is candesartan or losartan.

7. A method of monitoring progression of a CTGF-associated disorder associated with an elevated urinary CTGF level in a subject comprising: a) detecting a first urinary CTGF level in a sample; b) detecting a second urinary CTGF level in a sample at a time after the first level is detected; c) comparing the first urinary CTGF level and the second urinary CTGF level, and determining a difference, wherein detection of an increase in the second level of urinary CTGF as compared to the first level is indicative of progression of the disorder and detection of a decrease in the second level of urinary CTGF as compared to the first level is indicative of resolution of the disorder and detection of no change in the second level as compared to the first level indicates that the disorder has not substantially progressed or resolved.

8. The method of claim 7, wherein the CTGF-associated disorder is diabetic nephropathy or glomerulosclerosis.

9. The method of claim 7, wherein the first sample is detected prior to therapy.

10. The method of claim 7, wherein the second sample is detected following treatment with an angiotensin converting enzyme (ACE) inhibitor and an angiotensin II receptor blocker (ARB).

11. A method of identifying an agent that reduces urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF comprising: a) contacting a test animal or system with a test agent and determining urinary CTGF levels prior to and following contacting with the test agent, wherein an agent that reduces urinary CTGF levels following administration of the test agent identifies the test agent as an agent that can reduce urinary CTGF levels in a subject having a disorder associated with elevated urinary CTGF levels.

12. The method of claim 11 , wherein the test agent is selected from a peptide, polynucleotide, peptidomimetic, nucleic acid molecule or small organic molecule or any combination thereof.

13. An agent identified by the method of claim 12.

14. A method of treating diabetic nephropathy in a subject comprising administering to the subject an angiotensin converting enzyme (ACE) inhibitor and an angiotensin II receptor blocker (ARB), whereby the urinary CTGF level is reduced by a greater amount than the level of urinary CTGF reduction due to the ACE inhibitor, alone, and the level of urinary CTGF reduction due to the ARB, alone, thereby treating the nephropathy.