Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders

Definitions

• MRP8 MRP14 heterodimer or its individual components in combination, for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers
• the present invention relates to the use of an MRP8/MRP14 heterodimer, or of its individual components in combination, of at least one nucleic acid encoding the entire heterodimer or its individual components in combination, or of a cell which is expressing the entire heterodimer, or its individual components in combination, for treating and/or preventing skin diseases, wounds, and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 hetero- dimers, in particular diabetes-associated wounds, and to methods for identifying pharmacologically active substances which exert an influence on the function or expression of MRP8/MRP14 heterodimers.
• Blood platelets which release growth and coagulation factors, aggregate during coagulation. A fibrin matrix is formed, thus enabling cells to migrate within the wound. Subsequently, an inflammatory reaction develops approx. 5-7 days after the injury. In association with this, a variety of cell types, in particular neutro- philic granulocytes and monocytes, migrate into the wound and release mediators of the inflammatory reaction.
• the proliferation phase is required for restoring the blood vessels, regenerating damaged tissue and restructuring the regenerated tissue.
• the processes involved comprise, in particular, neovascularization, fibroblast proliferation and reepithelialization by means of the proliferation and differentia- tion of keratinocytes.
• the fibroblasts secrete several growth factors, such as PDGF and TGF-beta, which in turn regulate the synthesis and deposition of components of the extracellular matrix (ECM), such as fibronectin, laminin, glycosa- minoglycans and collagen.
• ECM extracellular matrix
• the ECM components particularly the collagen, are rearranged.
• the reepitheliahzed wound can mature, and a flat scar is formed within 2 years.
• a large number of growth factors and chemoattractants are required for reconstructing the tissue in a coordinated manner.
• Established forms of therapy are restricted to physical support of the wound healing (e.g. dressings, compresses and gels), to scraping out the necrotic tissue, and to the transplantation of skin cells which have been cultured from skin tissues and/or of matrix proteins. While the therapeutic use of growth factors has been tried out for improving wound healing in recent years, it has not improved conventional therapy in any decisive manner; only PDGF-BB has been authorized for treating venous foot ulcers.
• wound healing e.g. dressings, compresses and gels
• Decubitus ulcers are due to the continuous effect of pressure over long periods and are very deep wounds which are accompanied by necrosis, infection and maceration of the tissue.
• venous ulcers which are induced by venous stasis, are more superficial.
• arterial ulcers are frequently caused by arterial occlusion diseases.
• Diabetic ulcers for their part, are ulcers which occur frequently in diabetes patients.
• the late complications of diabetes also encompass characteristic skin changes such as frequent infections, trophic disturbances and necrobiosis lipoidica. These changes can then, frequently as a result of microangiopathic disturbances, develop into poorly healing ulcers.
• the epidemiological importance of these diseases is made clear by the following statistical data.
• diabetes type II diabetes frequently suffer from chronic ulcers (e.g. "diabetic foot"), about half of which require elaborate in-patient treat- ment and heal poorly.
• Diabetic foot on its own gives rise to more hospital admissions than any other complication associated with diabetes. The number of these cases associated with diabetes type I and type II is on the increase and represents approx. 2.5% of all hospital admissions.
• the object of the present invention is therefore to find a novel active compound which decisively improves the healing and/or prevention of skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular of diabetes-associated wounds which heal poorly.
• diabetes-associated wounds which heal poorly are to be understood as being skin wounds in mammals and humans suffering from diabetes. Examples of such skin wounds are ulcers which are caused by diabetes, for example ulcus cruris arteriosum or necrobiosis lipoidica.
• diabetes-associated wound-healing disturbances are distinguished by a decreased abundance, which is specific for these diseases, in the expression of the MRP8 and MRP14 polypeptides and consequently of the heterodimer MRP8/MRP14.
• compensating for the deficiency of MRP8/MRP14 in diabetic rabbits resulted in an extraordinary increase in the rate of wound healing.
• genetherapeutic treatment of diabetic rats with both MRP8 and MRP 14 genes resulted in significantly improved wound healing.
• an assay for human MRP8/MRP14 heterodimer function on cells could be made available for the first time.
• human MRP8/MRP14 heterodimer is known for a long time, no effect of the human polypeptides on cellular activity could be determined.
• the human MRP8/MRP14 heterodimer exhibits a positive effect on the migration of keratinocytes, thus providing a cell-based assay to test substances for their influence on MRP8/MRP14 heterodimer function.
• the assay can be used to screen for pharmacologically active substances which can be used to treat and/or prevent diseases characterized by a disregulated amount or activity of human MRP8/MRP14 heterodimer or its individual components in combination. It can also be used for monitoring the activity of purified or recombinantly ex- pressed MRP8/MRP14, e.g. in a quality assurance procedure.
• the invention also relates to a functional assay on the activity of human MRP8/MRP14 heterodimer or its individual components in combination, or of at least one nucleic acid encoding the heterodimer or its individual components in combination, comprising the steps of: 1) bringing the human MRP8/MRP14 heterodimer or its individual components in combination, or the at least one nucleic acid encoding the entire heterodimer or its individual components in combination into contact with at least one cell,
• the test substances exhibit a positive effect on migration.
• Such sub- stances can be used to treat and/or prevent skin diseases, wounds and/or wound- healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds.
• the in- vention also relates to a use of this assay for the identification of pharmacologically active substances.
• MRP8 and MRP 14 are polypeptides which have apparent molecular weights of 8 and 14 kDa respectively and which can form heterodimers in vivo, with both the monomeric and the heterodimeric forms being able to exhibit functional activities in mammals (see below).
• the monomers both belong to the family of the cal- granulin or SI 00 polypeptides (Kligmann and Hilt, Trends Biochem. Sci., 13: 437-447).
• the polypeptides of these families are Ca 2+ -binding and Ca 2+ - modulated polypeptides which form antiparallel, noncovalent dimers and which regulate a very wide variety of cellular functions such as cell growth, differentiation, energy metabolism and cytoskeleton-membrane interactions.
• the MRP8 and MRP 14 polypeptides are principally expressed in neutrophilic granulocytes and monocytes and also in activated macrophages (Hessian et al., 1993, J. Leukocyte Biol., 53: 197-204).
• Initial studies suggested that, in humans, the heterodimer MRP8/MRP14 acts as a factor inhibiting macrophage migration (Burmeister et al., 1986, Immunbiol., 171 : 461-474; EP 0162 812), a finding which it has not, however, been possible to confirm. To date, it has not been possible to demonstrate that the heterodimer or the monomers have any chemotactic activity (Hessian et al., see above).
• the murine homolog of MRP8 appears on its own to act as a powerful chemoattractant for neutrophilic granulocytes (Lackmann et al., 1992, J. Biol. Chem., 267: 7499-7504).
• the human MRP8/MRP14 heterodimer plays a role in the adhesion of leukocyte cells to vascular endothelium (Newton and Hogg; 1998, J. Immunol., 160: 1427-1435).
• MRP8 and MRP14 are expressed in keratinocytes which developed abnormally in cell culture (Olsen et al., 1995, Electrophoresis, 16: 2241-2248).
• Examples of biochemically characterized functions of the human MRP8/MRP14 heterodimer are those of binding fatty acid and calcium, with the calcium binding exerting an influence on the fatty acid binding (Kerkhof et al., 1999, J. Biol. Chem., 274: 32672-9).
• the following section shows that, while a number of publications have related to MRP8 or MRP 14 with diseases which are generally characterized by an increased quantity of MRP8 and MRP 14, MRP8 and MRP 14 have not been related to the treatment and/or prevention of skin diseases, wounds and/or wound-healing dis- turbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds which heal poorly.
• an MRP8/MRP14 heterodimer, or its individual components in combination at least one nucleic acid encoding the entire heterodimer or its individual components in combination, or a cell which is expressing the entire hetero- dimer, or its individual components in combination, can be used for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds.
• MRP8 and MRP 14 are greatly increased in association with inflammatory diseases, such as rheumatoid arthritis, inflammatory intestinal diseases, psoriasis, lung inflammation and the rejection of foreign implants (Brandtzaeg et al., 1987, Am. J. Clin. Pathol., 87: 700-707; Wilkinson et al., 1988, J. Cell Science, 91: 221-230; Kelly et al., 1991, Br. J. Dermatol., 124: 403-409; Madsen et al., 1992, J. Invest. Dermatol., 99: 299-305; Roth et al., 1992, Int. Arch. Allergy.
• MRP8 and MRP 14 by phagocytes are increased in association with contact dermatitis (Frantzen et al., 1993, Int. Arch. Dermatol. Immunol., 101: 182-189; Roth et al., 1992, Int. Arch. Dermatol. Immunol., 98: 140-145).
• the hetero- dimer has been repeatedly detected in the epidermis of patients suffering from lichen planus, lupus erythematosus and psoriasis vulgar is but not, however, in the epidermis of normal skin or in patients suffering from leukocytoclastic vasculitis (Kunz et al., 1992, Arch Dermatol.
• MRP14 is strongly ex-pressed in mononuclear phagocytes in granulomatous diseases whereas MRP8 is expressed in granuloma of the foreign body type, erythema nodosum and cat- scratch disease but not, or only weakly, in phagocytes in association with sar- coidosis and tuberculosis.
• MRP8 MRP14 heterodimers are dimers which are associated by way of noncovalent bonds and which contain a mouse or human MRP8 polypeptide as depicted in SEQ ID No. 1 or 2, or an active functional variant thereof, and a mouse or human MRP 14 polypeptide as depicted in SEQ ID No. 3 or 4, or a functional variant thereof.
• “individual components" of the MRP8/MRP14 heterodimer are mouse or human MRP8 polypeptides as depicted in SEQ ID No. 1 to 2, or functional variants thereof, or nucleic acids encoding them, or variants thereof, and mouse or human MRP 14 polypeptides as depicted in SEQ ID No. 3 to 4, or functional variants thereof, or nucleic acids encoding them, or variants thereof.
• the term "functional variants" is to be understood as denoting MRP8 polypeptides or MRP 14 polypeptides which form stable heterodimers with an MRP 14 polypeptide or an MRP8 polypeptide, respectively.
• the formation of the heterodimer can be determined, for example, by means of mass spectrometry (Strupat et al., 2000, J. Am. Soc. Mass Spectrom., 11 : 780-788).
• variants of said polypeptides possess at least approximately 70%, in particular at least approximately 80%, especially at least approxi- mately 90%, sequence identity with one of the sequences SEQ ID No. 1 to SEQ ID No. 4.
• Functional variants of the polypeptide can also be parts of the polypeptides used in accordance with the invention provided the function of the polypeptide is not significantly altered.
• Such amino acids can, for example, be identified by means of alanine scanning (see, e.g., Nagashima et al., 1993, J. Biol. Chem., 268: 2888-92).
• Examples of such functional variants are the polypeptides which are homologous to the polypeptides which can be used in accordance with the invention and which are derived, in particular, from organisms other than humans or mice, preferably from nonhuman mammals such as monkeys, pigs and rats.
• polypeptides which are encoded by different alleles of the gene, in different individuals or in different organs of an organism.
• a posttranslational or cotranslational modification of the polypeptide chain which is present in the native state can be lacking or be altered without this alteration sign- ficantly impairing the activity of the polypeptides.
• the invention also encompasses N-terminal and/or C-terminal, and/or internal, deletions of the polypeptide in the range of approx. 1-15, preferably of approx. 1-10, in particular of approx. 1- 5, amino acids.
• the first amino acid i.e. methionine
• Migration assays suitable for keratinocytes are known to the skilled person and comprise for example, the Boyden chamber assay, the colloidal gold assay, the scratch assay and an assay based on the migration in a fibrin matrix, which are described below in detail.
• the function of the MRP8/MRP14 heterodimer or the individual components of the MRP8/MRP14 heterodimer in combination is understood to encompass the activity of the heterodimer or of its individual components in combination, i.e. especially the activity the heterodimer or of its individual components in combination exert onto the migration of cells, especially onto skin cells such as keratinocytes.
• the activity of the MRP8/MRP14 heterodimer or of its individual components in combination further encompass the che- motrophic acitvity, cell-adhesion, binding activity with respect to fatty acid and calcium, influence onto cell growth, differentiation, energy metabolism and cyto- skeleton-membrane interactions.
• stringent hybridization conditions is to be understood, in particular, as meaning those conditions in which a hybridization takes place, for example, at 60°C in 2.5 ⁇ SSC buffer followed by several washing steps at 37°C in a lower buffer concentration and remains stable.
• the nucleic acids encoding MRP8 and MRP 14 polypeptides which can be used in accordance with the invention are preferably DNA or RNA, preferably DNA, in particular double-stranded DNA. Furthermore, the sequence of the nucleic acids can be characterized by the fact that it possesses at least one intron and/or a polyA sequence.
• a double-stranded DNA is preferred for expressing the relevant genes, both for preparing the polypeptides which can be used in accordance with the invention and in association with vectors which can be used in accordance with the invention and which are applicable in gene therapy, with the DNA region encoding the polypeptide being particularly preferred.
• this region begins with the first start codon (ATG) which is located in a Kozak sequence (Kozak, 1987, Nucleic. Acids Res. 15: 8125-48) and extends to the next stop codon (TAG, TGA or TAA) which is located in the same reading frame as the ATG.
• this region begins with the first AUG (or GUG) after a Shine- Dalgarno sequence and ends with the next stop codon (TAG, TGA or TAA) which is located in the same reading frame as the ATG.
• nucleic acids which have been prepared synthetically can be used according to the invention.
• the nucleic acids which are used in accordance with the invention can, for example, be synthetisized chemically, e.g. according to the phosphotriester method, with the aid of the DNA sequences described in Table 1 and/or with the aid of the polypeptide sequences which are likewise described in this table by referring to the genetic code (see, e.g., Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. 4).
• polypeptides which can be used in accordance with the invention can also be prepared synthetically.
• the entire polypeptide, or parts thereof can, for example, be synthesized by classical synthesis (Merrifield technique). Particularly preferred is the use of polypeptides which have been prepared recombinantly us- ing one of the previously described nucleic acids.
• MRP8 and MRP 14 polypeptides can be isolated from an organism or from tissue or cells and used in accordance with the invention.
• cell lines can be prepared from MRP8 and/or MRP14-expressing cells and then used for isolating MRP8 and/or MRP 14.
• the wounds are diabetes-associated wounds, in particular a diabetic ulcer. Furthermore, the wounds are preferably of large surface area .
• At least one nucleic acid which can be used in accordance with the invention and which encodes an MRP8 polypeptide is contained in an expression cassette in a vector, preferably in an expression vector or in a vector which is applicable in gene therapy, and at least one nucleic acid which can be used in accordance with the invention and which encodes an MRP 14 polypeptide is contained in another expression cassette in another vector, preferably in an expression vector or a vector which is applicable in gene therapy.
• vectors which can be used in accordance with the invention can then be employed, in combination, as drugs for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds which heal poorly (see Examples 3, 6).
• the vectors which are applicable in gene therapy preferably contain wound-specific, skin-specific or constitutively active regulatory sequences which are functionally connected to the previously described nucleic acids.
• At least one nucleic acid which can be used in accordance with the invention and which encodes an MRP8 polypeptide is contained, together with a nucleic acid which can be used in accordance with the invention and which encodes an MRP 14 polypeptide, in an expression cassette in a vector, preferably in an expression vector or in a vector which is applicable in gene therapy (see, e.g. Example 3).
• These vectors which can be used in accordance with the invention can then be employed as drugs for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular dia- betes-associated wounds which heal poorly.
• the vectors which are applicable in gene therapy preferably contain wound-specific, skin-specific or constitutively active regulatory sequences which are linked functionally to the previously described nucleic acids.
• the expression vectors which are used for preparing a polypeptide which can be used in accordance with the invention can be prokaryotic or eukaryotic expression vectors.
• prokaryotic expression vectors are the pGEM vectors or pUC derivatives, which are used for expression in E. coli
• eukaryotic expression vectors are the vectors p426Met25 or p426GALl (Mum- berg et al. (1994) Nucl.
• Acids Res., 22, 5767-5768 which are used for expression in Saccharomyces cerevisiae
• Baculovirus vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, which are used for expression in insect cells
• the vectors Rc/CMV and Rc/RSV, or SV40 vectors which are used for expression in mammalian cells, with all these vectors being generally available.
• Examples of preferred regulatable elements which permit constitutive expression in eucaryotes are promoters which are recognized by RNA polymerase III or viral promoters, CMV enhancer, CMV promoter (see also Example 3), SV40 promoter or LTR promoters, e.g. derived from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232) and other viral promoter and activator sequences which are derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV.
• promoters which are recognized by RNA polymerase III or viral promoters
• CMV enhancer CMV promoter
• CMV promoter see also Example 3
• SV40 promoter or LTR promoters e.g. derived from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232) and other viral promoter and activator sequences which are derived from, for example
• regulatable elements which permit inducible expression in eucaryotes are the tetracycline operator in combination with an appropriate repressor (Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20).
• regulatable elements which permit cell cycle-specific expression in eucaryotes are promoters of the following genes: cdc25, cyclin A, cyclin E, cdc2, E2F, B-myb or DHFR (Zwicker J. and M ⁇ ller R. (1997) Trends Genet. 13, 3-6).
• a regulatable element which permits keratinocyte-specific expression in skin is the FiRE element (Jaakkola et al., 2000, Gen. Ther., 7: 1640-1647).
• the FiRE element is an AP-1 -driven, FGF-inducible response element of the syn- decan-1 gene (Jaakkola et al., 1998, FASEB J., 12: 959-9).
• Examples of regulatable elements which permit metabolism-specific expression in eucaryotes are promoters which are regulated by hypoxia, by glucose deficiency, by phosphate concentration or by heat shock.
• the nucleic acid can be present as a plasmid, or as a part of a viral or non viral vector.
• viral vectors in this connection are: baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses.
• non viral vectors are for example: virosomes, liposomes, cationic lipids and polylysine-conjugated DNA.
• vectors which are applicable in gene therapy are viral vectors, for example adenoviral vectors or retroviral vectors (see Example 6; Lindemann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58).
• Eukaryotic expression vectors are suitable for use in gene therapy when present in isolated form since naked DNA can penetrate into skin cells when applied topically (Hengge et al., 1996, J. Clin. Invest. 97: 2911-6; Yu et al., 1999, J. Invest. Dermatol. 112: 370-5).
• Vectors which are applicable in gene therapy can also be obtained by complexing the nucleic acid used in accordance with the invention with liposomes, since this makes it possible to achieve a very high efficiency of transfection, particularly of skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279-85).
• lipo- fection small, unilamellar vesicles consisting of cationic lipids are prepared by subjecting the liposome suspension to ultrasonication.
• the DNA is bound ionically on the surface of the liposomes, specifically in a relationship which is such that a positive net charge remains and 100% of the plasmid DNA is complexed by the liposomes.
• DOTMA l,2-dioloyloxypropyl-3- trimethylammonium bromide
• DPOE dioleoylphosphatidylethanolamine
• lipid formulations are DOTAP N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium ethyl sulfate or DOGS (TRANSFECTAM; dioctadecylamidoglycylspermine).
• DOGS DOGS
• the Cytofectin GS 2888 cationic lipids have also proved to be very well suited for transfecting keratinocytes in vitro and in vivo (US 5,777,153; Lewis et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 3176-3181).
• Auxiliary substances which increase the transfer of nucleic acids into the cell can, for example, be proteins or peptides which are bound to DNA or synthetic peptide-DNA molecules which make it possible to transport the nucleic acid into the nucleus of the cell (Schwartz et al. (1999) Gene Therapy 6, 282; Branden et al. (1999) Nature Biotech. 17, 784).
• Auxiliary substances also encompass molecules which enable nucleic acids to be released into the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213).
• Liposomes are a pharmaceutically acceptable carrier within the meaning of the present invention. Liposomes comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) and large unilammellar vesicles (LUVs).
• liposomes encompasses, for example, liposomal compositions which are disclosed in US 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP 524,968 Bl.
• Liposomes can be used as a pharmaceutical carrier for either or both of the nucleic acids which can be used in accordance with the invention and the polypeptides which can be used in accordance with the invention; they are preferably used as a pharmaceutical carrier for the nucleic acids which can be used in accordance with the invention.
• the therapeutically active substance can be conjugated to the lipo- some or it can be conjugated to a hydrogel polymer, with it being possible for the hydrogel polymer (or a component of the hydrogel polymer) to be conjugated to a liposome or to be enclosed by a liposome.
• vectors for gene therapy can be obtained by applying the nucleic acid used in accordance with the invention to gold particles and using for example a Gene Gun to administer the charged particles topically by firing them into the skin or cells (Example 3; Wang et al., 1999, J. Invest. Dermatol., 112:775-81, Tuting et al., 1998, J. Invest. Dermatol. 111:183-8).
• Devices for performing intradermal injection using pressure have been disclosed, for example, in US 5630796.
• Another form of vector which is applicable in gene therapy can be prepared by introducing "naked" expression vectors into a biocompatible matrix, for example a collagen matrix.
• This matrix can, for example, be introduced into diabetes- associated wounds in order to transfect the immigrating cells with the expression vector and to express the polypeptides used in accordance with the invention in the cells (Goldstein and Banadio, US 5,962,427).
• the part of the nucleic acid which encodes the polypeptide contains one or more noncoding sequences, including intron sequences, preferably between the promoter and the start codon for the polypeptide (see Example 3) and/or a polyA sequence, in particular the naturally occurring polyA sequence or an SV40 virus polyA sequence, in particular at the 3' end of the gene since this makes it possible to stabilize the mRNA (Jackson, R.J. (1993) Cell 74, 9-14 and Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci.USA 88, 478-482).
• E. coli BL21 cells have, for example, proved to be suitable cells for expressing human MRP8 and MRP14 (Hunter and Chazin, 1998, J. Biol. Chem., 273: 12427-12435).
• the use of E. coli cells for preparing polypeptides which can be used in accordance with the invention constitutes a preferred em- bodiment.
• the invention furthermore relates to the use of an MRP8/MRP14 heterodimer, or of its individual components in combination, with at least one individual component being employed in the form of a fusion protein for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular of diabetes- associated wounds which heal poorly, with the fusion protein being prepared using a previously described nucleic acid.
• fusion proteins which contain the above-described MRP8 and/or MRP 14 polypeptides, with the fusion proteins themselves already being active or only becoming active after the fusion moiety has been eliminated.
• These fusion proteins include, in particular, fusion proteins having a content of approx. 1-300, preferably approx. 1-200, particularly preferably approx. 1-150, in particular approx. 1-100, especially approx. 1-50 foreign amino acids.
• Examples of such peptide sequences are prokaryotic peptide sequences which can be derived, for example, from E. coli galactosidase.
• peptide sequences for fusion proteins are peptides which facilitate detection of the fusion protein; examples of these are the green fluorescent protein or functional variants thereof.
• murine MRP 14 has al- ready been expressed as a fusion protein in mammalian cells (Nacken et al., 2000, Eur. J. Biochem. 267: 560-565).
• polypeptide tag for the purpose of purifying the previously described proteins.
• suitable protein tags enable the proteins which are to be purified to be absorbed with high affinity to a matrix. This is then followed, for example, by the following steps: stringent washing with suitable buffers without eluting the complex to any significant extent, and, subsequently, specific elution of the absorbed complex.
• nonembryonic eukaryotic cells in a mammal in accordance with the invention by providing, for example by means of transfection, suitable cells or organs with an expression vector which contains the nucleic acids which can be used in accordance with the invention.
• DEAE dextran or polyionic complexes to transfect the guinea pig lactiferous duct with an expression vector containing the hGH gene results, for example, in continuous expression of hGH (Hens et al., Biochem. Biophys. Acta, 2000, 1523: 161-171).
• polyclonal antibodies which are formed in an animal as a result of an immunological reaction can subsequently be readily isolated from the blood using well-known methods and be purified, e.g. by means of column chromatography.
• Monoclonal antibodies can be prepared, for example, using the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299).
• the cells can, for example, be labeled in vivo with radioactive calcium and the MRP8/MRP14 het- erodimers, or their individual components in combination, can be isolated, before or after treating the cells with the substances, and examined by means of scintillation for the presence of radioactively bound calcium.
• Another functional assay can consist in analyzing the binding of keratin in the cell before and after applying the substances, as described in Goebeler et al. (Biochem J., 1995, 309: 419-24).
• Fur- thermore it is possible, by means of double-staining untreated or treated cells with an anti-tubulin antibody and an anti-MRP8 antibody, an anti-MRP 14 antibody or an anti-MRP8/MRP14 antibody to examine, using a suitable detection system, whether substances exert an influence on tubulin binding, which is essen- tial for the polypeptides to be secreted. In addition, it is possible to test whether the quantity of extracellular, secreted MRP8/MRP14 heterodimers, or their individual components in combination, is altered by applying substances to the cells.
• this polypeptide can, for example, be purified, or partially purified, from the cells, after these substances have been applied, and the isolate can be examined for chemotactic activity, with this activity being compared with that of control MRP8 which has been obtained from untreated cells (Lackmann et al., 1992, J. Biol. Chem., 267, 7499-7504).
• the expression of the reporter gene can be greatly increased if the second fusion protein interacts with the investigated polypeptide according to the invention.
• This increase in expression can be used for identifying new interacting partners, for example by preparing a cDNA library from wound tissue for the pu ⁇ ose of constructing the second fusion protein.
• the interaction partner is an activator of MRP8/MRP14 This test system can also be used for screening substances which inhibit an interaction between the polypeptide according to the invention and an interacting partner.
• Such substances decrease the expression of the reporter gene in cells which are expressing fusion proteins of the polypeptide according to the invention and the interacting partner (Vidal and Endoh, 1999, Trends in Biotechnology, 17: 374-81).
• novel active compounds which can be employed for the therapy of and/or prevention of skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers; perferably for the therapy of and/or prevention of diabetes-associated badly-healing wounds.
• the invention also relates to a functional assay on the activity of human MRP8/MRP14 heterodimer or its individual components in combination, or of at least one nucleic acid encoding the heterodimer or its individual components in combination, comprising the steps of:
• At least one human MRP8/MRP14 heterodimer which can be used in accordance with the invention, or its individual components in combination are brought into contact with the at least one cell.
• the cells are skin cells, especially keratinocytes, fibroblasts or endothelial cells.
• cell function is proliferation, migration or differentiation, especially migration.
• a migration assay with keratinocytes using the MRP8/MRP14 heterodimer see Example 4). Migration can be established readily by means of the "migration index" test (Charvat et al., see above) and comparable test systems (Benestad et al., 1987, Cell Tissue Kinet. 20: 109-19; Junger et al., 1993, J. Immunol. Methods 160: 73- 9).
• Example for migration assays are well known to the skilled person and comprise for example the Boyden chamber method (Example 4), the scratch assay, the colloidal gold assay and an assay based on the migration in a fibrin matrix.
• a scratch assay cells are seeded on a tissue culture plate and are grown to conflu- ency. The confluent cell layer is then wounded under standard conditions with a plastic pipet tip to create a cellfree zone. Subsequently, test substances can be added after and migration into the cellfree zone can be monitored by photo documentation of identical locations in the scratch.
• cover- slips are coated with colloidal gold salts and covered with a suitable substratum, for example Collagen I.
• Cells for example keratinocytes are plated on the cover slip and allowed to migrate for several hours. Afterwards the cells are fixed in formaldehyde and migration tracks can be analysed using computer assisted im- age analysis.
• the assay based on the migration in a fibrin matrix cells are plated onto a fibrin matrix, that is obtained from freeze-dried surgical fibrinogen and distributed onto culture dishes before clotting.
• the fibrin matrix is transparent and therefore suitable for microscopic analysis of the cells.
• Suitable cells for example keratinocytes are incubated on the matrix for 24 hours, fixed with formaldehyde and tunnels generated by migrating cells in the matrix are examined by light microscopy.
• Test systems for the monitoring of differentiation depend on the cell types and are well known for skilled persons for many cell types.
• keratinocytes examples of suitable differentiation markers are keratin 6, 10 and 14 and also loricrin and involucrin (Rosen- thai et al., 1992, J. Invest. Dermatol. 98: 343-50) whose expression can be readily detected, for example, using generally available antibodies.
• suitable test systems for measuring proliferation can be established very rapidly by means, for example, of the inco ⁇ oration of labeled nucleotides into the DNA of the cells (see, e.g., Savino and Dardenne, 1985, J. Immunol.
• Suitable assays for examining a pharmacological effect of test substances on an MRP8/MRP14 heterodimer which is bound to a solid phase, or its individual components in combination are, for example, a fatty acid-binding assay for the MRP8/MRP14 heterodimer, known from Kerkhoff et al. (J. Biol. Chem., 1999, 274: 32672-32679), or a calcium-binding assay using 45 Ca 2+ .
• a wound-healing assay for example carried out in mice, whether the application of MRP8/MRP14 heterodimers, or their individual components in combination, or of at least one nucleic acid encoding an MRP8/MRP14 heterodimer, or its individual components in combination, or of a cell which is expressing the entire heterodimer, or its individual components in combination, together, or at different time points, with test substances, to a wound alters the healing of the wound.
• This can be done, for example, by measuring the rate of reepithelialization, the amount of collagen deposition or the determination of wound breaking strength.
• the invention also relates to the use of at least one nucleic acid encoding an MRP8/MRP14 heterodimer, or its individual components in combination, or of a cell which is expressing the entire heterodimer or its individual components in combination, in combination, for identifying at least one pharmacologically active substance which exerts an influence on the expression of MRP8/MRP14 hetero- dimers, mRNAs encoding them, and/or their individual components in combination, or mRNAs encoding them.
• cells which express MRP8 and/or MRP 14, for example granulocytes can be cultured as a test system for analyzing gene expression in vitro, with preference being given to skin cells, in particular keratinocytes, fibroblasts or endothelial cells.
• a possible test sys- tern is the human keratinocyte cell line HaCaT, which is available generally.
• Gene expression is analyzed, for example, at the level of the mRNA or of the polypeptides.
• RNA or mRNA can first of all be isolated from cells and the absolute quantity, or the relative proportion, of the MRP8 and/or MRP 14 mRNA can then be determined, for example by means of quantitative RT-PCR (see EP 0 200 362; Wittwer et al., 1997, BioTechniques 22: 130-8; Morrison et al., 1998, BioTechniques 24: 954-62) or by means of the RNAse protection assay (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York, chapter 7; EP 0 063 879).
• Another embodiment of the invention relates to the pharmacologically active substances which are identified with the aid of the screening methods.
• the invention furthermore relates to a drug which comprises pharmacologically active substances for treating skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds.
• At least one MRP8/MRP14 heterodimer which can be used in accordance with the invention, or its individual components in combination, is/are expressed by at least one cell and at least one substance is examined for its pharmacological activity.
• At least two substances are, for the pu ⁇ ose of identifying pharmacological substances, examined for their pharmacological activity, with the substances being selected from at least one library of substances.
• the invention furthermore relates to a process for producing a drug, with, in a first step, a pharmacologically active substance being identified using one of said methods for identifying such substances and, in a second step, the pharmacologically active substance which has been identified being brought into contact or combined with suitable auxiliary substances and/or additives.
• Table 1 shows the SEQ ID numbers and access numbers of the polypeptides which can be used in accordance with the invention and the cDNAs which encode them.
• Figure 1 Determination of MRP8/MRP14 polypeptide concentrations in the wound fluid of healthy patients, in the wound fluid of patients suffering from chronic diabetic wounds and in the wound fluid of patients suffering from venous ulcer, by means of sandwich ELISA.
• Figure 2 Results of the genetherapeutic treatment of diabetic rats with MRP8/MRP14.
• the plot depicts E/C values of the breaking strength, each value representing an E/C value of an individual treated wound compared to a control wound at day 7 after injury.
• Figure 3 Results of the genetherapeutic treatment of diabetic rats with MRP8/MRP14.
• the plot depicts E/C values of the breaking strength, each value representing an E/C value of an individual treated wound compared to a control wound at day 10 after injury.
• SEQ ID No. 1 to SEQ ID No. 8 show the sequences of the murine and human MRP8 and MRP 14 polypeptides and the cDNAs encoding them.
• SEQ ID No. 9 to SEQ ID No. 18 show the sequences of the oligonucleotides which were used for the experiments.
• Example 1 Localization of MRP 14 mRNA in human biopsies taken from healthy skin and from a wound in a healthy test subject, and in biopsies taken from a venous ulcer and a diabetic ulcer patient
• MRP14 is a component of the heterodimer MRP8/MRP14.
• the localization of the MRP 14 mRNA was investigated by means of nonradioac- tive in situ hybridization.
• a partial human MRP 14 cDNA fragment was amplified by PCR.
• the primers which were used in this context contained an RNA polymerase promoter, for preparing ribo- probes, in addition to the segment which was homologous to MRP 14 (antisense primer: Sp6-MRP-14 primer (ATTTAGGTGACACTATAGAATAC CCC GAG GCC TGG CTT ATG GT; SEQ ID No. 9); control-sense primer: T3-MRP-14 primer (AATTAACCCTCACTAAAGGGG GTG GCT CCT CGG CTT TGA CA; SEQ ID No. 10).
• “Aberrant regulation" of the MRP8/MRP14 heterodimers, and/or their individual components in combination, in association with skin diseases, wounds and/or wound-healing disturbances is defined as a strength of expression which turns out to be markedly reduced, as compared with that seen in normal wound healing, in the cells, in the body fluids, in the wound liquid and/or in the skin.
• a different result was su ⁇ risingly observed in the case of the labeling of the diabetic ulcer: only very weak labeling was observed at the edge of the wound. This shows that MRP 14 is only expressed to a decreased extent at the edge of the diabetic ulcer wound but not at the edge of the venous ulcer wound or in normally healing wounds.
• Example 2 Detecting MRP8 polypeptide in wound fluid from various wound- healing diseases
• Example 1 The aim now was to test the result obtained in Example 1 as far as the binding partner MRP8 was concerned.
• wound fluid was isolated, by means of drainage, from normally healing human day 1 and day 2 wounds, as was wound fluid from a poorly healing wound of a diabetes patient and of a patient with a venous ulcer and the quantity of MRP8 in the wound fluids was determined by means of Western blot analysis.
• Biopsy tissue from a normally healing human day 5 wound, which was isolated as described in Example 1 was used as a positive control for detecting the polypeptide.
• the polypeptide concentrations in the samples were determined by means of a BCA test (Sigma- Aldrich Chemicals) and the same quantities of polypeptide were loaded onto a 4-20% gradient gel (tris-glycine buffered, 1 mm, Novex) and the polypeptides were fractionated electrophoretically.
• an immunostaining was carried out using the polyclonal goat anti-human calgranulin A antibody (dilution 1 :100; Santa Cruz, sc8112), as the first antibody, and the donkey anti-goat IgG F(ab') fragment (dilution 1 :5000; Dianova, # 705- 036-147) as the second antibody.
• the signal was detected using the "Amersham ECL Western Blotting detection reagent" (Amersham, # RPN 2166) in accordance with the manufacturer's instructions.
• MRP8/MRP14 is in fact particularly suitable for treating and/or preventing skin diseases, wounds and/or wound-healing disturbances which are characterized by a reduced quantity of MRP8/MRP14 heterodimers, in particular diabetes-associated wounds which heal poorly
• the influence of MRP8/MRP14 on wound healing in vivo was investigated in untreated and dia- betic male New Zealand White rabbits.
• the alloxan administration-induced diabetic "New Zealand White rabbit model system” is suitable for simulating diabetes-associated wound-healing disturbances since it is a well investigated, scientifically established model system in which the state of health of the animals is comparable and wound closure is significantly retarded, namely by 50% (Davidson, 1998, Arch. Derm.
• pMHintMRP ⁇ and pMHintMRPH a suitable expression vector, pMHint, was first of all constructed, with this vector being prepared from vector pMH (S. Hoffmann-La Roche) by inserting intron II of the rat insulin gene into the Hindlll cleavage site between the CMV promoter and the multiple cloning site.
• the mur- ine MRP 8 or MRP 14 cDNA was then cloned into pMHint using the multiple cloning site.
• MRP14-primer 1 GAG AGA GGT ACC ATG GCC AAC AAA GCA (SEQ ID No. 13) and MRP14-primer 2: GAG ACC CGG GTT ACT TCC CAC AGC CTT TG (SEQ ID No. 14)).
• MRP14-primer 2 GAG ACC CGG GTT ACT TCC CAC AGC CTT TG (SEQ ID No. 14)
• the resulting product was cut with Kpnl and Smal and ligated to the expression vector pMHint, which had been cut with Kpnl and Pmll, thereby giving rise to the expression plasmid pMHintMRP14.
• MRP14-primer 3 The coding sequence of MRP 14 was amplified by PCR (MRP14-primer 3:
• Adrenaline solution (2% xylo- cain solution and epinephrine, 1 : 100,000) was subsequently injected intradermally in order to constrict the blood vessels and to separate the skin from the ear cartilage lying below it. Punches were then used to make four 8 mm wounds on the inside of the ear.
• Each wound was treated at a pressure of 500 psi using a Helios Gene Gun (BioRad), with 0.5 ⁇ g of control plasmid or expression plasmid, which had been immobilized on gold particles (BioRad) being used per shot.
• 0.5 ⁇ g of MRP8/MRP14 were used per shot in the case of the experiment using the vector pMRP8/MRP14; in the case of the experiment using pMHintMRP ⁇ and pMHintMRP14, 0.25 ⁇ g of expression plasmid was in each case immobilized on gold particles and shot into the wound together.
• the wounds were subsequently covered with a semiocclusive dressing. On day 10 after wounding, the wound surface which had still not completely reepithelialized was determined by means of EPICAM and compared with the value at day 0 (immediately after wounding).
• Example 4 In vitro assay for human MRP ⁇ /MRP14 suitable for the identification of pharmacologically active substances
• the extracellular function of human MRP ⁇ and MRP 14 in combination is unknown. Therefore, it is also object of the present invention to identify an assay, which allows the identification of pharmacologically active substances, which modulate the extracellular function of human MRP ⁇ and MRP 14 in combination.
• an assay may be used to identify substances which increase the activity of human MRP ⁇ and MRP 14 in combination, as such substances may be used for the treatment of diseases characterized by reduced levels of MRP ⁇ and/or MRP 14, especially diabetic wounds.
• Substances, which modulate the extracellular function of human MRP8 and MRP 14 in combination have the advantage that they need not enter the cell for exerting their influence, but may be applied easily topically.
• MRP8 in combination with MRP 14 have a positive effect on the migration of keratinocytes, which was previously unknown and not suggested in the prior art, although the polypeptides are known for a long time and although they have been investigated in many respects. Therefore, the problem was solved by a migration assay using human MRP8 and MRP 14 in combination as active substances and skin cells, especially keratinocytes, in particular HaCat cells.
• the migration assay may also be used to test whether variants of human MRP8 and MRP 14 in combination are functional variants according to the present invention.
• a migration assay using the Boyden chamber method was used. It is the principle of the Boyden chamber migration assay to measure a cell movement towards a polypeptide gradient.
• the classical Boyden chamber consists of two compartments, which are separated by a filter with defined pore size. The lower compartments is filled with a chemoattractant, the upper compartments with cell suspension. During incubation the cells of the upper chamber migrate through the filter towards the polypeptide gradient. At the end of the assay the cells on the lower side of the filter are quantified.
• the assay employing this prin- ciple used here is a Transwell Assay (Chemicon International, Inc.) which consists of chamber inserts with filters in a 24 well tissue culture dish.
• the lower compartment, of the Transwell chambers were filled with the chemoattractant (human MRP ⁇ in combination with MRP 14: non-phosphorylalted complex (isolated from human blood), phosphorylated complex (isolated from human blood), recombinant complex (expression in E. coli); concentration range:500ng/ml, 5 ⁇ g/ml)).
• BSA 5 ⁇ g/ml was used as negative control
• EGF Epidermatitis, N-derived growth factor
• the upper compartment was filled with the HaCaT cell suspension: 2.5 x 10 6 cells/well and incubated at 37°C and 10% CO 2 for 4 hours. After incubation the filters were stained following the manufacturers instructions: Briefly, medium was removed from the compartment and cells on the upper side of the filter were carefully removed without puncturing the filter. The filters were then placed in the staining solution (Transwell Kit, Chemicon International, Inc.) for 30 minutes to stain the cells on the lower side of the filter which have migrated through the fil- ter. Afterwards the filters were washed carefully with distilled water to remove residual staining solution from the filter.
• the staining solution Transwell Kit, Chemicon International, Inc.
• the staining solution absorbed by the cells was then extracted with extraction buffer (Transwell Kit, Chemicon International Inc.) by slowly shaking the filters on a shaker for 10 minutes. Finally, the absorbance of lOO ⁇ l of the extracted solution was measured in an ELISA reader at 570 nm. The intensity of the absorbance correlates with the number of cells migrated through the filter. In the case of human MRP ⁇ in combination with MRP 14, an induction of migration was detected for all three complexes, the highest induction being observable at a protein complex concentration of 5 ⁇ g/ml. The addition of the heterodimer resulted in a two-fold increase in the number of migrating cells compared to the negative control (BSA).
• BSA negative control
• the migration assay can be used for testing whether MRP ⁇ or MRP 14 variants in combination are functional variants according to the invention. For example, several migration assays may be performed in parallel.
• Example 5 Determination of MRP ⁇ /MRP14 concentrations by sandwich ELISA in wound fluids
• wound fluid samples from patients suffering from chronic diabetic wound were collected, venous ulcers as well as wound fluids from wounds of healthy persons. Wound fluids were isolated as described in Example 2 by means of drainage.
• Maxi-Sorb Immuno- plates (96-well, Nunc, Wiesbaden, Germany) were coated with polyclonal rabbit antisera against MRP 14 by incubating each well overnight at 4°C with 50 ⁇ l of coating solution (anti-MRP14 antibody, 2 ⁇ g/ml, in 0.1 M NaHCO3, pH 9.5).
• Bound antigen was detected by incubation (lh, 37°C) of each well with 50 ⁇ l of a biotinylated monoclonal antibody against MRP 14 (clone S36.4 ⁇ , Dianova, 125 ng/ml antibody in buffer A). After another washing procedure (3x buffer A) the biotinylated antibody was detected by adding 50 ⁇ l peroxidase- conjugated Streptavidin (Jackson ImmunoResearch; 1/25,000 in buffer A for 30 min (lh, 37°C).
• MRP ⁇ and MRP 14 spontaneously form noncovalently associated complexes to a quantitative extent which are detected by the ELISA system. Therefore, the ELISA is calibrated with the native MRP ⁇ /MRP14 complex and data are presented as microgram of MRP ⁇ /MRP14 per mg total polypeptide. The total polypeptide content per probe was determined using the BCA assay. Die results are shown in Figure 1.
• MRP ⁇ /MRP14 was almost absent in the wound fluid of the patient with the chronic diabetic wound, whereas the concentrations are clearly higher in wound fluids from normally healing wounds and even more elevated in wound fluids from venous ulcer patients.
• MRP ⁇ /MRP14 heterodimer In order to prove the suitability of the MRP ⁇ /MRP14 heterodimer for the treat- ment of diabetes-associated wounds, male diabetic Sprague Dawley rats (250-300 g) were treated with adenoviral constructs expressing murine MRP ⁇ and MRP 14, respectively (SEQ ID No. 5 and SEQ ID No. 7).
• the constructs were obtained using the AdEasyTM Technology (QBiogene) according to the manufacturer's protocol.
• the cDNAs are amplified by PCR using primers which introduce Kpnl and EcoRV sites at the end.
• PCR products are cut with Kpnl and EcoRV and are subsequently cloned into the Transfer vector resulting in the vectors pShuttle-m ⁇ and pShuttle-m ⁇ l4.
• recombinant adenovi- ruses were created according to the protocol, resulting in the constructs Ad- ⁇ up8 and Ad-n pl4. Expression of the polypeptides was verified by Western blot analysis of transfected QBI-293 cell lysate as described in the protocol.
• the adenoviral constructs were suspended and dialyzed in in HBS-buffer (20 mM HEPES/150 mM NaCl, pH 7,8).
• rats were anaesthetized with an inhalant mixture consisting of 2% O 2 (2L/min) + 1.25% Isoflurane. After shaving and marking the wound sites on the back, incisional wounds with 1 cm in length were made. A mixture of adenoviral constructs carrying MRP ⁇ (5*10 PFU in 50 ⁇ l HBS) and MRP 14 (5*10 8 PFU in 50 ⁇ l HBS) cDNA was injected intracutaneously into the skin along the margin of incision. Each rat was treated with duplicate MRP ⁇ +MRP14 genes and the control adenoviral construct expressing lacZ (1 *10 9 PFU).

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Pathology (AREA)
• Analytical Chemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Food Science & Technology (AREA)
• General Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Pharmacology & Pharmacy (AREA)
• Biochemistry (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Dermatology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Marine Sciences & Fisheries (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26009206

Family Applications (1)

Country Status (3)

Families Citing this family (14)

Family Cites Families (2)

• 2002
  • 2002-04-30 AU AU2002304710A patent/AU2002304710A1/en not_active Abandoned
  • 2002-04-30 EP EP02732688A patent/EP1390058A2/de not_active Withdrawn
  • 2002-04-30 WO PCT/EP2002/004765 patent/WO2002088181A2/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events