WO2004069795A2 - Diagnosis and treatment of chronic tissue damage - Google Patents

Abstract

This invention relates to use of proteins associated with chronic tissue damage, e.g., chronic dermal ulcers. The proteins identified are useful for clinical screening, diagnosis, prognosis, therapy, and prophylaxis, as well as for drug screening and drug development.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF CHRONIC TISSUE DAMAGE

Related Applications

This application claims the benefit of priority to U.S. Provisional Patent Application No. 60/444,600, filed on February 3, 2003, which application is hereby incorporated by reference in its entirety.

Field of the Invention

This invention relates to use of proteins associated with chronic tissue damage. The proteins identified are useful for clinical screening, diagnosis, prognosis, therapy, and prophylaxis, as well as for drug screening and drug development.

Background of the Invention

Chronic dermal ulcers (CDUs) are a major cause of morbidity in the aging population, and represent a significant economic burden on healthcare systems. Recent figures for CDUs, including pressure sores, diabetic and venous ulcers, indicate a total of about 3.75 million and 12 million patients in the US and world- wide respectively (Wound Healing Technological Innovations and Market Overview (1998) Technology Catalysts

International Corporation, NA, USA). Of these patients, approximately 70% are classified as moderate to severe. Despite recent advances in their treatment, the healing of these ulcers remains slow (typically 16 weeks for a venous ulcer). The clinical assessment of chronic wound healing spans a 12-24 week period, the outcome being determined by speed of healing or time to complete healing.

In a longitudinal study of wound healing (over 12 months), an increased expression of pro-matrix metalloproteinase-9 and neutrophil elastase were shown to be associated with greater severity of the wound site (Tarton et al. (1999) Wound Repair Regen. 7(5): 347- 355). Currently, no biomarkers are available for a rapid determination (significantly <20 weeks post treatment initiation) of healing status. Such biomarkers would enhance the rapid evaluation of pro wound healing agents resulting in decreased time to accurate evaluation of drug efficacy. Brief Summary of the Invention

The present invention rests in part on the identification of proteins associated with chronic tissue damage (CTD), termed "Tissue Repair-Associated Proteins" (TRPs). The proteins are useful as specific biomarkers of CTD and correlate with the presence of CTD. These proteins are also clinically useful as predictive of the occurrence and type of such lesions. The use of these proteins in assays will augment existing diagnostic methodologies in identifying subjects at risk for and suffering from CTD. They will also facilitate the development of therapeutic agents directed against CTD, while potentially highlighting new targets for such intervention. The present invention further identifies specific Tissue Repair- Associated protein isoforms (TRPIs). Protein isoforms can be expressed that differ in amino acid composition, for example, as a result of alternative splicing or limited proteolysis, or as a result of differential post-translational modification such as glycosylation, phosphorylation, acylation, or both, so that protein isoforms of identical amino acid sequence can differ in their pi, MW, or both. The TRPs and TRPIs of the present invention are useful in a number of ways, including in methods and compositions for screening, diagnosis and treatment of CTD and clinically related conditions, and for screening and development of agents for treatment of such conditions.

In one aspect, the present invention provides a method for assessing, screening, diagnosis or prognosis of CTD in a subject, for identifying a subject at risk of developing CTD, or for monitoring the effect of therapy administered to a subject having CTD, said method comprising detecting and/or quantifying in a biological sample from said subject a TRPI as defined in Tables IN to NI. hi one embodiment, the present invention provides a method for assessing, screening, diagnosis or prognosis of CTD in a subject, for identifying a subject at risk of developing CTD, or for monitoring the effect of therapy administered to a subject having CTD, said method comprising detecting and/or quantifying in a biological sample from said subject a TRP as defined in Tables I to HI. hi a further embodiment the present invention provides a method for determining the type and severity of CTD, comprising (a) analyzing a first biological sample from a subject for the presence of a TRP and/or TRPI, and (b) comparing the abundance of a TRP and/or TRPI in the body fluid with a control sample, wherein the presence and abundance of a TRP and/or TRPI indicates the type and severity of CTD. In a further embodiment a plurality, i.e. more than 2, more than 5 or more than 10, TRPIs are measured. In a further aspect, the invention provides antibodies specific for a TRP, TRPI or TRPI-related polypeptide of the invention, including polyclonal, monoclonal, humanized, chimeric, synthetic/recombinant, and bispecific antibodies capable of immunospecific binding to a TRP or TRPI of the invention. The antibodies of the invention are useful in a variety of ways, including in diagnostic assays for identifying the level of a TRP or TRPI in a biological sample, and as potential therapeutics. another aspect, the invention provides kits that may be used in the above-recited methods, and that may comprise single or multiple preparations, or capture reagents (e.g. antibodies), together with other reagents, labels, substrates, if needed, and directions for use. The kits maybe used, for example, to identify the presence and/or level of a TRP or TRPI in a biological sample, or may be used in assays for the identification of new diagnostic and/or therapeutic agents.

The TRPs and TRPIs of the instant invention may be therapeutically useful to a subject in need thereof. Accordingly, in a further aspect, the invention provides pharmaceutical compositions comprising one or more of the TRPIs listed in Tables IV- VI, and a pharmaceutically acceptable carrier, vehicle, or diluent. The invention also provides an isolated or recombinant TRPI, i.e. a TRPI substantially free from other proteins or protein isoforms.

The TRPIs of the invention are useful separately and in any suitable combination, as targets in screening assays for identifying agents capable of modulating the expression of one or more TRPIs. Such identified agents are useful therapeutics for treatment or prevention of CTD in a subject in need thereof. Accordingly, in another aspect, the invention provides a method for screening for or identifying an agent capable of modulating the expression of a TRPI or TRPI-related polypeptide, said method comprising: contacting a first population of cells expressing the TRPI or TRPI-related polypeptide with a candidate agent; contacting a second population of cells expressing the TRPI or TRPI-related polypeptide with a control agent; and comparing the level of the TRPI or TRPI related polypeptide in the first and second populations of cells, wherein an agent capable of modulating the expression of the TRPI or TRPI-related polypeptide is identified by a difference in the level of expression of the TRPI or TRPI- related polypeptide in the first and second populations of cells. In one embodiment, the level of the TRP or TRPI is greater in the first population of cells than in the second population of cells. In another embodiment, the level of the TRP or TRPI is less in the first population of cells than in the second population of cells. In a more specific embodiment, the level of the TRP or TRPI is determined by measurement of the corresponding mRNA.

In a further aspect, the invention provides a method of screening for or identifying agents capable of modulating the expression of a TRPI or a TRPI-related polypeptide, comprising: administering a candidate agent to a first mammal or group of mammals; administering a control agent to a second mammal or group of mammals; and comparing the level of expression of the TRPI or TRPI-related polypeptide in the first and second groups, wherein an agent capable of modulating the expression of the TRPI or TRPI-related polypeptide is identified by a difference in the level of expression of the TRPI or TRPI- related polypeptide in the first and second group of animals, hi one embodiment, the mammals are animal models for CTD. hi another embodiment, the level of expression of the TRPI or TRPI-related polypeptide is greater in the first group than in the second group. In another embodiment, the level of expression of the TRPI or TRPI-related polypeptide is less in the first group than in the second group. In yet another embodiment, the expression level of the TRPI or TRPI-related polypeptide in the first and second groups are further compared to the level of the TRPI or TRPI-related polypeptide in normal control mammals. In a more specific embodiment, administration of the candidate agent modulates the expression level of the TRPI or TRPI-related polypeptide in the first group towards the expression level of the TRPI or TRPI-related polypeptide in the second group, i a further embodiment, the mammals are human subjects having CTD or a related condition. h an additional aspect, the invention provides a method of screening for or identifying agents capable of modulating the activity of a TRPI or TRPI-related polypeptide, said method comprising: in a first aliquot, contacting a candidate agent with the TRPI or TRPI-related polypeptide; and comparing the level of the TRPI or TRPI-related polypeptide in the first aliquot after addition of the candidate agent with the level of the TRPI or TRPI-related polypeptide in a control aliquot, or with a previously determined reference range, wherein an agent capable of modulating the activity of the TRPI or TRPI-related polypeptide is identified by a difference in the activity of the TRPI or TRPI-related polypeptide in the first and second aliquot, hi a more specific embodiment, the TRPI or TRPI-related polypeptide is a recombinant polypeptide.

In a further aspect, the invention provides a method of identifying agents capable of modulating the activity of a TRPI or a TRPI-related polypeptide, said method comprising: contacting a first population of cells expressing the TRPI or TRPI-related polypeptide with a candidate agent; and comparing the activity of the TRPI or TRPI-related polypeptide in the first population of cells after addition of the candidate agent with the level of the TRPI or TRPI-related polypeptide in a second cell population treated with a control agent, or with a previously determined reference range, wherein an agent capable of modulating the activity of the TRPI or TRPI-related polypeptide is identified by a difference in the activity of the TRPI or TRPI-related polypeptide in the first and second population of cells.

In another aspect, the invention provides method for the treatment of chronic tissue damage comprising administering to a subject in need thereof, a TRPI, a TRPI-related polypeptide or an active agent that modulates the expression or activity of a TRPI or TRPI- related polypeptide.

Other objects and advantages will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular methods, compositions, and experimental conditions described, as such methods and compounds may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "Tissue Repair- Associated protein isoform (TRPI)" includes one or more of such proteins, reference to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications and patents mentioned are incorporated herein by reference in their entireties.

Definitions

"Chronic Tissue Damage" (CTD) encompasses a chronic disease in which an external wound, ulcer, sore or other tissue or skin disruption is chronic and wherein healing is deficient or otherwise impaired relative to a normal wound, ulcer, sore, or other tissue or skin disruption. CTD includes and encompasses any of: chronic wounds, chronic ulcers, chronic dermal ulcers, pressure sores, bed sores, diabetic ulcers and venous ulcers.

In the context of the present invention, a "biological sample" includes samples obtained from any source, such as and without limitation, a body fluid sample or a tissue sample, e.g. dermal tissue. "Body fluid" refers to any collectable or isolatable fluid or liquid, with or without cells, which can be collected or otherwise derived from a patient. The term body fluid includes but is not limited to blood, serum, plasma, urine, semen and saliva, and further includes wound exudate.

"Wound exudate" refers to the material, such as fluid, cells or cellular debris, which has escaped from blood vessels and has been deposited in tissues or on tissue surfaces, usually as a result of inflammation. An exudate is characterized by a high content of protein, cells or solid materials derived from cells.

The TRPs of the invention are identified as features detected in a 2D gel that are differentially present in a biological sample from a subject having CTD compared with a sample from a subject free from CTD. A TRP is characterized by its isoelectric point (pi) and molecular weight (MW) as determined by 2D gel electrophoresis, particularly utilizing the Preferred Technology described herein. A TRP, is identified as "differentially present" in a first sample with respect to a second sample when a method for detecting it (i.e. 2D electrophoresis) gives a different signal when applied to the first and second samples (i.e. the signal, or apparent amount of the feature present is different in the first versus second samples). "Tissue Repair-Associated Isoform" (TRPI) refers to an isoform that is characterized by one or more peptide sequences of which it is comprised, and may be further characterized by reference to a pi and MW as determined by 2D gel electrophoresis, particularly utilizing the Preferred Technology as described herein, hi one embodiment a TRPI is identified by the amino acid sequencing of a TRP. As depicted herein, a TRP may comprise one or more TRPIs, which have indistinguishable pi and MWs using the Preferred Technology, but which comprise distinct peptide sequences. The peptide sequence(s) of the TRPI can be utilized to search database(s) for polypeptides comprising such peptide sequence(s), for which TRPI it can be ascertained whether, for example, an antibody exists which may recognize the protein and/or a member of its protein family, and as such, may be used in the methods and compositions of the present invention.

A TRP or TRPI is "increased" in the first sample with respect to the second if the method of detection indicates that the TRP or TRPI is more abundant in the first sample than in the second sample, or if the TRP or TRPI is detectable in the first sample and substantially undetectable in the second sample, or if the TRP or TRPI is more frequently detectable in the first sample than in the second sample. Conversely, a TRP or TRPI is "decreased" in the first sample with respect to the second if the method of detection indicates that the TRP or TRPI is less abundant in the first sample than in the second sample, or if the TRP or TRPI is undetectable in the first sample and detectable in the second sample, or if the TRP or TRPI is detected less frequently in the first sample than in the second sample. Samples may be compared from a single subject or a group of subjects, wherein a subject or subjects having CTD are compared singly, in groups or in overlapping super groups to a subject or subjects relatively free from or not likely to have such condition.

The relative abundance of a TRP in two samples is determined in reference to its normalized signal, in two steps as follows: first, the signal obtained upon detecting the TRP in a sample is normalized by reference to a suitable background parameter, e.g. (a) to the total protein in the sample being analyzed (e.g. total protein loaded onto a gel); (b) to an expression reference protein i.e. a feature whose abundance is substantially invariant, within the limits of variability of the Preferred Technology, in the population of subjects being examined, or (c) more preferably to the total signal detected as the sum of each of all proteins in the sample. Secondly, the normalized signal for the TRP in one sample or sample set is compared with the normalized signal for the same TRP in another sample or sample set to identify TRPs that are "differentially present" in the first sample (or sample set) with respect to the second sample. "Fold change" includes "fold increase" and "fold decrease" and refers to the relative increase or decrease in abundance of a TRP or TRPI in a first sample (or sample set) compared to a second sample (or sample set). A TRP or TRPI fold change may be measured by any technique known to those of skill in the art, where the observed increase or decrease will vary depending upon the technique used. The skilled artisan will understand, based on the present description, how to select, apply and interpret any such technique(s). Preferably, fold change is determined herein as described in the Examples herein.

The term "modulate" when used herein in reference to expression or activity of a TRP or TRPI, includes the up-regulation or down-regulation of the expression or activity of the TRP or TRPI. Based on the present disclosure, such modulation can be determined by assays known to those of skill in the art and/or described herein. "Diagnosis" includes diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clmical trials, and identifying patients at risk for or having CTD, or having experienced a CTD, or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment. "Treatment" includes therapy, prevention, amelioration, and/or prophylaxis, and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis, or to cure or reduce the extent of (ameliorate) or likelihood of occurrence the infirmity or malady or condition or event in the instance where the patient is afflicted. For example, the TRPs and/or TRPIs of the invention are useful therapeutically as targets for the identification of agents able to modulate their expression or activity in order to prevent or treat a CTD, or to heal or ameliorate a CTD. A therapeutically effective amount of an agent, for example, a TRP or TRPI, or combination(s) thereof, is an amount sufficient to achieve the desired prophylactic, ameliorative, protective, or preventive result desired.

"TRPI-related polypeptide" includes TRPI homologs, TRPI analogs, TRPI orthologs and fragments of the foregoing. TRPI homologs are at least 60% preferably at least 70%o, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to a TRPI.

"Agent" refers to all materials that may be used to prepare pharmaceutical and diagnostic compositions, and may include compounds, small molecules, nucleic acids, polypeptides, fragments, isoforms, or other suitable materials that may be used independently or collectively for such purposes, all in accordance with the present invention.

Preferred Technology

The Tissue Repair- Associated proteins (TRPs) of the invention were identified by two-dimensional electrophoresis methodology of the Preferred Technology. As used herein, "two-dimensional electrophoresis" (2D-electrophoresis) means a technique comprising isoelectric focusing, followed by denaturing electrophoresis; this generates a two-dimensional gel (2D-gel) containing a plurality of separated proteins. Preferably, the step of denaturing electrophoresis uses polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Especially preferred are the highly accurate and automatable methods and apparatus ("the Preferred Technology") described in WO 98/23950 and U.S. 6,064,754, which publications are herein specifically incorporated by reference, with particular reference to the experimental protocol described therein. It should be recognized by the skilled artisan, however, that many recognized and known two- dimensional electrophoresis systems may be utilized in practicing the present invention. Using the Preferred Technology a two-dimensional array is generated by separating biomolecules on a two-dimensional gel according to their electrophoretic mobility and isoelectric point. A computer-generated digital profile of the array is generated, representing the identity, apparent molecular weight, isoelectric point, and relative abundance of a plurality of biomolecules detected in the two-dimensional array, thereby permitting computer-mediated comparison of profiles from multiple biological samples, as well as computer aided excision of separated proteins of interest. In accordance with an aspect of the present invention, the TRPs and TRPIs disclosed herein may be identified by detecting and or measuring the presence and expression of proteins in wound exudates from patients with CTD, and comparing with a matched sample obtained from control subjects, e.g. subjects with non-chronic tissue damage.

As those of skill in the art will readily appreciate, the MW and pi of an identified protein or protein isoform measured by the method described herein will vary to some extent depending on the precise protocol used for each step of the 2D electrophoresis and for landmark matching. As used herein, the terms "MW" and "pi" are defined, respectively, to mean the apparent molecular weight and the apparent isoelectric point of a feature or protein isoform as measured in exact accordance with the Reference Protocol, Wliere the Reference Protocol is followed and when samples are run in duplicate or a higher number of replicates, variation in the measured mean pi of a TRP or TRPI is typically less than 3% and variation in the measured mean MW of a TRP or TRPI is typically less than 5%. Where the skilled artisan wishes to deviate from the Reference Protocol, calibration experiments should be performed to compare the MW and pi for each TRP or TRPI as detected (a) by the Reference Protocol and (b) by the deviant protocol.

TRPs

Three groups of TRPs have been identified through the methods and apparatus of the Preferred Technology. The first group consists of TRPs that are decreased in a biological sample, particularly a body fluid sample e.g. wound exudate of subjects having CTD e.g. a chronic dermal ulcer as compared with the wound exudate of subjects with a healing dermal ulcer. These TRPs can be described by MW and pi as provided in Table I.

Table I. TRPs Decreased in Wound Exudate of Subjects Having a Chronic Dermal Ulcer

The second group consists of TRPs that are increased a biological sample, particularly a body fluid sample e.g. wound exudate of subjects having CTD e.g. a chronic dermal compared with the a biological sample from a control subject e.g. a subjects with a healing dermal ulcer. These TRPs can be described by MW and pi as provided in Table H

Table II. TRPs Increased in Wound Exudate of Subjects Having a Chronic Dermal Ulcer

The third group consists of TRPs that are significantly associated with CTD in a multivariate analysis. These TRPs can be described by MW and pi as provided in Table in. Table III. TRPs significantly associated with CTD

In one embodiment, a biological sample, particularly body fluid e.g. wound exudate, from a subject is analyzed for quantitative detection ofone or more of the TRPs as described in Table I or any suitable combination of them, wherein a decreased abundance of the TRP or TRPs (or any suitable combination of them) in the sample from the subject relative to a sample from a subject or subjects with a healing dermal ulcer (e.g. a control sample or a previously detennined reference range) indicates the presence of CTD e.g. a chronic dermal ulcer. h another embodiment of the invention, a biological sample, particularly body fluid e.g. wound exudate from a subject is analyzed for quantitative detection ofone or more of the TRPs as described in Table II or any suitable combination of them, wherein an increased abundance of the TRP or TRPs (or any suitable combination of them) in the sample from the subject relative to a sample from a subject or subjects with a healing dermal ulcer (e.g. a control sample or a previously detennined reference range) indicates the presence of CTD e.g. a chronic dermal ulcer.

In another embodiment of the invention, a biological sample, particularly body fluid e.g. wound exudate from a subject is analyzed for quantitative detection ofone or more of the TRPs as described in Table m or any suitable combination of them, wherein the expression of the TRP or TRPs (or any suitable combination of them) is significantly associated with CTD.

The TRPs of the invention can be qualitatively or quantitatively detected by any method known to those skilled in the art, including but not limited to the Prefened Technology described herein, kinase assays, enzyme assays, binding assays and other functional assays, immunoassays, and western blotting. In one embodiment, the TRPs are separated on a 2-D gel by virtue of their MWs and pis and are visualized by staining the gel. h one embodiment, the TRPs are stained with a fluorescent dye and imaged with a fluorescence scanner. Sypro Red (Molecular Probes, Inc., Eugene, Oregon) is a suitable dye for this purpose. Alternative dyes are described in US 6,335,446.

TRPIs h another aspect of the invention, a biological sample, particularly a body fluid e.g. wound exudate from a subject is analyzed for quantitative detection ofone or more Tissue Repair- Associated Protein Isoforms (TRPIs), e.g. for identification or treatment of CTD or for development of pharmaceutical products.

Three groups of TRPIs are described herein by the amino acid sequencing of TRPs. TRPIs were isolated, subjected to proteolysis, and analyzed by mass spectrometry using in this instance the methods and apparatus of the Prefened Technology, it being understood that the prefened technology is set forth as representative but not restrictive of the invention. One skilled in the art can identify sequence infonnation from proteins analyzed by mass spectrometry and/or tandem mass spectrometry using various spectral interpretation methods and database searching tools. Examples of some of these methods and tools can be found at the Swiss Institute of Bioinformatics web site at http://www.expasy.ch/, and the European Molecular Biology Laboratory web site at http://www.nanador.embl-heidelberg.de/GroupPages/PageLinlc/peptidesearchpage.html. Identification of TRPIs was performed using the SEQUEST search program (Eng et al, 1994, J. Am. Soc. Mass Spectrom. 5:976-989) and the method described in WO02/21139, which is incorporated herein by reference in its entirety.

The first group comprises of TRPIs that are decreased in a biological sample, particularly a body fluid e.g. wound exudate of subjects having CTD e.g. a chronic dennal ulcer as compared with a biological sample from a control subject e.g. a subject with a healing dermal ulcer. The amino acid sequences of peptides produced from these TRPIs by proteolysis using trypsin and identified by tandem mass spectrometry and database searching as described in the Examples, infra are listed in Table IV, in addition to their conesponding pis and MWs.

Table IV. TRPIs Decreased in Wound Exudate of Subjects Having a Chronic Dermal Ulcer

The second group comprises TRPIs that are increased in a biological sample, particularly a body fluid e.g. wound exudate of subjects having CTD e.g. a chronic dermal ulcer as compared with a biological sample from a control subjects e.g. subjects with a healmg dermal ulcer. The amino acid sequences of peptides produced from these TRPIs by proteolysis using trypsin and identified by tandem mass spectrometry and database searching are listed in Table V, in addition to their conesponding pis and MWs.

Table V. TRPIs Increased in Wound Exudate of Subjects Having a Chronic Dermal Ulcer

The third group consists of TRPIs that are significantly associated with CTD in a multivariate analysis. The amino acid sequences of peptides produced from these TRPIs by proteolysis using trypsin and identified by tandem mass spectrometry and database searching are listed in Table VI, in addition to their conesponding pis and MWs.

Table VI. TRPIs Significantly Associated with CTD

Determination and/or Quantification of a TRPI.

Those skilled in the art will understand, based upon the present description, that a given TRPI can be described according to the data provided for that TRPI in Table IV, V or VI. The TRPI is a polypeptide comprising one or more of the peptide sequence described for that TRPI and has a pi of about the value stated for that TRPI, or a pi value within about 10%, 5% , or 1% of the stated value, and has a MW of about the value stated for that TRPI, or within about 10%, 5%, or 1% of the stated value. h one embodiment, a biological sample, particularly body fluid e.g. wound exudate, from a subject is analyzed for quantitative detection ofone or more of the TRPIs as described in Table IV or any suitable combination of them, wherein a decreased abundance of the TRPI or TRPIs (or any suitable combination of them) in the sample from the subject relative to a sample from a subject or subjects with a healing DU (e.g. a control sample or a previously determined reference range) indicates the presence of CTD e.g. a chronic dennal ulcer.

In another embodiment of the invention, a biological sample, particularly body fluid e.g. wound exudate from a subject is analyzed for quantitative detection ofone or more of the TRPIs as described in Table N or any suitable combination of them, wherein an increased abundance of the TRPI or TRPIs (or any suitable combination of them) in sample from the subject relative to a sample from a subject or subjects with a healing DU (e.g. a control sample or a previously determined reference range) indicates the presence of CTD e.g. a chronic dermal ulcer. hi another embodiment of the invention, a biological sample, particularly body fluid e.g. wound exudate from a subject is analyzed for quantitative detection ofone or more of the TRPIs as described in Table NI or any suitable combination of them, wherein the expression of the TRPI or TRPIs (or any suitable combination of them) is significantly associated with CTD. The TRPIs of the invention can be qualitatively or quantitatively detected by any method known to those skilled in the art, including but not limited to the Prefened Technology described herein, kinase assays, enzyme assays, binding assays and other functional assays, immunoassays, and western blotting. In one embodiment, the TRPIs are separated on a 2-D gel by virtue of their MWs and pis and are visualized by staining the gel. hi one embodiment, the TRPIs are stained with a fluorescent dye and imaged with a fluorescence scanner. Sypro Red (Molecular Probes, Inc., Eugene, Oregon) is a suitable dye for this purpose. Alternative dyes are described in US 6,335,446.

Alternatively, TRPIs can be detected in an immunoassay. In one embodiment, an immunoassay is performed by contacting a sample with an anti-TRPI capture reagent, e.g. an antibody, under conditions such that immunospecific binding can occur if the TRPI is present, and detecting or measuring the amount of any specific binding by the capture reagent. Anti-TRPI capture reagents, e.g. antibodies, can be produced by the methods and techniques described herein. Particularly, the anti-TRPI capture reagent, e.g. antibody, preferentially binds to the TRPI rather than to other isoforms of the same protein. In a particular embodiment, the anti-TRPI capture reagent, e.g. antibody, binds to the TRPI with at least 2-fold greater affinity, more particularly at least 5-fold greater affinity, still more particularly at least 10-fold greater affinity, than to said other isoforms of the same protein. TRPIs can be transferred from a gel to a suitable membrane (e.g. a PNDF membrane) and subsequently probed in suitable assays that include, without limitation, competitive and non-competitive assay systems using techniques such as western blots and "sandwich" immunoassays using anti-TRPI capture reagents, e.g. antibodies raised against the TRPIs of interest as those skilled in the art will appreciate based on the present description. The immunoblots can be used to identify those anti-TRPI capture reagents, e.g. antibodies, displaying the selectivity required to immuno-specifically differentiate a TRPI from other isoforms encoded by the same gene.

In one embodiment, binding of capture reagent, e.g. antibody, in tissue sections can be used to detect TRPI localization or the level of one or more TRPIs. In a specific embodiment, a capture reagent, e.g. an antibody to a TRPI can be used to assay a tissue sample (e.g. a skin biopsy) from a subject for the level of the TRPI where a substantially changed level of TRPI is indicative of CTD.

Any suitable immunoassay can be used to detect a TRPI, including, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. For example, a TRPI can be detected in a biological sample (e.g. wound exudate, blood, urine, or tissue homogenate) by means of a two-step sandwich assay, hi the first step, a capture reagent (e.g. an anti-TRPI antibody) is used to capture the TRPI. The capture reagent can optionally be immobilized on a solid phase. In the second step, a directly or indirectly labeled detection reagent is used to detect the captured TRPI. In one embodiment, the detection reagent is a lectin. A lectin can be used for tins purpose that preferentially binds to the TRPI rather than to other isoforms that have the same core protein as the TRPI or to other proteins that share the antigemc determinant recognized by the antibody. In a prefened embodiment, the chosen lectin binds to the TRPI with at least 2-fold greater affinity, more preferably at least 5 -fold greater affinity, still more preferably at least 10-fold greater affinity, than to said other isoforms that have the same core protein as the TRPI or to said other proteins that share the antigenic determinant recognized by the antibody. In an alternative embodiment, the detection reagent is an antibody, e.g., an antibody that immunospecifically detects other post-translational modifications, such as an antibody that immunospecifically binds to phosphorylated amino acids.

Isolation of DNA Encoding a TRPI The nucleotide sequences which encode TRPIs, including DNA and RNA, may be synthesized using methods known in the art, such as using conventional chemical/ approaches or polymerase chain reaction (PCR) amplification. These nucleotide sequences also permit the identification and cloning of a gene encoding a TRPI-related polypeptide including, for example, by screening cDNA libraries, genomic libraries or expression libraries.

For example, to clone a gene encoding a TRPI by PCR techniques, anchored degenerate oligonucleotides (or a set of most likely oligonucleotides) can be designed for all TRPI peptide fragments identified as part of the same protein. PCR reactions under a variety of conditions can be performed with relevant cDNA and genomic DNAs (e.g. from skin biopsies) from one or more species. Also vectorette reactions can be performed on any available cDNA and genomic DNA using the oligonucleotides (which preferably are nested) as above.

Nucleotide sequences comprising a nucleotide sequence encoding a TRPI or TRPI fragment of the present invention are useful, for example, for their ability to hybridize with complementary stretches of the gene(s) encoding the TRPI. Nucleotide sequences comprising a nucleotide sequence encoding a TRPI or TRPI fragment of the present invention are additionally useful, for example, for their ability to hybridize selectively with complementary stretches of genes encoding other proteins, including TRPI-related proteins. Depending on the application, a variety of hybridization conditions may be employed to obtain nucleotide sequences at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, or 100% identical, to the sequence of a nucleotide encoding a TRPI. hi particular, the skilled artisan will recognize that some stretches of a gene may demonstrate a higher or most significant amount of identity, e.g. consensus domains or regions, enzymatic or active sites, such that, while overall identity for hybridization conditions may be for instance 30%), 35%>, 40%>, 45%, there will exist regions or areas of significantly higher identity such that the overall % identity will still indicate relatedness and permit selective hybridization with complementary stretches of genes encoding other proteins, including TRPI-related proteins or relevant variants of a TRPI.

For a high degree of selectivity (i.e. for hybridization to a relatively complementary or very similar or homologous gene), relatively stringent conditions are used to form the duplexes, such as low salt or high temperature conditions. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes, such as low salt or high temperature conditions. For some applications, less stringent conditions for duplex formation are required. Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be chosen depending on the desired results, as will be appreciated by those skilled in the art based on the present description, hi general, convenient hybridization temperatures in the presence of 50%) formamide are: 42°C for a probe which is 95 to 100% identical to the fragment of a gene encoding a TRPI, 37°C for 90 to 95% identity and 32°C for 70 to 90% identity. In the preparation of genomic libraries, DNA fragments are generated, some of which will encode parts or the whole of a TRPI. Any suitable method for preparing DNA fragments may be used in the present invention. For example, the DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The DNA fragments can then be separated according to size by standard techniques, including but not limited to agarose and polyacrylamide gel electrophoresis, column chromatography and sucrose gradient centrifugation. The DNA fragments can then be inserted into suitable vectors, including but not limited to plasmids, cosmids, bacteriophages lambda or T4, and yeast artificial chromosome (YAC). (See, e.g. Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. The genomic library may be screened by nucleic acid hybridization to labeled probe.

Based on the present description, the genomic libraries may be screened with labeled degenerate ohgonucleotide probes conesponding to the amino acid sequence of any peptide of the TRPI using optimal approaches well known in the art. Any probe used is at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, or at least 100 nucleotides. Preferably a probe is 15 nucleotides or longer.

TRPIs disclosed herein in Tables IV, V and NI include those that conespond to isoforms of known proteins or relate, e.g. as family members, to known proteins encoded by genes whose nucleic acid sequences are publicly known. To screen such a gene, any probe may be used that is complementary to the gene or its complement; preferably the probe is 10 nucleotides or longer, more preferably 15 nucleotides or longer. The SWISS -PROT and trEMBL databases (held by the Swiss Institute of Bioinformatics (SIB) and the European Bioinformatics Institute (EBI) which are available at http://www.expasy.ch/) and the GenBank database (held by the National Institute of Health (NTH) which is available at http://www.ncbi.nlm.nih.gov/) provide protein sequences comprising the amino acid sequences listed for the TRPIs in Tables IN, V and VI under the following accession numbers and each sequence is incorporated herein by reference:

Table VII. Nucleotide sequences encoding TRPIs, TRPI-Related Proteins

For any TRPI, degenerate probes, or probes taken from the sequences described above by accession number may be used for screening. In the case of degenerate probes, they can be constructed from the partial amino sequence information obtained from tandem mass spectra of tryptic digest peptides of the TRPI. To screen such a gene, any probe may be used that is complementary to the gene or its complement; the probe is 10 nucleotides or longer, preferably 15 nucleotides or longer. When a library is screened, clones with insert DNA encoding the TRPI of interest or a fragment thereof will hybridize to one or more members of the conesponding set of degenerate ohgonucleotide probes (or their complement). Hybridization of such ohgonucleotide probes to genomic libraries is carried out using methods known in the art. For example, hybridization with one of the above-mentioned degenerate sets of ohgonucleotide probes, or their complement (or with any member of such a set, or its complement) can be performed under highly stringent or moderately stringent conditions as defined supra, or can be carried out in 2X SSC, 1.0%> SDS at 50°C and washed using the washing conditions described supra for highly stringent or moderately stringent hybridization.

Production of Antibodies to TRPs and TRPIs According to the invention a TRP, TRPI, or a TRPI-related polypeptide may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The tenn "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e.g. IgG, IgE, IgM, IgD and IgA ) or subclass of immunoglobulin molecule. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognize a specific domain of a TRPI, one may assay generated hybridomas for a product which binds to a TRPI fragment containing such domain. For selection of an antibody that specifically binds a first TRPI-related polypeptide but which does not specifically bind to (or binds less avidly to) a second TRPI- related polypeptide, one can select on the basis of positive binding to the first TRPI-related polypeptide and a lack of binding to (or reduced binding to) the second TRPI-related polypeptide. Similarly, for selection of an antibody that specifically binds a TRPI but which does not specifically bind to (or binds less avidly to) a different isoform of the same protein (such as a different glycoform having the same core peptide as the TRPI), one can select on the basis of positive binding to the TRPI and a lack of binding to (or reduced binding to) the different isoform (e.g. a different glycoform). Thus, the present invention provides an antibody (particularly a monoclonal antibody) that binds with greater affinity (particularly at least 2-fold, more particularly at least 5-fold still more particularly at least 10-fold greater affinity) to a TRPI than to a different isoform or isoforms (e.g. glycoforms) of the TRPI. Polyclonal antibodies which may be used in the methods of the invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Unfractionated immune serum can also be used. Various procedures known in the art may be used for the production of polyclonal antibodies to a TRP, TRPI or a TRPI- related polypeptide. For preparation of monoclonal antibodies (mAbs) directed toward a TRP, TRPI or a TRPI-related polypeptide any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. The mAbs include but are not limited to human mAbs and chimeric mAbs (e.g. human-mouse chimeras). Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g. U.S. 5,585,089). Chimeric and humanized monoclonal antibodies (mAbs) can be produced by recombinant DNA techniques known in the art, for example using methods described in, for example, WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; U.S. 4,816,567; EP 125,023. Completely human antibodies (antibodies derived solely from human antigenic material) are particularly desirable for therapeutic treatment of human subjects. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with selected antigens, e.g. all or a portion of a TRPI or TRPI-related polypeptide. mAbs directed against the antigen can be obtained using conventional hybridoma technology. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, h t. Rev. Immunol. 13:65-93). Methods of Diagnosis

TRPs and TRPI can be used for the diagnosis of CTD or for drug development, hi one embodiment of the invention, a biological sample, particularly body fluid e.g. wound exudate from a subject is analyzed by 2D electrophoresis for quantitative detection ofone or more of the TRPs or TRPIs listed in Tables I to NI. Where the differential expression of a TRP or TRPI indicates the presence of CTD. Other diagnostic methods include those where the proteins are initially separated according to molecular weight e.g. by one dimensional electrophoresis. Such methods include but are not limited to immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoassays e.g. western blots.

The methods of diagnosis according to the present invention may be also performed using a number of methods known to those skilled in the art, including, without limitation, i munocytochemistry, immunohistochemistry, immunoassays, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. h an additional aspect, the invention provides the use of a capture reagent, e.g. an antibody, that binds to a TRP or a TRPI in the diagnosis CTD in a subject and/or monitoring the effectiveness of CTD therapy in a subject. Preferably, the capture reagent is used for detecting and/or quantifying the amount of a TRP or a TRPI in a biological sample obtained from said subject, hi another embodiment of the invention, labeled antibodies, derivatives and analogs thereof, which specifically bind to a TRPI can be used. Preferably, CTD is identified in an animal, more preferably in a mammal and most preferably in a human. In accordance with the present invention, suitable first (test) biological samples, e.g. of wound exudate, obtained from a subject suspected of having or known to have CTD can be used for diagnosis. In one embodiment, a decreased abundance ofone or more TRPs or TRPIs (or any combination of them) in a first sample relative to a second sample (from a subject or subjects without CTD) or a previously determined reference range indicates the presence of CTD; TRPs and TRPIs suitable for this purpose are identified in Tables I and IN, respectively, as described in detail above, h another embodiment of the invention, an increased abundance ofone or more TRPs or TRPIs (or any combination of them) in a first sample compared to a second sample or a previously determined reference range indicates the presence of CTD; TRPs and TRPIs suitable for this purpose are identified in Tables II and N, respectively, as described in detail above, h another embodiment, the relative abundance ofone or more TRPs or TRPIs (or any combination of them) in a first sample compared to a second sample or a previously determined reference range indicates a subtype of CTD. In yet another embodiment, the relative abundance ofone or more TRPs or TRPIs (or any combination of them) in a first sample relative to a second sample or a previously determined reference range indicates the degree or severity of the CTD. hi another embodiment the expression of one or more TRPs or TRPIs whose expression is significantly related to CTD is determined in a first biological sample, where the expression level of the TRP or TRPI indicates the extent of CTD. The TRPs and TRPIs described in Table m and VI are suitable TRPs and TRPIs for this purpose. In any of the aforesaid methods, detection ofone or more TRPIs described herein may optionally be combined with detection ofone or more additional biomarkers for CTD. h cases where a TRPI has a known function, an assay for that function may be used to measure TRPI expression. In a further embodiment, a decreased abundance of mRΝA encoding one or more TRPIs identified in Table IV (or any combination of them) in a first sample relative to a second sample or a previously determined reference range indicates the presence of CTD. In yet a further embodiment, an increased abundance of mRΝA encoding one or more TRPIs identified in Table V (or any combination of them) in a first sample relative to a second sample or previously determined reference range indicates the presence of CTD. h another embodiment the expression of mRΝA encoding one or more TRPs or TRPIs whose expression is significantly related to CTD is determined in a first sample, where the abundance of RNA encoding the TRP or TRPI indicates the extent of CTD, The TRPs and TRPIs described in Table m and VI are suitable TRPs and TRPIs for this purpose. Any suitable hybridization assay can be used to detect TRPI expression by detecting and/or visualizing mRΝA encoding the TRPI (e.g. Northern assays, dot blots, in situ hybridization, etc.).

Kits

The invention also provides diagnostic kits, comprising an anti-TRPI capture reagent. In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the anti-TRPI capture reagent for identification of the type of CTD, prognosis, therapeutic monitoring or any suitable combination of these applications;

(2) a labeled binding partner to the capture reagent; (3) a solid phase (such as a reagent strip) upon which the anti-TRPI capture reagent is immobilized; and

(4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any suitable combination thereof. If no labeled binding partner to the capture reagent is provided, the anti-TRPI capture reagent itself can be labeled with a detectable marker, e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety. In a prefened embodiment the capture reagent is an antibody that recognizes the TRPs or TRPIs.

Kits are also provided which allow for the detection of a plurality of TRPIs or TRPI- related polypeptides. A kit can optionally further comprise a predetennined amount of an isolated TRPI protein, e.g. for use as a standard or control.

Screening Assays

The present invention provides methods for identifying agents that are capable of modulating the expression ofone or more TRPs or TRPIs. Agents identified through the screening method of the invention are potential therapeutics for use in treating CTD in a subject in need thereof.

Examples of agents, including active agents, candidate agents and control agents include, but are not limited to: TRPIs, TRPI-related polypeptides, nucleic acids (e.g. DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. 5,738,996; and U.S. 5,807,683, each of which is herein specifically incorporated by reference in its entirety).

Examples of suitable methods based on the present description for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;

Zuckermami et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Canell et al. (1994) Angew. Chem. hit. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. hit, Ed. Engl. 33:2061; and Gallop et al (1994) J. Med. Chem. 37:1233, each of which is herein incorporated by reference in its entirety. Libraries of compounds may be presented, for example, presented in solution (e.g.

Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82- 84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. 5,223,409), spores (U.S. 5,571,698; 5,403,484; and U.S. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378- 6382; and Felici (1991) J. Mol. Biol. 222:301-310), each of which is herein specifically incorporated by reference in its entirety.

In one embodiment, agents that modulate the expression of one or more TRPIs or TRPI-related polypeptides are identified in a cell-based assay system. In accordance with this embodiment, cells expressing one or more TRPIs or TRPI-related polypeptides are contacted with a candidate agent or a control agent and the ability of the candidate agent to alter expression of the TRPI or TRPI-related polypeptide is determined. This assay may also be used to screen a plurality (e.g. a library) of candidate agents. The cell, for example, can be of prokaryotic origin (e.g. E. coli) or eukaryotic origin (e.g. yeast or mammalian). Further, the cells can express the TRPI or TRPI-related polypeptide endogenously or be genetically engineered to express the TRPI or TRPI-related polypeptide. The ability of the candidate agent to alter expression ofone or more TRPI or TRPI-related polypeptides can be determined by methods known to those of skill in the art, for example, by flow cytometry, a scintillation assay, immunoprecipitation or western blot analysis. In another embodiment, agents that modulate the expression ofone or more TRPI or

TRPI-related polypeptides are identified in a cell-free assay system. In accordance with this embodiment, a native or recombinant TRPI or TRPI-related polypeptide is contacted with a candidate agent or a control agent and the ability of the candidate agent to modulate one or more TRPIs or TRPI-related polypeptides is determined. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate agents. hi another embodiment, a cell-based assay system is used to identify agents capable of modulating the activity of a TRPI or TRPI-related polypeptide when the TRPI or TRPI- related polypeptide is an enzyme, hi another embodiment, a cell- free assay system is used to identify agents such as an enzyme, or a biologically active portion thereof, which is responsible for the production or degradation of a TRPI or TRPI-related polypeptide, or is responsible for the post- translational modification of a TRPI or TRPI-related polypeptide. In a primary screen, a plurality (e.g. a library) of agents are contacted with cells that naturally or recombinantly express: (i) a TRPI or TRPI-related polypeptide, and (ii) a protein that is responsible for processing of the TRPI or TRPI-related polypeptide, in order to identify agents that modulate the production, degradation, or post-translational modification of the TRPI or TRPI-related polypeptide. If desired, agents identified in the primary screen can then be assayed in a secondary screen against cells naturally or recombinantly expressing the specific TRPI or TRPI-related polypeptide of interest. The ability of the candidate agent to modulate the production, degradation or post-translational modification of a TRPI or TRPI-related polypeptide can be determined by methods known to those of skill in the art, including without limitation, flow cytometry, a scintillation assay, immunoprecipitation and western blot analysis.

In a specific embodiment, agents that modulate (e.g. upregulate or downregulate) the expression of a TRPI or TRPI-related polypeptide are identified by contacting cells (e.g. cells of prokaryotic origin or eukaryotic origin) expressing the TRPI or TRPI-related polypeptide with a candidate agent or a control agent (e.g. phosphate buffered saline (PBS)) and determining the expression of the mRNA encoding the TRPI or TRPI-related polypeptide. The level of expression of a selected mRNA encoding the TRPI or TRPI- related polypeptide in the presence of the candidate agent is compared to the level of expression of the mRNA encoding the TRPI or TRPI-related polypeptide in the absence of the candidate agent (e.g. in the presence of a control agent). The candidate agent can then be identified as a modulator of the expression of the TRPI or TRPI-related polypeptide based on this comparison. For example, when expression of the TRPI or TRPI-related polypeptide is significantly greater in the presence of the candidate agent than in its absence, the candidate agent is identified as a stimulator of expression of the TRPI or TRPI- related polypeptide. Alternatively, when expression of the TRPI or TRPI-related polypeptide is significantly less in the presence of the candidate agent than in its absence, the candidate agent is identified as an inhibitor of the expression of the TRPI or TRPI- related polypeptide. The level of expression of a TRPI or TRPI-related polypeptide or the encoding mRNA can be determined by methods known to those of skill in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR, and protein levels can be assessed by western blot analysis. h another embodiment, agents that modulate (e.g. upregulate or downregulate) the expression of a TRPI or TRPI-related polypeptide are identified in an animal model.

Examples of suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. Preferably, the animal used represents a model of CTD. hi accordance with this embodiment, the candidate agent or a control agent is administered (e.g. orally, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the expression of the TRPI or TRPI-related polypeptide is determined. Changes in the expression of a TRPI or TRPI-related polypeptide can be assessed by the methods outlined above.

Therapeutic Uses of TRPIs The invention provides for treatment and/or prevention of CTD by administration of an active agent. Such agents include but are not limited to: TRPIs, TRPI-related polypeptides, TRPI fusion proteins or TRPI fusion protein fragments; antibodies to the foregoing; nucleic acids encoding TRPIs, TRPI-related polypeptides, TRPI fusion proteins or TRPI fusion protein fragments; antisense nucleic acids to a gene encoding a TRPI or TRPI-related polypeptide; modulators (e.g. agonists and antagonists) of a gene encoding a TRPI or TRPI-related polypeptide and modulators of the expression or activity of a TRPI or TRPI-related polypeptide. CTD can be treated (e.g. to ameliorate symptoms or to retard onset or progression) or prevented by administration of an active agent that promotes function or expression ofone or more TRPIs that are decreased in a biological sample from subjects having CTD, or by administration of an active agent that reduces function or expression ofone or more TRPIs that are increased in the biological sample from subjects having CTD. In one embodiment, one or more capture reagents each specifically binding to a TRPI or TRPI-related polypeptide are administered alone or in combination with one or more additional therapeutic agents or treatments. Preferably, a biological product, e.g. a capture reagent, is allogeneic to the subject to which it is administered, hi one embodiment, a human TRPI or a human TRPI-related polypeptide, a nucleotide sequence encoding a human TRPI or a human TRPI-related polypeptide, or an antibody to a human TRPI or a human TRPI-related polypeptide, is administered to a human subject for therapy (e.g. to ameliorate symptoms or to retard onset or progression or for prophylaxis).

CTD can be treated or prevented by administration to a subject suspected of having or known to have CTD or to be at risk of developing CTD of an active agent that modulates (e.g. increases, decreases, blocks or inhibits) the level or activity (i.e. function) ofone or more TRPIs (or ofone or more TRPs) that are differentially present in biological samples of subjects having CTD compared to control subjects. In one embodiment, CTD is treated by administering to a subject suspected of having or known to have CTD or to be at risk of developing CTD an active agent that upregulates (e.g. increases) the level or activity (i.e. function) ofone or more TRPIs (or one or more TRPs) that are decreased a biological sample from subjects having CTD.

CTD is also treated or prevented by administration to a subject suspected of having or known to have CTD or to be at risk of developing CTD of an active agent that downregulates the level or activity ofone or more TRPIs (or one or more TRPs) that are increased in a biological sample from subjects having CTD. i a prefened embodiment, therapy or prophylaxis is tailored to the needs of an individual subject. Thus, in specific embodiments, agents that promote the level or function ofone or more TRPIs, (or one or more TRPs) are therapeutically or prophylactically administered to a subject suspected of having or known to have CTD, in whom the levels or functions of said one or more TRPIs (or one or more TRPs), are absent or are decreased relative to control subjects or a normal reference range. In further embodiments, active agents that decrease the level or function ofone or more TRPIs, are therapeutically or prophylactically administered to a subject suspected of having or known to have CTD in whom the levels or functions of said one or more TRPIs, (or one or more TRPs) are increased relative to control subjects or to a reference range. The change in TRPI function or level, or TRP level, due to the administration of such active agents can be readily detected, e.g. by obtaining a sample (e.g. a sample of wound exudate, blood or urine or a tissue sample such as dermal tissue) and assaying in vitro the levels of said TRPs or the levels or activities of said TRPIs, or the levels of mRNAs encoding said TRPIs. or any combination of the foregoing. Such assays can be performed before and after the administration of the agent as described herein.

Therapeutic Formulations and Methods of Administration

The invention provides methods of treatment comprising administering to a subject an effective amount of an active agent, as defined above. The active agent will usually be formulated as a pharmaceutical composition in which the active agent can optionally be combined with a pharmaceutically acceptable carrier, vehicle or diluent. In a prefened aspect, the active agent is substantially purified (e.g. substantially free from substances that limit its effect or produce undesired side-effects).

In a further embodiment, the invention also provides the use of a TRPI, a TRPI- related polypeptide or an active agent that modulates the expression or activity of a TRPI or TRPI-related polypeptide in the preparation of a medicament for the treatment of CTD. Formulations and methods of administration that can be employed are described below and can be determined and selected by the skilled artisan based on the present description. Various delivery systems are known and can be used to administer an agent of the invention, e.g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the active agent, receptor-mediated endocytosis (see, e.g. Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g. by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it maybe desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g. as an ointment or cream, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, fibers, or commercial skin substitutes. In one embodiment, administration can be by direct application at the site of the CTD.

In another embodiment, the active agent can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet another embodiment, the active agent can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). hi another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drag Bioavailability, Drug Product Design and

Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al, 1985, Science 228:190; During et al., 1989, Ann. Neural. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e. the CTD, thus requiring only a fraction of the systemic dose (see, e.g. Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138 (1984)).

In another embodiment where the active agent of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g. by use of a retroviral vector (see, e.g. U.S.4,980,286), or by direct injection, or by use of microparticle bombardment (e.g. a gene gun; Biolistic™, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g. Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88:1864- 1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions, Such compositions comprise a therapeutically effective amount of an active agent, and a pharmaceutically acceptable carrier. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which,the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a prefened carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid caniers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral fonnulation can include standard caniers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. hi a prefened embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or salt fonns. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the active agent of the invention which will be effective in the treatment of CTD can be determined by standard clinical techniques based on the present description. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human admimstration, (b) directions for use, or both.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention, including uses thereof. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental enors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1. Identification of Proteins Differentially Expressed in the Wound

Exudate of Patients with Chronic Dermal Ulcers.

Sample Selection and Preparation. Wound fluid samples were collected from venous ulcer (CDU) patients and from acute wound samples of control subjects. Wound fluid samples from CDU patients were collected at baseline, followed by sequential collection over 16 weeks. Clinical data on healing status was also reported and collected. A protein assay (Pierce BCA 23225) was performed on each wound exudate sample as received. Prior to protein separation, each sample was processed for selective depletion of certain proteins, in order to enhance and simplify protein separation and facilitate analysis by removing proteins that may interfere with or limit analysis of proteins of interest. See WO 99/63351, which is herein specifically incorporated by reference in its entirety, with particular reference to pages 3 and 6.

Removal of albumin, haptoglobin, transfenin, alpha- 1-antitrypsin and immunoglobin G (IgG) from wound exudate ("wound exudate depletion") was achieved by an affinity chromatography purification step in which the sample was passed through a series of Hi-Trap™ columns containing immobilized antibodies for selective removal of albumin, haptoglobin, alpha- 1-antitrypsin and transfenin, and protein G for selective removal of immunoglobin G. Two affinity columns in a tandem assembly were prepared by coupling antibodies to protein G-Sepharose™ contained in Hi-Trap™ columns (Protein G-Sepharose Hi-Trap™ columns (1 ml) Pharmacia 17-0404-01). This was done by circulating the following solutions sequentially through the columns: (1) Dulbecco's Phosphate Buffered Saline (Gibco BRL 14190-094); (2) concentrated antibody solution; (3) 200 mM sodium carbonate buffer, pH 8.35; (4) cross-linking solution (200 mM sodium carbonate buffer, pH 8.35, 20 mM dimethylpimelimidate); and (5) 500 mM ethanolamine, 500 mM NaCl. A third (un-derivatised) protein G Hi-Trap column was then attached to the lower end of the tandem column assembly. The chromatographic procedure was automated using an Akta Fast Protein Liquid Chromatography (FPLC) System. The samples were passed through the series of 3 Hi-Trap™ columns in which the affinity chromatography media selectively bind the above proteins thereby removing them from the sample. Fractions (typically 3 ml per tube) were collected of unbound material ("Flowthrough fractions") that eluted through the column during column loading and washing stages and of bound proteins ("Bound/Eluted fractions") that were eluted by step elution with h nunopure Gentle Ag/Ab Elution Buffer (Pierce 21013). The eluate containing unbound material was collected in fractions which were pooled, and desalted/concentrated by centrifugal ultrafiltration. The sample was recovered in 2D Sample Buffer (see below) containing a cocktail of protease inlήbitors (Sigma P2714) and stored at -70°C to await further analysis by 2D PAGE. An aliquot of the stored sample was prepared for 2D analysis by adding Resolytes 3.5-10 (BDH 44338 2x) to 2% (v/v), as well as a trace of

Bromophenol Blue and further 2D Sample Buffer in a final volume of 370 microl. 2D

Sample Buffer: 8M urea (BDH 452043w), 2M thiourea (Fluka 88810), 4% CHAPS (Sigma

C3023), 65mM dithiotheitol (DTT).

Isoelectric Focusing. Isoelectric focusing (DBF), was performed using the hnmobiline™ DryStrip Kit (Pharmacia BioTech), following the procedure described in the manufacturer's instructions, see Instructions for hnmobiline™ DryStrip Kit, Pharmacia, 18-

1038-63, Edition AB. (incorporated herein by reference in its entirety). Immobilized pH Gradient (IPG) strips (18cm, pH3-10 non- linear strips; Pharmacia 17-1235-01) were rehydrated overnight at 20°C with the 370 microl. of sample prepared as described above.

The reswelled IP G strips were then transfened to a Multiphor II Electrophoresis Unit (Pharmacia 18-1018-06), covered with mineral oil (Pharmacia 17-3335-01) and subjected to first dimension isoelectric focussing using a Pharmacia EPS3500XL power supply (19- 3500-01) according to the following profile: Initial voltage = 300N for 2 hrs; Linear Ramp from 300N to 3500V over 3 hrs; Hold at 3500V for 19 hrs. For all stages of the process, the current limit was set to 10 mA for 12 gels, and the power limit to 5W. The temperature was held constant at 20°C throughout the run.

Gel Equilibration and SDS-PAGE. After the final 19 hr step, the strips were immediately removed and immersed for 10 mins at 20°C in a solution of the following composition: 6M urea; 2% (w/v) DTT; 2% (w/v) SDS; 30% (v/v) glycerol (Fluka 49767); 0.05M Tris/HCl, pH 6.8 (Sigma T-1503). After removal from the solution, the strips were loaded onto supported gels for SDS-PAGE according to Hochstrasser et al., 1988, Analytical Biochemistry 173: 412-423, with modifications as specified below.

Preparation of Supported Gels. The gels were cast between two glass plates of the following dimensions: 23cm wide x 24cm long (back plate); 23cm wide x 24cm long with a 2cm deep notch in the central 19cm (front plate). To promote covalent attachment of SDS- PAGE gels, the back plate was treated with a 0.4% solution of γ-methacryl- oxypropyltrimethoxysilane in ethanol (BindSilane™; Pharmacia 17-1330- 01).(BindSilane™; Pharmacia 17-1330-01). The front plate was treated with a 2% solution of dimethyldichlorosilane dissolved in octamethyl cyclo-octasilane (RepelSilane™ Pharmacia 17-1332-01) to reduce adhesion of the gel. Excess reagent was removed by washing with water, and the plates were allowed to dry. At this stage, both as identification for the gel, and as a marker to identify the coated face of the plate, an adhesive bar-code was attached to the back plate in a position such that it would not come into contact with the gel matrix.

The dried plates were assembled into a casting box with a capacity of 13 gel sandwiches. The front and back plates of each sandwich were spaced by means of 1mm thick spacers, 2.5 cm wide. The sandwiches were interleaved with acetate sheets to facilitate separation of the sandwiches after gel polymerization. Casting was then carried out according to Hochstrasser et al., op. cit. A 9-16%) linear polyacrylamide gradient was cast, extending up to a point 2cm below the level of the notch in the front plate, using the Angelique gradient casting system (Large Scale Biology Corporation, Germantown, MD). Stock solutions were as follows. Acrylamide (40% in water) was from Serva (10677). The cross-linking agent was PDA (BioRad 161-0202), at a concentration of 2.6%> (w/w) of the total starting monomer content. The gel buffer was 0.375M Tris/HCl, pH 8.8. The polymerization catalyst was 0.05%> (v/v) TEMED (BioRad 161-0801), and the initiator was 0.1% (w/v) APS (BioRad 161-0700). No SDS was included in the gel and no stacking gel was used. The cast gels were allowed to polymerize at 20°C overnight, and then stored at 4°C in sealed polyethylene bags with 6ml of gel buffer, and were used within 4 weeks.

SDS-PAGE. A solution of 0.5% (w/v) agarose (Fluka 05075) was prepared in running buffer (0.025M Tris, 0.192M glycine (Fluka 50050), 0.1% (w/v) SDS), supplemented also by a trace of bromophenol blue. The agarose suspension was heated to 70°C with stirring, until the agarose had dissolved. The top of the supported 2nd D gel was filled with the agarose solution, and the equilibrated IPG strip was placed into the agarose, and tapped gently with a palette knife until the IPG strip was intimately in contact with the 2nd D gel. The gels were placed in the 2nd D running tank, as described by Amess et al. (1995) Electrophoresis 16: 1255-1267 (incorporated herein by reference in its entirety). The tank was filled with running buffer (as above) until the level of the buffer just exceeded the top of the slab gel, so as to achieve efficient cooling of the active gel area. Running buffer was added to the top buffer compartments formed by the gels, and then voltage was applied immediately to the gels using a Consort E-833 power supply. The gels were run at lOmA/gel for 10 mins. The power limit was set to 150W for a tank containing 6 gels, and the voltage limit was set to 600V. After 10 mins, the gels were then run at 30mA/gel, with the same voltage and power limits as before, until the bromophenol blue line was 0.5 cm from the bottom of the gel. The temperature of the buffer was held constant at 16°C throughout the run.

Staining. Upon completion of the electrophoresis run, the gels were immediately removed from the tank for fixation. The top plate of the gel cassette was carefully removed, leaving the gel bonded to the bottom plate. The bottom plate with its attached gel was then placed into a staining apparatus, which can accommodate 12 gels. The gels were completely immersed in fixative solution of 40% (v/v) ethanol (BDH 28719), 10% (v/v) acetic acid (BDH 100016X), 50% (v/v) water (MilliQ-Millipore), which was continuously circulated over the gels. After an overnight incubation at room temperature, the fixative solution was drained from the tank, and the gels were primed by immersion in 7.5%> (v/v) acetic acid, 0.05% (w/v) SDS, 92.5% (v/v) water for 30 mins. The priming solution was then drained, and the gels were stained by complete immersion for 4 hours in a staining solution of Sypro Red (Molecular Probes, Inc., Eugene, Oregon). Alternative dyes which can be used for this purpose are described in US patent application number 09/412168, filed October 5 1999.

Gel Imaging. A computer-readable output was produced by imaging the fluorescently stained gels with the modified version of the prefened scanner described above. This scanner has a gel carrier with four integral fluorescent markers (Designated Ml, M2, M3, M4) that are used to conect the image geometry and are a quality control feature to confirm that the scanning has been performed conectly. For scanning, the gels were removed from the stain, rinsed with water and allowed to air dry briefly, before they were scanned. After imaging, the gels were sealed in polyethylene bags containing a small volume of staining solution, and then stored at 4^°C.

Digital Analysis of the Data. The data were processed as described in U.S. Patent No 6,064,754, (published as WO 98/23950) at Sections 5.4 and 5.5 (incorporated herein by reference).

Assignment of pi and MW Values. Landmark identification was used to determine the pi and MW of features detected in the images. Twelve landmark features, designated WEI to WEI 1, were identified in a standard wound exudate image. These landmark features are identified and were assigned the pi and/or MW values identified in Table IX.

Table IX. Landmark Features Used in this Study

As many of these landmarks as possible were identified in each gel image of the dataset. Each feature in the study gels was then assigned a pi value by linear interpolation or extrapolation (using the MELANIE®-iI software) to the two nearest landmarks, and was assigned a MW value by linear interpolation or extrapolation (using the MELANIE®-II software) to the two nearest landmarks.

Results. These initial experiments identified 64 features that were decreased and 67 features that were increased in tissue repair (CDU) wound exudate as compared with healing wound exudates. Details of these TRPs are provided in Tables I and H, the fold changes seen for these TRPs are given in Tables X and XI below. Each TRP was differentially present in tissue repair wound exudate as compared with normal wound exudate. h addition, these experiments also identified 69 features that were found to be significantly associated with wound healing in multivariate analyses. Details of these MCIs are provided in Table HI. Some more highly prefened TRPs were identified using several analysis methods: TRP-61 and TRP-125. Partial amino acid sequences were determined for the TRPIs present in these TRPs. Details of these TRPIs are provided in Tables TV, V and VI.

Table X - TRPs Decreased in Wound Exudate of Subjects Having a CDU

Table XI - TRPs Increased in Wound Exudate of Subjects Having a CDU

EXAMPLE 2. Evaluating Serum Protein Profiles for Biomarker Discovery.

A study was conducted to examine the use of two-dimensional electrophoresis (2- DE) to profile proteins from different samples for use as markers of dennal ulcers. The protocol was to determine markers at 4 weeks that are predictive of clinical status at 12 weeks. A total of 100 MCIs were chosen, and samples were taken from serum, biopsy and wound fluid, with a fourth group being chosen based on mapping to previous results, such as putative fransthyretins, etc. The procedures and analysis described above were followed in the processing of the samples and the review of the results. Based on an optimization study and experience, all samples were run at a pH ranging from 4 to 7. It was found that the acid range was more finely resolved, but that there was little loss at the basic range, so that the latter was prefened and chosen.

Results.

From among 21 patients, 4 patients were observed to have wounds that healed ('healers'). This result was not predicted by pre-screening clinical examination. These 4 patients were grouped and compared to 8 non-healers, and proteins were selected where their feature presence > 20%, and their fold change > 2 -fold. Based on these results, the ability of the present invention to yield meaningful information and to identify markers of dennal ulcers is established and confirmed.

Image analysis software was used to compute master gels, allowing the expression of a subset of 25 proteins to be monitored. Clustering software was applied to identify protein responses to treatment throughout the study time course. Two proteins were observed to decrease in abundance in the treated cluster set (2/3 subjects), yet remained either stable, or increased in the control, untreated, set (2/3 subjects). These proteins conespond to TRP-110/TRPI-llO and TRP104/TRPI-104 which been demonstrated herein to be increased in patients with CDUs compared to wound exudate from healing dermal ulcers. An additional protein was essentially unchanged in the control set, while it showed variable expression after treatment. These results, demonstrate that enriched serum samples can be used with 2-DE to observe downstream physiological responses to drag treatment and that the biomarkers which form part of the invention may also be used to monitor treatment and healing in patients.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing, screening, diagnosis or prognosis of chronic tissue damage in a subject, for identifying a subject at risk of developing chronic tissue damage, or for monitoring the effect of therapy administered to a subject having chronic tissue damage, said method comprising detecting and/or quantifying in a biological sample from said subject a TRPI as defined in Tables TV to VI.

2. The method of claim 1 wherein the chronic tissue damage is a chronic dermal ulcer.

3. The method of claim 1 or claim 2 wherein the subj ect is a hurnanΛ

4. The method of any one of claims 1 to 3 wherein the level of the TRPI is further compared to a control or a pre-determined reference range.

5. The method of any one of claims 1 to 4 wherein the biological sample is a body fluid.

6. The method of claim 5 wherein the body fluid is wound exudate.

7. An isolated or recombinant TRPI selected from the proteins listed in

Tables iN-NI.

8. A pharmaceutical composition comprising a TRPI as defined in claim 7, and a pharmaceutically acceptable ca ier, vehicle or diluent.

9. A method for screening for or identifying an agent capable of modulating the expression aTRPI as defined in claim 7 or a TRPI-related polypeptide, said method comprising:

(a) contacting a first population of cells expressing the TRPI or TRPI-related polypeptide with a candidate agent;

(b) contacting a second population of cells expressing the TRPI or TRPI-related polypeptide with a control agent; and (c) comparing the level of the TRPI or TRPI related polypeptide in the first and second populations of cells, wherein an agent capable of modulating the expression of the TRPI or TRPI-related polypeptide is identified by a difference in the level of expression of the TRPI or TRPI- related polypeptide in the first and second populations of cells.

10. A method of screening for or identifying agents capable of modulating the activity of a TRPI as defined in claim 7 or a TRPI-related polypeptide, said method comprising:

(a) in a first aliquot, contacting a candidate agent with the TRPI or TRPI-related polypeptide; and

(b) comparing the level of the TRPI or TRPI-related polypeptide in the first aliquot after addition of the candidate agent with the level of the TRPI or TRPI-related polypeptide in a control aliquot, or with a previously determined reference range, wherein an agent capable of modulating the activity of the TRPI or TRPI-related polypeptide is identified by a difference in the activity of the TRPI or TRPI-related polypeptide in the first and second aliquot.

11. The method of claim 10, wherein the TRPI or TRPI-related polypeptide is a recombinant polypeptide.

12. A method of identifying agents capable of modulating the activity of a TRPI as defined in claim 7 or TRPI-related polypeptide, said method comprising:

(a) contacting a first population of cells expressing the TRPI or TRPI-related polypeptide with a candidate agent; and (b) comparing the activity of the TRPI or TRPI-related polypeptide in the first population of cells after addition of the candidate agent with the level of the TRPI or TRPI-related polypeptide in a second cell population treated with a control agent, or with a previously determined reference range, wherein an agent capable of modulating the activity of the TRPI or TRPI-related polypeptide is identified by a difference in the activity of the TRPI or TRPI-related polypeptide in the first and second population of cells.

13. The method of claim 12, wherein the cells recombinantly express the TRPI or TRPI-related polypeptide.

14. A pharmaceutical composition comprising an active agent that modulates the expression and/or activity of a TRPI as defined in claim 7 or a TRPI-related polypeptide and a pharmaceutically acceptable carrier, vehicle or diluent.

15. A method for the treatment of chronic tissue damage comprising administering to a subject in need thereof, a TRPI as defined in claim 7, a TRPI-related polypeptide or an active agent that modulates the expression or activity of a TRPI or TRPI-related polypeptide.