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
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to the use of trefoil factor 1 (TFF1) and trefoil factor 3 (TFF3) dimers and a pharmaceutical composition comprising TFF dimers for increasing the viscosity of mucin in mucus layers and the repair of damaged mucus layers in the gastrointestinal tract (mouth, oesophagus, stomach, small and large intestine, colon) the respiratory passages, the eye, the urinary system (including the bladder) and the cervis uteri.
• TFF1 trefoil factor 1
• TFF3 trefoil factor 3
• TFFs Mammalian trefoil factors
• TFF1 , TFF2 and TFF3 Mammalian trefoil factors
• TFF1 and TFF3 Mammalian trefoil factors
• TFF2 and TFF3 Mammalian trefoil factors
• a trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which six cysteines are disulphide-linked in a 1-5, 2-4 and 3-6 configuration.
• the trefoil peptides are expressed in the gastrointestinal tract in a tissue specific manner.
• TFF1 and TFF2 are expressed in mucus producing cells in the stomach and duodenum, whereas TFF3 is primarily expressed in goblet cells in the small and large intestine.
• TFF3 is primarily expressed in goblet cells in the small and large intestine.
• trefoil peptides In the case of gastric ulceration or inflammatory bowel disease the expression of trefoil peptides is highly upregulated. This suggest that trefoil peptides may have a repair function for damages in the gastrointestinal tract thus acting as natu- rally occurring healing factors.
• the importance of TFFs for normal mucosal function have also been investigated by two gene knock-out studies in which the genes encoding TFF1 and TFF3, respectively, were deleted by gene-targeting techniques.
• TFF3 knock-out mice had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulphate a situation that could be circumvented by luminal administration of re- combinant TFF3.
• trefoil peptides together with mucins form stable gel complexes resistant to mechanical stress and gastrointestinal proteases.
• no direct evidence for such gel formation has so far been given some studies have indicated an interaction/binding between trefoil peptides and mucins.
• the present invention relates to the use of human TFF1 dimer and TFF3 dimer peptides for improving Theological properties of mucin solutions.
• TFF dimer peptides have by the present inventors been found to increase the viscosity and elasticity of different mucins solu- tions, which are correlated to physiological and pathophysiological conditions.
• the present invention discloses the mechanism by which the TFF dimer peptides exerts their biological activity, which are documented by a direct effect of TFF dimer peptides on the viscosity and elasticity of mucin solutions.
• the TFF dimer peptides significantly increases the viscosity of mucin solutions.
• the net effect is an increase in the viscosity of sev- eral times and can be visualised by the fact that the liquid mucin solution is converted into a more viscous gel-like substance.
• TFF dimer peptides have by the present inventors been found to be usefull for increasing the viscosity and elasticity of mucus layers, which can be used in the treatment of many different indications, where abnormalities in existing mucus layers are present.
• the advantage over known therapies is, that treatment with TFF dimer peptides represent a specific treatment at the site of injury without major side effects.
• TFF dimer peptides can increase the viscosity and elastic properties of mucin in mucus layers, which may be usefull in many different indications: 1) For the treatment of the oral mucosa. TFF dimer peptides may be given alone or together with mucus-like preparations to patients with reduced secretion of saliva caused by irradiation therapy, treatment with anticholinergics or in patients with Sj ⁇ grens syndrome.
• TFF dimer peptides may also be used for parenteral applications: Parenteral TFF dimer is taken up by cells associated with stem cells in the gastroin- testinal tract. It can be used for protection of the stomach against stress-induced damage and the stomach and intestine against damage following irradiation or chemotherapy or in the treatment of acute excerbations in ulcerative colitis, irritable bowel syndrome or Crohn's disease.
• Injected TFF dimer peptide is excreted intact in urine and may increase the defence mechanism of the urinary bladder by binding to the layer of mucopolysaccharids that coat the urothelium and thereby interfere with the adherence of bacteria in chronic bladder infections, in patients with catheter or interstitial cystitis, or interfere with the binding of urinary growth factors in papillomas or cancer of the bladder.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of mucus layers in mammals, the composition comprising a TFF dimer peptide or a pharmaceutically acceptable salt thereof.
• TFF dimer peptides or "a TFF dimer peptide” is meant a protein that is substantially homologous to human TFF1 or human TFF3 in dimer forms.
• Fig. 1 shows TFF1 and TFF3 in the monomer form.
• the TFF1 dimer consists of two TFF1 monomers linked together by a disulfide bond between cysteine amino acid residue at position 58 of each TFF1 mono- mer.
• the TFF3 dimer consists of two TFF3 monomers linked together by a disulfide bond between cysteine amino acid residue at position 57 of each TFF3 monomer.
• the term TFF dimer peptides also includes analogs of naturally occurring TFF dimer peptides.
• Analogs can differ from naturally occurring TFF dimer by amino acid sequence differences or by modifications that do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even 99%, homology with a naturally occurring TFF dimer sequence.
• Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and proc- essing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes.
• TFF dimer peptide includes biologically active fragments of the polypeptides.
• fragment as applied to a polypeptide, will ordinarily be at least 10 contiguous amino acids, typically at least 20 contiguous amino acids, more typically at least 30 contiguous amino acids, usually at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids in length.
• TFF dimer peptide The ability of a candidate fragment to exhibit a biological activity of a TFF dimer peptide can be assessed by methods known to those skilled in the art. Also included in the term "fragment" are biologically active TFF dimer peptides containing amino acids that are normally removed during protein processing, including additional amino acids that are not required for the biological activity of the polypeptide, or including additional amino acids that result from alternative mRNA splicing or alternative protein processing events.
• a TFF dimer peptide, including a fragment, or analog is biologically active if it exhibits a biological activity of a naturally occurring TFF dimer, e.g., the ability to alter viscosity or elasticity of mucin in mucus layers in a mammal.
• glycosylation means the post-translational modification of a peptide, wherein a carbohydrate molecule is covalently attached to the peptide.
• the glycosylation may take place in a eucaryotic host cell, such as a yeast cell or it may be done by chemical linkage in vitro after production of the peptide in a cell, e.g. the peptide could be produced in a bacteria and glycosylated in vitro afterwards.
• the present invention relates to the use of a TFF dimer peptide for the preparation of a medicament for increasing the viscosity of mucus layers in mammals.
• the present invention relates to a method for in vivo increase in viscosity of mucus layers in a subject, the method comprising administering to the subject a composition comprising a) a pharmaceutically acceptable carrier or diluent, b) a therapeutically effective amount of a TFF dimer peptide, and optionally c) a mucin glycoprotein preparation,
• the present invention relates to the use of a TFF dimer peptide for the treatment of conditions with increased viscosity of mucus layers in mammals.
• treatment means the administration of an effective amount of a therapeutically active compound of the invention with the purpose of preventing any symptoms or disease state to develop or with the purpose of curing or easing such symptoms or disease states already developed.
• treatment is thus meant to in- elude prophylactic and protective treatment.
• the symptoms or disease state includes but is not limited to diseases, e.g. gastric ulcers or asthma, inherited biological disorders or conditions induced by damaging by external stimuli, e.g. Inhalation of toxic or acidic chemical.
• the mammal is human.
• Another embodiment of the present invention relates to a pharmaceutical composi- tion for local application.
• the present invention relates to a pharmaceutical composition for luminal application.
• the present invention relates to a pharmaceutical composition for parenteral administration. In a further embodiment the present invention relates to a pharmaceutical composition for oral administration.
• the present invention relates to a pharmaceutical composition further comprising a mucin glycoprotein preparation.
• the present invention relates to a pharmaceutical composi- tion for the treatment of oral mucosa.
• the present invention relates to a pharmaceutical composition for the treatment of patients with reduced secretion of saliva.
• the reduced secretion of saliva is caused by irradiation therapy, treatment with anticholinergics or Sj ⁇ grens syndrome.
• the present invention relates to a pharmaceutical composition for the treatment of patients receiving irradiation therapy.
• the present invention relates to a pharmaceutical composition for the treatment of patients treated with anticholinergics.
• the present invention relates to a pharmaceutical composi- tion for the treatment of patients with Sj ⁇ grens syndrome.
• the present invention relates to a pharmaceutical composition for the treatment of the respiratory passages.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of nasal secretions in rhinorrhoea in common cold or allergic rhinitis.
• the present invention relates to a pharmaceutical composition for the treatment of patients with common cold.
• the present invention relates to a pharmaceutical composition for the treatment of patients with allergic rhinitis. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of the respiratory tract. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of the respiratory tract following accidental inhalation of irritants.
• the present invention relates to a pharmaceutical composition for the treatment of the respiratory tract following accidental inhalation of gases, dusts or fumes.
• the present invention relates to a pharmaceutical composition for the treatment of oesophagus. In one embodiment the present invention relates to a pharmaceutical composition for the treatment of the distal part of the oesophagus.
• the present invention relates to a pharmaceutical composi- tion for protection against acid secretions from the stomach.
• the present invention relates to a pharmaceutical composition for protection against acid secretions from the stomach in reflux oesophagi's.
• the present invention relates to a pharmaceutical composition for protection against acid secretions from the stomach in hiatus hernia. In a further embodiment the present invention relates to a pharmaceutical composition for protection against acid secretions from the stomach in Barrets oesophagus.
• the present invention relates to a pharmaceutical composition for the treatment of the stomach.
• the present invention relates to a pharmaceutical composi- tion for treatment of stress induced gastric ulcers.
• the stress induced gastric ulcers is secondary to trauma.
• the stress induced gastric ulcers is secondary to shock.
• the stress induced gastric ulcers is secondary to large operations.
• the stress induced gastric ulcers is secondary to renal diseases.
• the stress induced gastric ulcers is sec- ondary to lever diseases.
• the stress induced gastric ulcers is secondary to treatment with aspirin, other non-steroidal anti-inflammatory drugs (NSAIDS), steroids or alcohol.
• NSAIDS non-steroidal anti-inflammatory drugs
• the present invention relates to a pharmaceutical composition for the treatment of diarrhoea. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of the small intestinal mucosa.
• the present invention relates to a pharmaceutical composition for the treatment of the colonic mucosa.
• the present invention relates to a pharmaceutical composi- tion for the treatment of Crohns disease. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of irritable bowel syndrome.
• the present invention relates to a pharmaceutical composition for the treatment of ulcerative colitis. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of the eye.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of lacrimal fluid.
• the present invention relates to a pharmaceutical composi- tion for increasing the viscosity of lacrimal fluid in patients with keratoconjunetivitis sicca.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of lacrimal fluid in patients with Sj ⁇ gren's syndrome.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of lacrimal fluid in patients with dry eyes.
• dry eyes means any condition where the eyes feels dry.
• the present invention relates to a pharmaceutical composition in eye droplets.
• the present invention relates to a pharmaceutical composition for the treatment of the knee joints. In a further embodiment the present invention relates to a pharmaceutical composition for increasing the viscosity of the synovial fluid.
• the present invention relates to a pharmaceutical composition for increasing the viscosity of the synovial fluid in osteoarthritis.
• the present invention relates to a pharmaceutical composi- tion for increasing the viscosity of the synovial fluid following joint replacement.
• the present invention relates to a pharmaceutical composition for the treatment of the bladder.
• the present invention relates to a pharmaceutical composition for the treatment of patients with catheter. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of infections. In one embodiment the infection is a cronic infection of the bladder.
• the present invention relates to a pharmaceutical composition for the treatment of interstitial cystitis. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of papillomas. In a further embodiment the present invention relates to a pharmaceutical composition for the treatment of cancer.
• the TFF dimer peptide is recombinant human TFF1. In a further embodiment of the invention, the TFF dimer peptide is recombinant human TFF3.
• the TFF dimer peptide is glycosylated.
• TFF dimer peptides are typically produced by recombinant DNA techniques.
• a DNA sequence encoding the TFF dimer peptide may be isolated by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
• the DNA sequence encoding the peptide is preferably of human origin, i.e. derived from a human genomic DNA or cDNA library.
• the DNA sequences encoding the TFF dimer peptides may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
• phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
• DNA sequences may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202, Saiki et al., Science 239 (1988), 487 - 491 , or Sambrook et al., supra.
• the DNA sequences encoding the TFF dimer peptides are usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• the vector is preferably an expression vector in which the DNA sequence encoding the TFF dimer peptide is operably linked to additional segments required for transcription of the DNA.
• the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
• operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.
• the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
• Suitable promoters for directing the transcription of the DNA encoding the TFF dimer peptide in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
• a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
• promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the IPh (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
• suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpjA promoter.
• suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A.
• triose phosphate isomerase or A. nidulans acetamidase.
• Preferred are the TAKA-amylase and gluA promoters. Suitable promoters are mentioned in, e.g. EP 238 023 and EP 383 779.
• the DNA sequence encoding the TFF dimer peptides may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093- 2099) terminators.
• the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
• the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
• a DNA sequence enabling the vector to replicate in the host cell in question.
• An example of such a sequence is the SV40 origin of replication.
• suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
• the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
• selectable markers include amdS, pyrG, arqB, niaD or sC.
• a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
• the secretory signal sequence is joined to the DNA sequence encoding the TFF dimer peptide in the correct reading frame.
• Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide.
• the secretory signal sequence may be that, normally associated with the peptide or may be from a gene encoding another secreted protein.
• the secretory signal sequence may encode any signal peptide, which ensures efficient direction of the expressed TFF dimer peptide into the secretory pathway of the cell.
• the signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the ⁇ -factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289. 1981 , pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al., Cell 48, 1987, pp.
• a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the TFF dimer peptide.
• the function of the leader peptide is to allow the expressed peptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e.
• the leader peptide may be the yeast ⁇ -factor leader (the use of which is described in e.g. US 4,546,082, US 4,870,008, EP 16 201 , EP 123 294, EP 123 544 and EP 163 529).
• the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
• the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.
• the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
• Suitable signal peptides are disclosed in, e.g. EP 238 023 and EP 215 594.
• the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. US 5,023,328).
• the host cell into which the DNA sequence encoding the TFF dimer peptide is introduced may be any cell, which is capable of producing the posttranslational modified TFF dimer peptide and includes yeast, fungi and higher eucaryotic cells.
• suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.
• Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol. Appl. Genet. I (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci.
• yeasts cells include cells of Saccharomyces spp. or
• Schizosaccharomyces spp. in particular strains of Saccharomyces cerevisiae or Saccharomyces reteyveri.
• Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in US 4,599,311 , US 4,931 ,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference.
• Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
• a preferred vector for use in yeast is the POT1 vector disclosed in US 4,931 ,373.
• the DNA sequence encoding the TFF dimer peptide may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above.
• suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279).
• Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger.
• Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438
• the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.
• the transformation of Tnchoderma spp. may be performed for instance as described in EP 244 234.
• a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell.
• This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell.
• Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4,879,236; US 5,155,037; 5,162,222; EP 397,485) all of which are incorporated herein by reference.
• the insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Tnchoplusia ni cells (cf. US 5,077,214).
• Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.
• the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting expression of the TFF dimer peptides after which all or part of the resulting peptide may be recovered from the culture.
• the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
• the TFF dimer peptides produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaqueous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
• the TFF dimer peptides may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences. 1985.
• the composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution.
• the compositions may be sterilized by conventional sterilization techniques, which are well known in the art.
• the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
• the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
• the pharmaceutical composition of the present invention may also be adapted for nasal, transdermal or rectal administration.
• the pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
• the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
• the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
• the concentration of the TFF dimer peptides in the composition may vary widely, i.e. from from about 5% to about 100% by weight. A typical concentration is in the range of 50- 100% by weight.
• a unit dosage of the composition may contain from about 1 mg to about 200 mg, typically from about 25 mg to about 75 mg, such as about 50 mg, of the peptide.
• a therapheutically effective amount is the effective dose to be determined by a qualified practitioner, who may titrate dosages to achieve the desired response. Factors for consideration of dose will include potency, bioavailability, desired pharmacoki- netic/pharmacodynamic profiles, condition of treatment (e.g. trauma, ulcerative colitis, gastric ulcers), patient-related factors (e.g. weight, health, age, etc.), presence of co-administered medications, time of administration, or other factors known to a medical practitioner.
• the dosage of a TFF dimer peptide administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of 0.1-1.0 mg/kg body weight.
• subject as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term “patient”.
• FIG. 1 The mammalian Trefoil Factors (TFFs), TFF1 and TFF3 in monomer form.
• TFFs Trefoil Factors
• TFF1 and TFF3 in monomer form.
• the figure shows the human sequences. Dimers of TFF1 and TFF3 are formed by disulfide linkage of the cysteine amino acid residues with indicated free sulfhydryl group on two different molecules of TFF1 or TFF3 respectively.
• FIG. 3 Oscillatory measurement of mucin solution (a) and mucinTFF3 dimer peptide gellike material (b). 2 ml of 10% (w/v) mucin I dissolved in 0.05% (w/v) sodiumazide was added 0.4 ml of water (a) or 0.4 ml of water containing 10 mg TFF3 dimer peptide (b). After 30 min at 20°C a sinosoidally varying stress was applied and the strain response was detected at different frequencies. The complex viscosity ( ⁇ * ) 0 0, the elastic modulus (G') D D and the viscous modulus (G”) O O was calculated and plotted as a function of different frequencies.
• FIG 6. The effect of luminal TFF3 in experimental colitis in rats. A significant effect on the overall colitis score is demonstrated in this figure.
• Figure 7. The effect of luminal TFF3 in experimental Dextran colitis in rats. The effect was predominantly in the midsection of the colon close to the site where the TFF3 had been introduced into the colonic lumen.
• Mucin I Crude mucin, type II from porcine stomach (Sigma, St. Louis, MO, USA).
• the dimer form of recombinant human TFF3 was prepared as previously described (Thim, L. et al. (1995) Biochemistry 34, 4757-4764).
• the dimer form of recombinant human TFF1 is prepared as previously described (Kannan, R. et al. (2001 ) Protein Expression and Purification 21 , 92-98)
• Mucin solutions A 10% (w/v) solution of mucin I was prepared and TFF dimer peptides were dissolved in water and added to the mucin solution. After mixing the sample (Vortex mixer), the sample was allowed to stand for 5 min. and the viscosity was visually assessed in relation to a control solution of mucin added water without TFF dimer peptides. The detailed experimental conditions for the rheometer measurement are given in the figure legends.
• Rheological measurements were measured by the use of a rotational Reologica Rheometer (Reologica Instriments AB, Lund, Sweden).
• the instrument is equipped with a stainless steel C40 4 cone-plate (40 mm diameter plate with an angle of 4 degree) requiring a sample volume of at least 1.2 ml.
• the instrument was operated using instrument standard software (Version 3.6) allowing several different types of measurements.
• Constant Rate vis- cosity and stress as a function of shear rate
• Oscillation complex viscosity, elastic modulus and viscous modulus at different frequencies
• Oscillation Stress Sweep to identify the stress range inside which the measurement results are linear i.e.
• n ⁇ 1 the solution is called shear-tinning, which is the characteristics of dispen- sions with asymmetric particles or emulsions. However, since the n value is close to 1 the solution is not far from being Newtonian. As can also be seen from fig. 2 the viscosity varies from 0.34 Pa s at low shear rates to 0.12 Pa s at high shear rates.
• Fig. 3 shows the result from the oscillatory measurement of the mucin solution alone (Fig. 3a) and the mucin/TFF3 dimer peptide gel-like material (Fig. 3b).
• Table 2 Elastic and viscous modules of mucin solution as compared to the mucin solution after addition of TFF3 dimer (Experimental details is given in legend to fig.3).
• Mitomycin C colitis In 16 rats colitis was induced by means of Mitomycin C, 3,75 mg/kg . Eight of the rats were given TFF3, 5 mg/kg in 0,5 ml H 2 O, two times each day from day 4 - 7 into the colonic tube. The 8 controls received NaCI. The rats were sacrificed by means of an overdose of barbiturate 8 days after mitomycin administration. The colon was fixed by intraluminal injection of 10% formalin, and after 10 minutes opened and suspended on a polyethylene plate. After further 24 hours fixation the specimens were flushed with water and surface stained with 0,3% Alcian Green 3BX.
• the colonic specimens were investigated by means of a Wild Photomacroscope - the extent of disease and the number of ulcerations were quantitated.
• Histologic analysis specimens were taken out (in a blinded way) from the proximal, middle and distal colon and embedded in paraffin. Histologic sections of 5 ⁇ m were stained with PAS-hematoxylin-aurentia. The severety of colitis was scored by means of a histologic colitis score (Fig. 5, Williams KL. et al. Gastroenterology 2001 ;120:925-37).
• Dextran colitis In 16 rats colitis was induced by means of dextran sulphate sodium 5% administered in the drinking water. Eight of the rats were given TFF3, 5 mg/kg in 0,5 ml H 2 O into the colonic tube, two times each day from the day before the initiation of dextran supplementation till day 9, where the rats were sacrificed. The 8 controls recieved NaCI. The rats were sacrificed by means of an overdose of barbiturate. The colon was fixed by intraluminal injection of 10% formalin, and after 10 minutes opened and suspended on a polyethylene plate. After further 24 hours fixation the specimens were flushed with water and surface stained with 0,3% Alcian Green 3BX.

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