GB2459099A - Genetic markers of wound development - Google Patents

Abstract

A method of diagnosis or prognosis of wound development, comprising detecting in a sample of a nucleic acid for the presence of a polymorphism in one or more genes selected from the ferroportin gene (preferably FPN-1-8CG and FPN-1-98GC) and the transferrin gene (preferably C2). The wound may be a chronic wound consisting of: diabetic ulcers, decubitis ulcers or most preferably venous ulcers. The method may also comprise detecting additional polymorphisms selected from: HFE C282Y; HD63; Factor XIII-V34L; and MMP12-82AG. Also claimed are: a diagnostic device for use in the diagnosis/prognosis of wound development; a diagnostic test kit; a wound treatment system comprising oxidised cellulose; a method of preventing a wound; a method of treating a wound; and a method of treating a patient utilising iron chelators, or steroids, or calcium antagonists, or NSAIDS and mesoglycan.

Description

GENETIC MARKERS OF WOUND DEVELOPMENT

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to monitoring patients for the potential onset or development of a wound by measuring in a sample of nucleic acid for the presence of a polymorphism in an identified gene. The present invention provides methods of diagnosis, prognosis and treatment of wounds; and also provides wound treatment systems and devices and kits for use in such methods.

BACKGROUND ART

io In mammals, injury triggers an organised complex cascade of cellular and biochemical events that result in a healed wound. Wound healing is a complex dynamic process that results in the restoration of anatomic continuity and function; an ideally healed wound is one that has returned to normal anatomic structure, function and appearance.

A chronic wound is a wound that does not heal in an orderly series of stages or in a predicted amount of time. Wounds that do not heal within three months are often considered chronic. Common types of chronic wounds include, but are not limited to venous leg ulcers, diabetic foot ulcers, decubitis ulcers, arterial leg ulcers, those of mixed aetiology (venous and arterial) or those with no known aetiology (Mustoe T. 2005. Dermal ulcer healing: Advances in understanding.

Presented at meeting: Tissue repair and ulcer/wound healing: molecular mechanisms, therapeutic targets and future directions. Paris, France, March 17-18, 2005).

Early diagnosis of the potential onset of a wound may help to prevent a wound from forming. In the situation where a wound has already developed, knowledge of the prognosis of a wound may enable patients to receive maximum benefit from therapy.

A particularly common type of wound is a venous leg ulcer (VLU). Most VLUs are associated with chronic venous disease (CVD), but other causes or contributing factors include immobility, obesity, trauma, arterial disease, vasculitis, diabetes and neoplasia.

CVD is a common condition that affects 20%-50% of the Caucasian population. In most cases it is a minimally disabling disease, but in approximately 10% of cases it progresses toward chronic venous leg ulceration, the overall prevalence of which is 1% to 2% of the population in the United States and Europe.

Patients who develop VLU often develop a pre-ulcer state lipodermatosclerosis (LDS), which appears as brown or red patches and is thought to occur by an inflammatory reaction affecting the skin and subcutaneous tissues. Without treatment, LDS will most likely progress to ulceration.

If the LDS progresses to ulceration, additional complications may also occur, such as infection and other clinical procedures will need to be initiated such as debridement of the wound, use of appropriate dressings or further surgery.

The earlier either LDS or the ulcer itself can be treated, the greater chance that the treatment will be successful.

Most leg ulcers could be healed if patients were admitted to hospital for continuous leg elevation, However, shortage of hospital beds and the high cost of inpatient care mean that this once popular approach is now rarely practical.

Previous work has demonstrated that a single nucleotide substitution in the hemochromatosis gene (HFE-C282Y) increased by seven-fold the risk of a patient developing a venous ulcer (Zamboni et a!., J. Vasc. Surgery 2005; 42:309-314).

Another single nucleotide substitution in the hemochromatosis gene (HD63) has been shown to correlate with the earlier onset of ulcers with regard to age. The hemochromatosis gene codes for a membrane receptor involved in iron metabolism and it is thought that this gene may lead to the body's inability to control iron levels, in the serum, which leads to ulceration.

It would be beneficial to know which patients are susceptible to developing ulcers and furthermore, if an ulcer does develop, how likely the patient is to respond to therapy. Thus a critical objective is to identify a prognostic factor for ulcer onset to provide earlier and improved choices of treatment. Present techniques, such as duplex scanning and plethysmographic investigations, fail to predict ulcer appearances and are insufficient for categorizing patients by the clinical severity of the disease. If patients at risk of developing a wound could be identified, suitable preventative measures could be used in a targeted program of potentially great effectiveness.

There therefore remains a need in the art for a method for the early diagnosis and prognosis of wound development, in particular of the development of chronic wounds such as venous leg ulcers, and for devices and wound treatment systems for use in carrying out such methods.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a method of diagnosis or prognosis of wound development.

In a first aspect of the invention, a method of diagnosis or prognosis of wound development is provided, which method comprises the step of detection in a is sample of nucleic acid of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene.

Any type of wound may be diagnosed for treatment using the method of the present invention. In one embodiment, the wound is a chronic wound, such as a venous ulcer, diabetic ulcer, decubitis ulcer and a chronic ulcer of unknown aetiology. Suitably, the chronic wound is a venous leg ulcer.

The diagnostic and prognostic methods of the present invention may be performed on a sample that has been removed from the body, that is, in vitro, for example as a clinical swab or as a fluid sample but can also be performed in situ.

The sample of nucleic acid may be derived from blood, urine, lymph or any suitable body fluid. Suitably, the sample of nucleic acid is derived from a sample of blood.

The sample of nucleic acid may be selected from one of genomic DNA, cDNA, RNA or mixtures thereof. Suitably, the sample of nucleic acid is genomic DNA. It will be appreciated that the sample may be a nucleic acid sequence corresponding to the sequence in the sample. That is to say that all or part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis for the presence of a polymorphism.

In one embodiment, the method according to the present invention further comprises genotyping the sample of nucleic acid. Recent advances in genetic screening have improved doctors' ability to diagnose and treat certain illnesses.

For example, genetic tests can identify a particular problem gene. Once a particular problem gene has been identified, alterations in this gene may provide a marker that would be useful for diagnosis. Furthermore alterations in this gene may be useful for the identification of proteins for further investigation as agents implicated in the particular disease in question.

The present inventors have discovered that genetics and gene derived products may play an important role in whether an individual patient may develop a wound such as an ulcer and in determining that patient's ability to heal.

A polymorphism is a genetic variant that appears in at least 1% of a population.

Developments in DNA sequencing have allowed the study of allelic versions of a gene by sequencing samples of the gene taken from different members of a population. Alleles whose sequence reveals only a single changed nucleotide are called single nucleotide polymorphisms or SNPs.

Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases. The following references give background details on uses of polymorphism detection: Linder et a!. (1997) Clinical Chemistry, 43, 254; Marshall (1997) Nature Biotechnology, 15, 1249; Schafer et a!. (1998), Nature Biotechnology, 16, 33.

The Ferroportin cDNA (GenBank Accession Nos. AF231121 and AF226614) and genomic sequences (GenBank Accession No. NT 0221 97) consists of eight exons and flanking regions. Complete sequencing of the promoter region and the eight exons of the ferroportin gene has identified five single nucleotide polymorphisms and one microsatellite in the promoter region of the gene as shown in Table 1 (Lee et a!, Blood Cells, Molecules, and Diseases (2001) 27(5) 783-802).

Table 1:

Location Polymorphism Promoter Microsatellite (CGG)7 (CGG)8 (CGG)9 5'UTR-98 G-*C 5-UTR-8 C-*G IVSI-24 C-*G Ex 6 nt 663 C-T Ex8nt1681 A-*G All positions herein relate to the position in the appropriate GenBank Accession number unless stated otherwise or apparent from the context.

In one embodiment, the polymorphism of the Ferroportin gene is selected from any one of the polymorphisms as listed in Table 1.

Mutations in the Ferroportin gene at nucleotide -8 (-8CG) and at nucleotide -98 (- 98GC) relative to the start of translation, are polymorphisms which occur in the 5' UTR flanking the iron-responsive element and are found in patients with problems in iron homostasis (prone to iron overload) (Lee et a!., Blood Cells, Molecules and Diseases, 2001, 27(5) 783-802). These single nucleotide polymorphisms occur at higher frequency in patients with high iron levels.

In a further embodiment of the invention, the polymorphism of the Ferroportin gene is selected from one or more of FPN-1-8CG and FPN-1-98GC.

Human serum Transferrin (Tf) is an iron binding protein, in the blood (Gomme and is McCann, DDT, 2005, Vol. 10, No.4, 267-273). Tf is divided into two evolutionary related lobes, designated the N-lobe (336 amino acids) and the C-lobe (343 amino acids), which are linked by a short spacer sequence.

The Tf molecule is characterised by a significant degree of genetic polymorphism (Welch and Langmead; 1990; Int. J. Biochem. 22, 275-282; Tf cDNA GenBank Accession No NM 001063; Tf genomic sequence GenBank Accession No. NT_005612). There are three major isotypes known as B, C and D. The majority of people carry the C allele, in particular Cl. The If C2 variant arises when a proline in the C-terminal lobe of native Tf Cl, in position 570, is replaced by a serine (Pro57OSer mutation). The Tf C2 allele is thought to be expressed in patients with diseases thought to be caused by free radicals.

In a further embodiment, the polymorphism of the Transferrin gene is the C2 polymorphism.

The inventors have found that the presence of a polymorphism in one or more genes selected from the group consisting of FPN-l-8CG and FPN-l-98GC of the Ferroportin gene and Tf C2 of the Transferrin gene correlates to an increased risk of developing chronic wounds such as venous leg ulcers.

In one embodiment, the polymorphism is indicative of a predisposition to developing a wound. Thus the likelihood of developing a wound is between 1.5-15 fold more likely in cases in which a polymorphism of the present invention is detected. Suitably, the likelihood of developing a wound is between 1.5-11.5 fold higher when a polymorphism of the present invention is detected.

In a further embodiment, the polymorphism is indicative of the likelihood of responding to therapy (for example surgery intervention together with treatment) if a wound develops. Thus the likelihood of responding to therapy is between 2-10 foid more likely in cases in which a polymorphism of the present invention is detected. Suitably, the likelihood of responding to therapy is between 4-10 fold more likely when a polymorphism of the present invention is detected.

The present invention allows the identification of a high-risk population of patients or the identification of potentially difficult to treat patients by means of a simple blood test that would act as a genetic screening device. The identified patients could then be treated as appropriate before the development of the wound or before the wound has progressed and thus become more difficult to treat.

The present invention thus provides an opportunity to selectively provide suitable preventative measures. Examples of such preventative measures include: (i) Increased monitoring (e.g. frequency of visits) of susceptible patients. If problems can be identified early and treatment regimes set into place, there will be an increased chance of a wound responding even with conventional simple treatments.

(ii) Implementing prevention methods in susceptible patients. For example enhancing mobility in younger patients (maintaining function of the calf muscle pump), compression therapy (i.e. a high risk patient could be put on preventative programs) or good diet. Good nutrition facilitates healing of wounds whilst malnutrition inhibits and complicates the process.

(iii) Targeting complex diagnostics (for example, ultrasound) to the high risk' patients which may save both cost and time and should prioritise high risk patients.

(iv) Enabling earlier referral to a vascular surgeon. Surgical intervention, if early enough, can stop the progression to an ulcer or even reverse it. Early intervention is critical as surgery is more successful in mobile patients and thus early diagnosis or prognosis would be vital to the chance of success. Early surgical treatment can reduce the complexity, risk and costs involved compared to later surgical treatments such as skin grafts which would result in increased hospital stays and therefore cost.

(v) Using various non-surgical approaches to aid the management of wounds.

The earlier provision of suitable pharmaceutical compounds, alone or in combination, will be advantageous in the management of wounds. Certain steroids, including but not limited to stanozolol and danazol, iron chelators and the like as disclosed in W003002119 have been reported to improve lipodermatosclerosis, which if treated will prevent the progression to ulceration. In addition pharmaceutical compounds such as pentoxyfylline improve perfusion of the peripheral vascular bed and therefore improve the microcirculation for the treatment of vascular ulcers. lioprost, a prostacyclin analogue, is also useful for treating ischaemic limbs and venous leg ulcers that are secondary to connective tissue diseases. Other drugs such as calcium antagonists (e.g. diltiazem and nifedipine) have also been reported to be useful for treating leg ulcers that are secondary to Raynaud's disease or connective tissue diseases (See Enoch, Grey and Harding, BMJ 2006; 332; 900-903).

In addition to the above advantages of early identification of high-risk patients, the identification of the polymorphism may also provide guidance on what type of therapy is likely to work. For example, if the polymorphism indicates it is to do with iron hemostasis and control of plasma iron levels (as the Ferroportin FPN1 -8CG, and/or FPN-98GC) or Transferrin (Tf C2) then therapy that chelates or reduces iron levels may be effective (see W0030021 19). Identification of polymorphisms that increase the risk of ulcer formation (or severity) may also provide guidance on what new therapies to develop.

The identification of one of the polymorphisms of the present invention may additionally allow the identification of patients that may be more likely to get recurrent ulcers.

It will be appreciated that the presence of more than one genetic marker may be measured.

A further aspect of the method of the present invention comprises a second step of detection in a sample of nucleic acid for the presence of one or more additional polymorphisms in one or more genes selected from the group consisting of the Hemochromatosis gene, Factor XIII gene or the family of matrix metalloproteinase genes.

The hemochromatosis gene (cDNA; GenBank Accession No NM_i 39009) codes for a membrane receptor involved in iron metabolism. The HFE-C282Y and H63D mutations of the hemochromatosis gene are common genetic defects of this gene.

Thus, in one embodiment, the polymorphism of the second step of detection is selected from one or more of HFE-C282Y and H63D.

Coagulation factor XIII (FXIII) is a plasma transglutaminase circulating as an inactive tetramer of two catalytic A-subunits and two accessory B-subunits (A2B2).

The FXIII gene (FXIII-A cDNA; GenBank Accession No NM_000i29 and FXIII-B cDNA; GenBank Accession No NM_001 994) is highly polymorphic and these polymorphisms are outlined in Table 2 (Tognazzo et. a!., J. Vasc. Surg. 2006; 44:815-9). In one embodiment, the polymorphism of the second step of detection is selected from any one of those listed in Table 2.

Table 2: Factor XIII genotypes Location Polymorphism Codon 34 (FXIII-A) Val-Leu Codon 564 (FXIII-A) Pro-*Leu Codon 204 (FXIII-A) Tyr-*Phe Codon 95 (FXIII-B) His-Arg In a further embodiment, the polymorphism of the second step of detection is Factor XIII V34L.

The matrix metalloproteinases (MMP5) are a family of secreted zinc metalloproteases that degrade the collagens and other extracellular matrix components important in tissue remodelling and repair during development and inflammation. Exemplary polymorphisms of the MMPs are outlined in Table 3 (Su et. a!., Carcinogenesis 2006; 27:5: 1024-1029; Zhai Y et al., Gut 2007; 56:445- 447). In one embodiment, the method of the invention additionally comprises the step of measuring in a sample of nucleic acid for the presence of a polymorphism of one of the MMP family of genes, for example one selected from any one of those listed in Table 3.

Table 3:

Location Polymorphism MMPI -1607 1GI2G MMP2 -1306 CIT MMP2 -735 CIT MMP3 -1171 5A16A MMP9 -1562 CIT MMP12-82 AIG MMP13-77 G/A In a further embodiment, the polymorphism of the second step of detection is MMP1 2-82AG.

In a particular embodiment, the polymorphisms of the second step of detection are selected from the group consisting of HFE C282Y, HD63, Factor Xlll-V34L and MMPI 2-82AG.

In certain embodiments, at least two, three, four, five, six or more markers are monitored.

Both the Factor Xlll-V34L and MMPI2-82AG polymorphisms have been shown to be associated with smaller ulcers and increased healing time (Gemmati et a!., J. Vasc. Surg. 2006; 44: 554-62).

Standard genetic technologies (i.e. P.C.R. based methods and enzyme restriction analysis) may be used to identify the selected polymorphisms or gene markers (SNP5)(Gemmati D, eta!, Mol Med. 2007;13:112-20; Gemmati eta!, J Vasc Surg.

2006;44:554-62; Zamboni et a!, J Vasc Surg. 2005;42:309-14). The informative SNPs may be processed by DNA micro-chip based techniques using automated hybridization and detection protocols as set out further below.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures, which may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Table 4 lists a number of mutation detection techniques, some based on the PCR. These may be used in combination with a number of signal generation systems, a selection of which is listed in Table 5. Further amplification techniques are listed in Table 6. Many current methods for the detection of allelic variation are reviewed by Nollau eta!., Olin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. By U. Landegren, Oxford university press, 1996 and "PCR", 2 Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations ALEXTM Amplification refractory mutation system linear extension APEX Arrayed primer extension ARMSTM Amplification refractory mutation system b-DNA Branched DNA CMC Chemical mismatch cleavage bp base pair COPS Compeititive oligonucleotide priming system DGGE Denaturing gradient gel electrophoresis FRET Fluorescence resonance energy transfer LCR Ligase chain reaction MASDA Multiple allele specific diagnostic assay NASBA Nucleic acid sequence based amplification OLA Oligonucleotide ligation assay PCR Polymerase chain reaction PIT Protein truncation test RFLP Restriction fragment length polymorphism SDA Strand displacement amplification SNP Single nucleotide polymorphism SSCP Single-strand conformation polymorphism analysis SSR Self sustained replication TGGE Temperature gradient gel electrophoresis Table 4 -Mutation Detection Techniques General: DNA sequencing, Sequencing by hybridisation Scanning: PTT (not useful for detection of promoter polymorphisms), SSCP, OGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage Hybridisation Based: Solid phase hybridization: Taqman TM -US-5210015 & US-5487972 (Hoffmann-La Roche); Molecular Beacons -Tyagia et al (1996), Nature Biotechnology, 14, 303; WO 95/1 3399 (Public Health Inst., New York).

Extension Based: ARMSTM, ALEXTM -European Patent No. EP 332435 Bi (Zeneca Limited), COPS -Gibbs et al (1989) Nucleic Acids Research, 17, 2347.

Incorporation Based: Mini-sequencing, APEX Restriction Enzyme Based: RFLP, Restriction site generating PCR Ligation Based: OLA Other: Invader assay Table 5-Signal Generation or Detection Systems Fluorescence: FRET, Fluroescence quenching, Fluorescence polarisation -UK Patent No. 2228998 (Zeneca Ltd) Other: chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetirc, Hybrid isation protection assay, Mass spectorometry.

Table 6-Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA General molecular biology procedures can be followed from any of the methods described in "Molecular Cloning -A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989).

In a second aspect, the present invention provides a diagnostic device for use in diagnosis or prognosis of wound development by measuring for the presence in a sample of nucleic acid, of at least one marker indicative of wound development, wherein said marker is a polymorphism in the Ferroportin gene or the Transferrin gene. In one embodiment the polymorphism is selected from the group consisting of FPNI -8CG and FPNI -98GC of the Ferroportin gene and C2 of the Transferrin gene.

In one embodiment, the diagnostic device comprises a genotyping device for measurement of the presence of at least one marker of the present invention indicative of wound development.

In a further embodiment, the diagnostic device, in addition to its measuring for a polymorphism in one or more of the Ferroportin gene or the Transferrrin gene, additionally measures in a second step for the presence of one or more polymorphisms selected from the group consisting of HFE C282Y, H63D, Factor XIII V34L and MM12 -82AG.

In one embodiment the device may take the form of a miniaturised Point of Care (Poc) device for near patient testing.

In a further embodiment, the device may comprise of four modules. Module one is a DNA extraction module for a selected SNP or a high purification module system for multiplex detection. Module two is a DNA amplification module (such as PCR) using specific primer design. In one embodiment the primers as set out in Table 7 may be used:

Table 7:

Gene sequence (5'-3') *FPN1 -8CG (Fw) BIOTIN-CCAGTTCCTTGCACTCCTG FPN1 -8CG (Rv) CATCCTCTCTGGCGGTTG FPN -98GC (Fw) CTGCTGAGCCTCCCAAACCGCT FPN -98GC (Rev) CATCCTCTCTGGCGGTTG TF Cl C2 (Fw) GAAAAAGACTATGAGTTGCTGTGC TF Cl C2 (Rv) BIOTIN-GTGTGACCACAGCGTGATTC *The polymorphism at FPN1 -8CG is in complete linkage disequilibrium with the polymorphism -98GC and thus the detection of one of the two provides simultaneous information about the other.

Module three is a DNA recognition module (in which patients' DNA is attached to an array and successively hybridized to complementary specific allele probes) and module four is a signal detection module in which the hybridized DNA is visualized by optical tools (such as fluorescent detection), set to discriminate adequately between the two different alleles by means of optimal background/signal optimization.

The DNA extraction module may contain elements for collecting a sample of blood, and all the reagents necessary for extraction, purification and concentration of nucleic acid. The DNA recognition module may take a format, or formats, similar to that discussed in the literature such as in the EZI DNA Handbook, Qiagen, second edition, February 2004. For example the probe DNA may be attached to a solid support such as glass, plastic or silicon. Alternatively, new lateral flow microarrays have recently been described for specific nucleic acid detection (see for example Carter and Cary, Nucleic Acids Research (2007) 1-11). The probe must be designed in such a way that it strongly distinguishes between the polymorphic and wild type sequence. The nature and number of probes must be selected to obtain the appropriate specificity and selectivity with ratio OD values set at diagnostic (1/5) or research (1/3) purpose depending on the target desired.

The signal detection module may utilise a number of technologies that can be used to determine if the patients DNA has hybridized to the probe or reporter sequences.

In a further aspect, the present invention provides a diagnostic test kit comprising a diagnostic device according to the present invention.

The test kit may comprise, in addition to a diagnostic device according to the io present invention, one or more components selected from: a colour chart or reader for interpreting the output of the diagnostic device, a sampling device for collecting a sample of blood, a wash liquid for carrying a sample of blood through the device, directions for use of the test kit and a pre-treatment solution containing a reagent for pre-treatment of the blood sample. In one embodiment, the components of the test kit are arranged in an easily accessible manner, for example in a package or case for the patient or health care provider to access. In a further embodiment, the test kit is enclosed in a package or case, which may be carried, for example by way of a handle or alternative holding means.

In a further aspect, the invention also provides a system for use in the diagnosis and treatment of wounds comprising a diagnostic device according to the invention and a wound dressing comprising an oxidized cellulose.

The term "oxidized cellulose" refers to any material produced by the oxidation of cellulose, for example with dinitrogen tetroxide. Such oxidation converts primary alcohol groups on the saccharide residues to carboxylic acid groups, forming uronic acid residues within the cellulose chain. The oxidation generally does not proceed with complete selectivity, and as a result hydroxyl groups on carbons 2 and 3 are occasionally converted to the keto form. These keto units introduce an alkali labile link, which at pH 7 or higher initiates the decomposition of the polymer via formation of a lactone and sugar ring cleavage. As a result, oxidized cellulose is biodegradable and bioabsorbable under physiological conditions.

The preferred oxidized cellulose for practical applications is oxidized regenerated cellulose (ORC) prepared by oxidation of a regenerated cellulose, such as rayon.

It has been known for some time that ORC has haemostatic properties. ORC has been available as a haemostatic product called SURGICEL (Registered Trade Mark of Johnson & Johnson Medical, Inc.) since 1950. This product is produced by the oxidation of a knitted rayon material. A modification of porosity, density and knit pattern led to the launch of a second ORC fabric product, INTERCEED (Registered Trade Mark of Johnson & Johnson Medical, Inc.), which was shown to reduce the extent of post-surgical adhesions in abdominal surgery.

io In preferred embodiments of the present invention, the oxidized cellulose in the wound dressing material is complexed with collagen and/or chitosan to form structures of the kind described in W098/00180, EP-A-1153622 and/or WO-A- 2004/026200. For example, the oxidized cellulose may be in the form of milled ORC fibres that are dispersed in a freeze-dried collagen or chitosan sponge. This provides for sustained release of the oxidized cellulose to the wound, together with certain therapeutic and synergistic effects arising from the complexation with collagen. Suitably, the weight ratio of oxidized cellulose to collagen and/or chitosan in the wound contacting material is from about 10:1 to about 1:10, for example from about 70:30 to about 30:70. Suitably, the wound contacting material comprises at least 75% on a dry weight basis of oxidized cellulose, collagen and chitosan, more suitably at least 90% and most suitably it consists essentially of oxidized cellulose, collagen and/or chitosan.

The wound treatment system may be in the form of a kit, and the device and the wound dressing(s) may be packaged together in a single package.

The wound dressing materials used in these aspects of the invention may for example be provided in the form of beads, flakes, powder, and suitably in the form of a film, a fibrous pad, a web, a woven or non-woven fabric, a freeze-dried sponge, a foam or combinations thereof. In certain embodiments, the dressing material is selected from the group consisting of woven fabrics, knitted fabrics, and nonwoven fabrics, all of which may be made by conventional methods. In other embodiments, the material may comprise (or consist essentially of) a freeze-dried sponge or a solvent-dried sponge.

The wound dressing material may be in the form of a solid, or a semi-solid ointment or gel. Suitably, the wound dressing material comprises only up to 20% by weight, suitably less than 10% by weight of water. The relatively low water content improves the stability of the material and makes it possible to sterilize by heat or irradiation without loss of activity. The material may also contain 0-40% by weight, suitably 0-25% by weight of a plasticiser, suitably a polyhydric alcohol such as glycerol. All of the above percentages are on a dry weight basis.

In a further aspect, the invention also provides a system for use in the diagnosis and treatment of wounds comprising a diagnostic device according to the invention and a pharmaceutical composition selected from iron chelators such as deferoxamine, deriprone and deferasirox, N-acetl-cysteine, steroids such as stanozolol and danazol, pentoxyfylline, Iloprost, calcium antagonists such as diltiazem and nifedipine, NSAIDS, mesoglycan and combinations thereof (Arosio, E., et a!., Eur J. Vasc Endovasc Surgery (2001) 22(4), 365-372).

In a further aspect, the present invention provides a method for preventing a (a) establishing the presence of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene in a sample of nucleic acid; and (b) providing one or more preventative strategies selected from; improving the nutritional care, enhancing the mobility of the patient, increased monitoring of the patient, prioritise the patient for preventative surgery or ultrasound diagnostics or suitable drug treatment.

The present invention also provides a method for treating a wound comprising the steps of: (a) establishing the presence of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene in a sample of nucleic acid; and (b) applying a wound dressing comprising oxidized cellulose to the wound; or (c) treating with one or more compounds selected from the group consisting of iron chelators such as deferoxamine, deriprone and deferasirox, N-acetl-cysteine, steroids such as stanozolol and danazol, pentoxyfylline, Iloprost, calcium antagonists such as ditiazem and nifedipine, NSAIDS, mesoglycan and combinations thereof (Arosio, E., et a!., Eur J. Vasc Endovasc Surgery (2001) 22(4), 365-372).

The present invention also provides a method for treating a patient using a pharmaceutica! composition se!ected from iron chelators such as deferoxamine, deriprone and deferasirox, N-acetl-cysteine, steroids such as stanozolol and danazol, pentoxyfylline, Iloprost, calcium antagonists such as diltiazem and nifedipine, NSAIDS, mesoglycan and combinations thereof, wherein the patient is from a subgroup carrying a polymorphism in the Ferroportin gene or the Transferrin gene.

In one embodiment the patient is from a subgroup carrying a polymorphism selected from the group consisting of FPN-1-8CG and FPN-1-98GC of the Ferroportin gene and C2 of the Transferrin gene.

Suitably, the wound is a chronic wound. For example, it may be selected from the group consisting of venous ulcers, diabetic ulcers, decubitis ulcers and chronic ulcers of unknown aetiology.

MODES FOR CARRYING OUT THE INVENTION

Selection of cases and controls 300 patients affected by severe CVD (CEAP clinical classes 04-6) from different areas of Italy were selected from an initial cohort of 1200 patients who were referred to the Center for Vascular Disease, University of Ferrara. For purposes of patient selection, these subjects underwent clinical and duplex scanning examination. Patients affected by peripheral arterial disease were identified and primary cases were separated from post-thrombotic cases. For patient selection the following exclusion criteria were strictly applied in order to exclude any other co-morbidity factor potentially involved in wound etiology: * diabetes; * peripheral arterial disease and/or ankle brachial index <0.9; * hemolytic anemia and/or iron-deficiency anemia and/or malnutrition; * inability to walk; * severe cardiac and/or hepatic and/or renal and/or pulmonary insufficiency; or * chronic administration of cortisones for chronic inflammatory disease and/or ,1,.

dU&01l lull lul u U Applying the above-listed exclusion criteria, 250 cases were selected, which constituted group A. These patients were subsequently divided into two groups: group B, 149 subjects having ulcers in the lower limbs of certain venous origin (clinical classes 05-6), and group C, 101 subjects who had never had skin lesions, of which 98 were primary and 3 were post-thrombotic cases (class C4). Finally, the ulcer group B was subdivided, in accordance with the pathogenesis as well as is the patho-physiology, into primary (subgroup F) and post-thrombotic ulcers (subgroup E).

DNA analysis Specific P.C.R. generated ampkcons were genotyped by Nanogen microchip system. A volume of 5 to 10 p1 containing the HFE C282Y, H63D, FPN1 -8CG was mixed with histidine buffer to 50 mmol/L final concentration in 60 p1 final volume.

About 150-200 ng of each amplicon were addressed onto the Nanochip� Cartridge (H2-type) by means of Nanochip� Loader using default parameters and protocois obtained by the machine's own software. A chemical denaturation of amplicons, by means of NaOH 0.1 N for 5 mm of treatment, ended the addressing protocol. Each reporter mix contained probes specific for each SNP considered, stabilizers and reporter oligonucleotides specific for each SNP considered (see Table 8), and hybridization step and fluorescence scan of the cartridge were carried out by the Nanochip� Reader (details published on www.nanogen.com).

Table 8:

Oligos sequence FPN1 -8CG Discriminator wild type 5'-CTGAGTCCGAACATTGAG-AAAGGTCATGACACTAG -3' FPN 1 -8CG Discriminator mutant 5'-GCAGTA TA TCGCTTGACA-AAAGGTCATGACACTAC-3 FPN1 -8CG Stabilizer 5'-GCGACCCCGCTGGCTCTTCTGCGGCTGCTA-3' HFE C282Y Discriminator wild type 5'-CTGAGTCCGAACATTGAG AGAGATATACGTG-3' HFE C282Y Discriminator mutant 5'-GCAGTCTATCGC]TGACA CAGAGATATACGTA-3' HFE C282Y Stabilizer 5'-CCMGGTGGAGCACCCAGGCCT-3' HFE H63D Discriminator wild type 5'-CTGAGTCCGAACATTGAG TCGTGTTCTATGATC -3' HFE H63D Discriminator mutant 5'-GCAGTATATCGCTTGACA TCGTGTTCTATGATG -3' HFE H63D Stabilizer 1 5'-ATGAGWGTCGCCGTGTGGAGCCCCGAA-3' HFE H63D Stabilizer 2 5'-TGGGCTACGTGGATGACCAGCT-3' TF C2 Discriminator wild type 5-CTGAGTCCGAACATTGAG-ATAGGTACCAGGAAAC-3' LTF 02 Discriminator mutant 5-GCAGTATATCGCTTGACA-MTTGGTACCAGG.A4T -3' TF C2 Stabilizer I 5'-CTGTGGAGGAGTATGCGAACTGCCACCTGG-3' I Italic nucleotides are universal sequence recognized by universal probes labelled with Cy3 and Cy5. The stabiliser' oligo sequences are not labelled with fluorescent probes. They are added to ensure the nucleic acid remains stable during the course of the experiment.

An optimized hybridization touch-down protocol was determined for the analysis of each polymorphism considered. Details of the instrumentation and general protocols of previous reports were followed (Methods Mo! Med 2005; 114: 93-1 06; and Psychiatr Genet 2002; 12: 181-92). About 20% of samples were loaded and analysed in duplicate and heterozygous controls were always included in each assay. Quantitative analysis and genotype designation were realized by using dedicated softwares supplied by the Nanogen� Company and both were set at a diagnostic level (mm. A-ratio between allele signal=5:1).

A standard PCR cycle (94°C130 sec; 56°C/30 sec; 72°/60 sec. Total 35 cycles) was used to genotype the FPN -98CC polymorphism. An amplicon fragment of 308bp in length was obtained and digested by Avail restriction enzyme (GGWCC). Two fragments of 170 and 138 base pairs were obtained when FPN - 98GC was present.

Results The ulceration risk in the presence of either FPN1 -8CG genotype or FPN -98CG genotype is about 4.0-5.0 fold.

The combination of either FPN1 -8CG or FPN -98CG with the HFE C282Y genotype yields appreciable risk value of about 2.7 fold.

The ulceration risk in the presence of TF C2 genotype is 3.6 fold (1.3-1 1.3).

P=O.004 for genotype distribution difference between cases and controls.

The combination of IF C2 with the HFE C282Y genotype yields appreciable risk value of about 3.03 fold (0.65-1 5.2). P=NS due to the shortage of double carriers in both cases and controls.

The above examples have been described by way of example only. Many other examples failing within the scope of the accompanying claims will be apparent to to the skilled reader.

Claims (28)

1. CLAIMS: 1. A method of diagnosis or prognosis of wound development, comprising the step of detection in a sample of nucleic acid for the presence of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene.
2. 2. A method as claimed in claim 1 wherein the polymorphism is selected from FPN-1-8CG and FPN-1-98GC of the Ferroportin gene and C2 of the Transferrin gene.
3. 3. A method as claimed in any preceding claim which method is an in vitro io method.
4. 4. A method according to any preceding claim wherein said step of measuring comprises genotyping the sample of nucleic acid.
5. 5. A method as claimed in any preceding claim wherein the nucleic acid is selected from the group consisting of genomic DNA, cDNA and RNA.
6. 6. A method as claimed in claim 5 wherein the nucleic acid is genomic DNA.
7. 7. A method according to any preceding claim wherein said sample is a sample of blood.
8. 8. A method according to any preceding claim wherein the wound is a chronic
9. 9. A method according to claim 8 wherein the chronic wound is selected from the group consisting of diabetic ulcers, venous ulcers and decubitis ulcers.
10. 10. A method according to claim 9 wherein the wound is a venous ulcer.
11. 11. A method according to any preceding claim, which comprises a second step of detection in a sample of nucleic acid for the presence of one or more additional polymorphisms selected from the group consisting of HFE C282Y, HD63, Factor Xlll-V34L and MMP12-82AG.
12. 12. A method as claimed in any preceding claim wherein the polymorphism is indicative of a predisposition to developing a wound.
13. 13. A method as claimed in any preceding claim wherein the polymorphism is indicative of the likelihood of responding to therapy if a wound develops.
14. 14. A diagnostic device for use in diagnosis or prognosis of wound development by measuring for the presence in a sampie of nucleic acid, or at least one marker indicative of wound development, wherein said marker is a polymorphism in a gene selected from the group consisting of the Ferroportin gene and the Transferrin gene.
15. 15. A diagnostic device as claimed in claim 14 wherein the polymorphism is selected from FPN-1-8CG and FPN-1-98GC of the Ferroportin gene and C2 of the Transferrin gene.
16. 16. A diagnostic device as claimed in claim 14 or claim 15 which comprises a genotyping device for measurement of the presence of the said markers.
17. 17. A diagnostic device as claimed in any one of claims 14 to 16 which measures for the presence of one or more additional polymorphisms selected from the group consisting of HFE C282Y, HD63, Factor Xlll-V34L and MMP12-82AG.
18. 18. A diagnostic test kit comprising a diagnostic device according to any one of claims 14-17 in combination with one or more components selected from the group consisting of: a colour chart for interpreting the output of the diagnostic device, a sampling device for collecting a sample of blood, a wash liquid for carrying a sample of blood through the device, a pretreatment solution containing a reagent for pretreatment of the blood sample, and combinations thereof.
19. 19. A wound treatment system for use in the diagnosis and treatment of wounds comprising: a diagnostic device as claimed in any one of claims 14-17 and one or more treatments selected from (i) a wound dressing comprising an oxidized cellulose; (ii) a pharmaceutical composition selected from iron chelators such as deferoxamine, deriprone and deferasirox, N-acetl-cysteine, steroids such as stanozolol and danazol, pentoxyfylline, Iloprost, calcium antagonists such as di!tiazem and nifedipine, NSA!DS and mesoglycan.
20. 20. A wound treatment system according to claim 19, wherein the wound to dressing comprises oxidized regenerated cellulose.
21. 21. A wound treatment system according to claim 19 or 20, wherein the wound dressing comprises a combination of oxidized regenerated cellulose with collagen and/or chitosan in the dry weight ratio of from about 10:1 to about 1:10.
22. 22. A method for preventing a wound comprising the steps of: is (a) establishing the presence of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene in a sample of nucleic acid; and (b) providing one or more preventative strategies selected from; improving the nutritional care, enhancing the mobility of the patient, increased monitoring of the patient, prioritise the patient for preventative surgery or ultrasound diagnostics or suitable drug treatment.
23. 23. A method for treating a wound comprising the steps of: (a) establishing the presence of a polymorphism in one or more genes selected from the group consisting of the Ferroportin gene and the Transferrin gene in a sample of nucleic acid; and (b) applying a wound dressing comprising oxidized cellulose to the wound.
24. 24. The method according to claim 23, where the oxidized cellulose is oxidized regenerated cellulose.
25. 25. The method according to claim 24, where the wound dressing further comprises collagen or chitosan.
26. 26. The method according to any one of claims 22-25, where the wound is a chronic wound selected from the group consisting of diabetic ulcers, venous ulcers and decubitis ulcers.
27. 27. A method for treating a patient using a pharmaceutical composition io selected from iron chelators such as deferoxamine, deriprone and deferasirox, N-acetl-cysteine, steroids such as stanozolol and danazol, pentoxyfylline, Iloprost, calcium antagonists such as diltiazem and nifedipine, NSAIDS and mesoglycan wherein the patient is from a subgroup carrying a polymorphism in the Ferroportin gene or the Transferrin gene.
28. 28. A method as claimed in claim 27 wherein the polymorphism is selected from FPN-1-8CG and FPN-1-98GC of the Ferroportin gene and C2 of the Transferrin gene.