EP1343522A2 - Procollagen (iii) propeptides and related substances for treating fibrotic diseases - Google Patents

Abstract

The invention relates to the use of procollagen (III) propeptides and related substances for treating fibrotic diseases and to a method for producing and renaturing recombinant N and/or C-terminal procollagen (III) propeptides. Said procollagen (III) propeptides and related substances are suitable for treating fibrosis of any type, of any organ manifestation. The invention also relates to a method for producing renatured N-terminal procollagen (III) propeptide and/or C-terminal procollagen (III) propeptide.

Description

 Procollagen (Ill) propeptides and related substances for the treatment of fibrotic diseases

A number of documents are mentioned in the description. The disclosure content of these documents, including the manufacturer's instructions for use, is hereby incorporated by reference.

The invention relates to the use of procollagen (III) propeptides and related substances for the treatment of fibrotic diseases, and to a method for producing and renaturing recombinant N- and / or C-terminal procollagen (III) propeptide. The Procollagen (Ill) propeptides and related substances are suitable for the treatment of fibrosis of any genesis and organ manifestation. The invention also relates to a method for producing renatured N-terminal procollagen (III) propeptide and / or C-terminal procollagen (III) propeptide.

State of the art: collagen biosynthesis

Type I and III collagens are synthesized as prepropetides and are extensively modified post-translationally. The intracellular modifications include, inter alia, glycosylations and enzymatic hydroxylation reactions which relate to lysine amino acid residues and proline amino acid residues in positions 3 and 4 of the imidazole ring. In the case of type III collagen, the modified propeptides spontaneously assemble into (oc ₁ (NI)) ₃ homotrimers. In collagen type I, mainly (ι (l)) ₂ α ₂ (l) heterotrimers are formed and to a lesser extent also (αι (lll)) ₃ Homotrimer. After exocytosis, the propeptides are cleaved at the C-terminal and then N-terminal on the nascent collagen by specific endoproteases. The C-terminal procollagen (III) propeptide (PIIICP) is cleaved by the procollagen C proteinase, which is identical to Bone Morphogenetic Protein-1 (BMP-1) (Kessler 1996). Tissue-specific expression patterns of different splicing variants of the BMP-1 protein were found (Janitz 1998). The N-terminal procollagen propeptide of collagen I (PINP) is cut by the same N-proteinase that also degrades the N-terminal procollagen propeptide of type II collagen. In contrast, the hydrolysis of the N-terminal procollagen (III) propeptide (PIIINP) is due to an enzymatic activity that is separate from that of the N-proteinase (I and II). The enzyme responsible for this is called procollagen-N-proteinase of type (III).

State of the art: PIIICP

PIIICP occurs as a trimer consisting of three identical monomeric PIIICP subunits. The subunits are connected by intermolecular disulfide bridges. Theoretical structural considerations and targeted mutagenesis experiments with so-called collagen minigens have shown that at least 4 and probably even 6 of the 8 cysteine residues of a monomeric PIIICP subunit are involved in the formation of intramolecular disulfide bridges. Probably only the cysteine residues in positions 51 and 68 are involved in the formation of intermolecular disulfide bridges. However, it was observed that the region around these two cysteine residues is also critical for the correct formation of intramolecular disulfide bridges, because Cys → Ser mutations in this region lead to a disturbed formation of these disulfide bridges. On the other hand, it has been described that the trimerization of type III procollagen also takes place when the formation of intermolecular disulfide bridges was made impossible by a Cys → Ser mutation in position 51. So far, no methods for quantifying PIIICP have been described, except in the patent application: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1).

PIIICP does not have any pharmacokinetic studies. Clearance and elimination mechanisms were only investigated for PICP. For ¹²⁵ l-labeled PICP, elimination from the serum by the mannose-6-phosphate receptor was described by competition experiments (Smedsrod 1990). Endocytosis through the mediation of this receptor would also be possible with PIIICP, since PIIICP has a glycosylation site in Asn173 (Dion 1987). However, this mechanism can be ruled out for recombinant PIIICP from E. coli because the protein is present in this host without glycosylations due to the expression.

There is no information in the literature regarding the physiological role of PIIICP. Various effects have been described in the literature regarding the biological effectiveness of the C-terminal propeptide of type I collagen.

The nucleotide sequence of the human PIIICP is stored in the genebank (Accession No. X14420, Ala-Kokko 1989, and X01742, Loidl 1984). The amino acid sequences of this propeptide are given by way of example in FIG. 1 (I). The propeptide sequence has been identified in the system in the overall sequence of the corresponding procollagen sequences C-terminal from the procollagen-C-proteinase interface.

In fibroblast cell culture, a 40% reduction in collagen biosynthesis was measured using 40 nM intact PICP, and a reduction of 30% at 10 nM (Wu 1986). However, these changes in protein biosynthesis correlated very well measured changes at the transcription level. As a result, PICP has been speculated about a regulatory inhibitory effect at the transcriptional level. For natural intact rat PICP, isolated from fibroblasts, an inhibition of collagen biosynthesis was also shown in cell cultures with hepatic star cells (Ma 1997). A 66% inhibitory effect was measured in concentrations of 33.3 nM, which increased to 83% at a concentration of 133.2 nM. The mRNA concentration changes under the influence of PICP were not examined. However, it was shown in these experiments that the inhibitory effect strongly depends on the structure of the protein. A covalent modification of PICP by acetaldehyde significantly weakened the effect on the biosynthesis rate.

The literature data are contradictory when using overlapping synthetic polypeptides from PICP in the fibroblast cell culture model.

An inhibitory effect at the level of the biosynthesis rate was determined with a polypeptide consisting of 22 amino acid residues (amino acid residues 225-246) (Katayama 1991).

In contrast, studies of another synthetic polypeptide consisting of 45 amino acid residues (amino acid residues 197-242) - in contrast to all previous results - showed an increase in the biosynthesis rates of the collagens type I and type III, as well as of fibronectin (Katayama 1991). At a concentration of 45 μM in human lung fibroblasts, collagen biosynthesis increased by 3.3 times and fibronectin biosynthesis by 6.1 times after 4 h. After 8 h, maximum stimulation of collagen biosynthesis was measured 6 to 8 times.

However, the stimulating effect was highly dependent on the degree of confluence of the cells. While an effect could be shown in subconfluent fibroblast cells, this was no longer detectable in confluent cells. The effects were neither line-specific nor species-specific. In further investigations, the polypeptide sequence necessary for the stimulation of the collagen biosynthesis rate could be reduced to a pentapeptide (amino acid residues 212-216), whereby 80% of the original maximum stimulation occurred (Katayama 1993). However, fibronectin with factor 5-11 showed a significantly stronger stimulation of the synthesis rate compared to type I collagen (factor 4-7). In addition to examining the changes at the protein level, the mRNA concentrations of the affected genes were also examined. No changes in concentration were measured at the mRNA level in either the collagens or fibronectin (Katayama 1991). The influence of the synthetic polypeptides on the stimulation of collagen biosynthesis was therefore at the post-transcriptional level.

In v / Vo studies with PIIICP or related substances have so far not been reported in the literature.

State of the art: PIIINP

PIIINP occurs as a trimer, which consists of three identical monomeric PIIINP subunits, which are interconnected by intermolecular disulfide bridges. The PIIINP molecule is structurally divided into three domains. The most N-terminal domain (CoM) has a globular structure and contains several intramolecular cysteine bridges. The so-called Col3 domain adjoining the C-terminal has a collagen-like structure that contains periodically recurring Gly and Pro residues. It forms a characteristic collagen triple helix structure. The Col 2 domain contains those parts of the procollagen telopeptide region which are N-terminal to the interface of the N-proteinase (III). In this region, the monomeric PIIINP strands are arranged parallel to one another. The presence of two cysteine residues, which form both intermolecular disulfide bridges and which are of crucial importance for the cohesion of the trimeric PIIINP, is characteristic of the Col2 domain. An oligosaccharide glycosylation site is located near the N-proteinase (III) interface. Eight amino acid residues are located at the C-terminal of the propeptide cleavage site, one of the four lysine residues which are oxidatively deaminated to aldehydes by the lysyl oxidase before the covalent cross-linking of the collagens.

A special feature of type III collagen is that some of the N-terminal propeptides in the procollagens are not split off. This so-called pN collagen type III is nevertheless built into fibrils (Fessler 1979). The shape of the fibrils is described differently: as thin fibrils associated with type I collagen (Keene 1987) or as thin pearl-like fibrils that associate with network-like structures (Amenta 1986). When viewed under electron microscopy, pN collagen type III has the appearance of a 'barbed wire' due to the still present PIIINP (Davis 1987). The presence of pN collagen type III on the outside of collagen fibrils could play a role in the regulation of fibril diameters (Schuppan 1991).

With regard to the stability of the PIIINP protein in the organism, half-lives between 2 min and 239 min have been reported (e.g. Smedsrβd 1988; Jensen 1989, intermediate values have been reported by Brooks 1993 and by Melkko 1994). The values determined differed greatly depending on the model and / or labeling of the antigen. In rats, clearance of the N-terminal propeptides of the collagens type III and type I from the serum - regardless of the antigen species - by the scavenger receptor of the liver endothelial cells was described (Melkko 1994). Endocytosis occurred in both proteins with the mediation of the same receptor.

The amino acid sequence of the human PIIINP is stored in the Genebank database with the accession number X14420 (Ala-Kokko 1989). The example is Amino acid sequences of this propeptide are given in Figure 1 (II). The propeptide sequence has been identified in the plant in the overall sequence of the corresponding PIIINP sequence N-terminai in the vicinity of the type III procollagen-N proteinase interface.

PIIINP has so far been primarily described as a fibrosis marker (Boigk, 1997). A framework of possible therapies for liver fibrosis can be used as a non-invasive course parameter (Afdahl, 1997).

Studies on their role as feedback inhibitors of collagen biosynthesis in cell culture systems and in cell-free lysates have been published for PIIINP. For the N-terminal propeptides of the collagens type I and type III and the Col 1 domain of the PINP, a concentration-dependent inhibition of the collagen production of the α-ι (l) and α ₂ (l) chains was measured cell-specifically in the fibroblast cell culture system ( Kühn 1982). Protein concentrations from 0.5 μM to 6 μM were used. The inhibition ranged from 30% to 50% compared to control experiments. An influence on the translation rate by the propeptides and their fragments was demonstrated. The findings were supported by the localization of the internalized proteins near the endoplasmic reticulum.

Investigations with the Col 1 domains of the two N-terminal propeptides were also carried out in the cell-free reticulocyte system (Paglia 1979). Protein concentrations of 0.4 to 3.2 μM were used. A concentration-dependent inhibition of collagen (I) synthesis was measured. In both cases, an inhibition between 38% and 71% was measured at the translation level compared to control experiments. When using very high concentrations of proteins (8 μM) it was shown that the inhibitory effect on collagen translation in this system could not be further increased. When examining the mechanism of action of PINP, a system for the recombinant cytosolic expression of PINP in fibroblast cell culture was also examined (Fouser 1991). While no changes in the collagen (I) mRNA concentrations were measured, there was a reduced biosynthesis rate of the corresponding protein and thus regulation at the post-transcriptional level.

The results for PINP are supported by experiments with skin fibroblasts from homozygous dermatosparactic sheep (Kühn 1982). In the homologous human disease, the Ehlers-Danlos syndrome type Vlla or Vllb, there is a mutation in the N-proteinase interface of the type I procollagen, so PINP cannot be split off (Byers 1997). In cell culture, these cells, without a PINP feedback mechanism, had a share of collagen biosynthesis in the total cellular biosynthesis of 15-20% compared to heterozygous control fibroblasts (control fibroblasts 2 to 4%).

State of the art: recombinant production of procollagen (IΙI) propeptides

The recombinant production of procollagen (III) propeptides has been described in a number of publications. Lee, 1990, describes the recombinant production of a collagen α ₂ (l) mutant in so-called A2 cells, which are derived from the epithelial liver cell line W8, which in turn is deficient in the production of collagen α ₂ (l). The recombinant expression of collagen ι (III) Minigene is described in two recent publications (Lees 1994; Bulleid 1996).

In a recent work, the production of PIIICP in Zf9 cells is described as a trimer protein (Zafarullah 1997). However, recombinant procollagen could only be produced in very small quantities for analysis purposes. The recombinant expression of PIIICP in E. coli was described in the patent applications: An immunoassay for procollagen-III-C-terminal propeptide. (WO09924835A2 and EP00988964A1) and in Burchardt, 1998. The yields in this expression process were more than 20 mg / l liquid culture medium. However, the recombinant proteins in E. coli were almost exclusively in the form of inclusion body proteins and had to be purified using denaturing digestion methods. In addition, the expressed proteins contained an N-terminal His tag, so that they could also be purified in denaturing solvents using a Ni-NTA column. These proteins were less suitable for chronic in wVo applications because the His day is potentially immunogenic and the biological half-life of the recombinant protein can therefore be shortened. They were predominantly in monomeric form under the methods given in this application, and their solubility in aqueous solutions was too low for most therapeutic applications. Exceeding concentrations of approx. 10 μg / ml led to the precipitation of the recombinant PIIICP from aqueous solutions.

The recombinant expression of human PIIINP in E. coli was described in the patent application: Monoclonal antibody and assay for detecting PIIINP (WO09961477A2) and that of murine PIIINP in Kauschke, 1999. The yields in this expression process were each about 5 mg / l liquid culture medium. In addition, these proteins also contained an N-terminal His tag, so that they could be purified in denaturing solvents using a Ni-NTA column. These proteins were also less suitable for chronic in wVo applications because the His day is potentially immunogenic and the biological half-life of these proteins can therefore be shortened. The solubility of PIIINP in aqueous solutions was too low for most therapeutic applications. State of the art: Fibrotic diseases

Fibrotic diseases are understood to mean a diverse group of diseases that are associated with a qualitatively altered collagen production or with an increased deposition of collagen in the extracellular space. This group of diseases includes systemic or local scleroderma, liver fibrosis of various origins, e.g. alcoholic cirrhosis of the liver, biliary cirrhosis, hepatitis viral or other genesis, the so-called veno-occlusive diesease, idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, acute pulmonary fibrosis, "acute respiratory distress syndrome" (ARDS), perimuscular fibrosis, pericentric fibroids, pericentric fibroids , diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scarring, joint adhesions, arthrosis, myelofibrosis, scarring of the cornea, cystic fibrosis, muscular fibrosis, Duchenne muscular dystrophy, esophageal strictures, ulcerative colitis, morbid oresophageal morbid conditions, ulcerative disease, morbus oresophageal morbid disorders, oesophageal strictures, ulcerative disease, morbus orotic disease pulmonary hypertension, angiopathies of the arteries and veins, aneurysms of the large vessels.

Further fibrotic diseases can be initiated or caused by surgical scar revisions, plastic surgery, glaucoma, cataract fibrosis, scarring of the cornea, the so-called "graft versus host disease", surgical interventions on tendons, nerve pinching syndromes, Dupuytren's contracture, adhesions as a result of gynecology Interventions, Pelvic adhesions, infertility, peridural fibrosis, diseases of the thyroid or parathyroid glands, due to metastatic bone attack, multiple myeloma or restenosis. Therapy for many of these diseases has so far not been successful. Others have a need for improved approaches or for reducing side effects. From the foregoing, there is a further need to provide effective drugs against fibrotic diseases. This object is achieved by the embodiments characterized in the examples.

Description of the invention

The present invention thus relates to a composition, preferably a medicament, comprising (a) a (poly) peptide which is the N-terminal procollagen (III) propeptide and / or the C-terminal procollagen (III) propeptide or (b) a fragment or derivative thereof having essentially the antifibrotic activity of (poly) peptide (a) and / or (c) a peptide which contains the recognition sequence of the type III procollagen C protease and / or a peptide which comprises the recognition sequence of Type III procollagen N proteinase, in combination with a pharmaceutically acceptable carrier or diluent.

The term "a (poly) peptide which is the N-terminal procollagen (III) propeptide and / or the C-terminal procollagen (III) propeptide" means in the sense of the invention that the (poly) peptide is the N- is terminal or the C-terminal procollagen (III) propeptide, to which further amino acid sequences can optionally be connected N-terminal or C-terminal, so that a longer (poly) peptide results. The additional sequences can come from procollagen or can be heterologous or artificial sequences. (Poly) peptides are preferred which, in addition to the (human) PIIICP or PIIINP amino acid sequence, also have possible procollagen C or procollagen N proteinase interface recognition sequences.

According to the invention, the term “a fragment or derivative thereof having essentially the antibiotic activity of the (poly) peptide (a)” means that this fragment or derivative is at least 50%, preferably 75%, more preferably 85%, particularly preferably 90% of the antifibrotic Has activity of the N-terminal or C-terminal procollagen / propeptide. Derivatives of the (poly) peptide may be other than that contain natural amino acids or substituted amino acids. Derivatives can be made, for example, by peptidomimetics.

When the embodiments below are usually discussed in the context of drugs, these apply mutatis mutandis to the compositions.

The term “(poly) peptide” relates to both polypeptides and peptides. A peptide usually contains no more than 30 amino acids.

Possible procollagen C proteinase recognition sequences have been identified in FIG. 1 (I).

Possible procollagen N proteinase recognition sequences have been identified in the PIIINP sequence in FIG. 1 (II).

Examples of suitable pharmaceutically acceptable carriers and / or diluents are known to the person skilled in the art and include, for example, phosphate-buffered saline solutions, water, emulsions, such as, for example, oil / water emulsions, various types of wetting agents or detergents, sterile solutions, etc. Carriers can be formulated using known conventional methods. These drugs can be administered to an individual in an appropriate dose. Administration can be oral or parenteral, for example intravenous, intraperitoneal, subcutaneous, intramuscular, local, intranasal, intrabronchial, oral or intradermal, or via a catheter at one location in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic ester compounds such as ethyl oleate, which are suitable for injections. Aqueous carriers include water, alcoholic-aqueous solutions, emulsions, suspensions, Saline solutions and buffered media. Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, and bound oils. Intravenous carriers include, for example, liquid, nutrient and electrolyte supplements (such as those based on Ringer's dextrose. The drug may also include preservatives and other additives such as antimicrobial compounds, antioxidants, complexing agents and inert gases. Furthermore, , depending on the intended specific use, other active ingredients such as interleukins, growth factors, differentiation factors, interferons, chemotactic proteins or a non-specific immunomodulatory agent may be included.

The treating doctor determines the type of dosage according to the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, such as, for example, the body size or weight, the body surface, the age, gender or general health of the patient, but also on the agent to be administered specifically, the duration and mode of administration, and other medications that may be administered in parallel. A typical dose can be, for example, in a range between 0.001 and 1000 μg, doses below or above this exemplary range being conceivable, especially taking into account the factors mentioned above. In general, with regular administration of the composition according to the invention, the dose should be in a range between 1 μg and 10 mg units per day. The active substances in these preparations will usually be present in a concentration of greater than 10 μg / ml of a physiological buffer. However, they can also be present in solid form in a concentration of 0.1 to 99.5% by weight of the total mixture. In general, it has proven to be advantageous to use the active ingredient (s) in total amounts of about 0.001 to 100 mg / kg, preferably in total amounts of about 0.01 to 10 mg / kg body weight per 24 hours, optionally as a continuous infusion or in the form of several Single doses to be administered to achieve the desired result. If the composition is administered intravenously, the dose should range between 1 μg and 10 mg units per kilogram of body weight per day. The drug can be administered locally or systemically.

Surprisingly, it was found according to the invention that the above-mentioned (poly) peptides and in particular PIIICP were taken up in vivo in the liver and showed antifibrotic effects. The mechanism of action of recombinant PIIICP described in the patent application by reducing the connective tissue growth factor mRNA was also surprising and unexpected. The role of PIIICP as a natural feedback inhibitor of fibrotic matrix deposition was described for the first time. The findings according to the invention are also surprising because PIIICP / PIIINP have so far been discussed predominantly only as diagnostic markers for monitoring the effectiveness of other medicaments, but not in connection with them as therapeutic agents for use in humans. The achievement of an antifibrotic therapeutic effect was therefore completely surprising.

In v / Vo studies on the reduction of fibrosis by using recombinant PIIINP have so far not been described in the literature. Furthermore, due to the lack of glycosylation of recombinant PIIINP from E. coli, the reduction in the fibrotic area described in this patent application was completely surprising and unexpected.

In a preferred embodiment, the (poly) peptide or fragment or derivative and / or the peptide derives from or is derived from human procollagen (III).

In a further preferred embodiment, the (poly) peptide comprising the recognition sequence comprises 10 to 15 amino acids N-terminal from the cleavage site.

In an additional preferred embodiment, the (poly) peptide comprising the recognition sequence comprises 10 to 15 amino acids C-terminal from the cleavage site. In another preferred embodiment, the (poly) peptide is a fusion protein.

In a particularly preferred embodiment, the (poly) peptide contains a His tag. The His tag can be attached at the C-terminal or N-terminal.

In a further particularly preferred embodiment, the His tag 6 His tag and N-terminal is added.

Furthermore, the present invention relates to the use of a (poly) peptide or fragment or derivative thereof described above for the manufacture of a medicament or medical device for the treatment or prevention of fibrotic diseases.

In a preferred embodiment, the fibrotic diseases are selected from systemic or local scleroderma, liver fibrosis of various origins, such as alcoholic cirrhosis of the liver, biliary cirrhosis, hepatitis viral or other genesis, the so-called "veno-occlusive disease", idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, acute pulmonary fibrosis, the "acute respiratory distress syndrome" (ARDS), perimuscular fibrosis, pericentral fibrosis, dermatofibromas, kidney fibrosis, diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scarring, joint adhesions, arthrosis, corneal fibrosis, myelofibular fibroids muscular fibrosis, Duchenne muscular dystrophy, esophageal strictures, Ormond 's disease, Crohn' s disease, ulcerative colitis, atherosclerotic changes, pulmonary hypertension, angiopathies of the arteries and veins and aneurysms of the large vessels or are caused or initiated by surgical scar revisions, plastic surgery, glaucoma, cataract fibrosis, scarring of the cornea, the so-called "graft versus host disease", surgical procedures on tendons, nerve trapping syndromes, Dupuytren's contracture, adhesions due to pelvic surgery Infertility epidural fibrosis, diseases of the thyroid or parathyroid glands, due to metastatic bone infestation, multiple myeloma or restenosis.

In addition, the present invention relates to processes for the production of renatured N-terminal procollagen (III) propeptide and / or C-terminal procollagen (III) propeptide, where (a) recombinantly produced with E. coli by conventional methods, the / dissolve the inclusion bodies containing propeptide (s) in a 0.5 to 8 M denaturing buffer; (b) the buffer according to (a) is added dropwise to a limit dilution buffer which buffers around a neutral pH, contains L-arginine in a final concentration of 200 to 1000 mM and a disulfide bridge-reducing coupled redox system until a volume ratio of the denaturing buffer to limit dilution buffer reached at most 1: 3; (c) dialyzing the buffer mixture according to (b) for at least 2 hours against a physiological buffer containing L-arginine in a final concentration of 50 to 200 mM and a disulfide bridge-reducing coupled redox system; (d) dialyzing the buffer mixture according to (c) for at least 2 hours against a physiological buffer containing a disulfide bridge-reducing coupled redox system; and (e) dialyzing the buffer mixture according to (d) for at least 2 hours against a physiological buffer.

Thus, the buffer in step (d) contains no arginine and in step (e) neither arginine nor the redox system.

The possibility of being able to dissolve recombinant PIIICP in higher concentrations than previously described in physiological buffer was completely surprising and unexpected, because it is known to the person skilled in the art that collagens are difficult to dissolve in physiological buffers.

By this method the therapeutic application of the recombinant

Procollagen propeptides and related substances in therapeutically relevant

Concentrations. Example 4 shows the use of after this Methods of renatured procollagen propeptides in animal models of liver fibrosis are described.

In a preferred embodiment, the buffer is in at least one of the steps

(b) to (d) a chelator is added.

The chelator is preferably EDTA, for example in a final concentration of 10 mM.

In a further preferred embodiment, the redox system glutathione is reduced - glutathione is oxidized.

It is particularly preferred that the components of the redox system have the concentrations given in Example 2.

In another preferred embodiment, a further protease inhibitor is added to the buffer in at least one of steps (b) to (d). The protease inhibitor Pefabloc SC is preferred.

In an additional preferred embodiment, the buffer is in one of the steps

(c) and / or (d) a salt is added to a final concentration of about 10 mM. The salt is preferably NaCl.

It is particularly preferred that the limit dilution and dialysis buffers have the concentrations given in Example 2.

In a further preferred embodiment, the denaturing buffer contains in

Step (a) urea at a final concentration of 0.5 to 8 M.

The buffer preferably contains urea in a final concentration of about 6 M.

In another preferred embodiment, Trizma base is used as the buffering agent. In an additional preferred embodiment, the dialysis steps are carried out at about 4 ° C.

In another preferred embodiment, dialysis is carried out in steps (c) to (e) against at least 100 times the dialysis buffer volume.

In addition, the present invention relates to a method for producing a medicament, wherein the renatured N-terminal procollagen (III) propeptide and / or C-terminal procollagen (III) propeptide produced by the method according to the invention is concentrated or lyophilized by and with conventional methods added to a pharmaceutically acceptable carrier or with a pharmaceutically acceptable diluent.

Useful pharmaceutical carriers and diluents have been discussed above.

Furthermore, the present invention relates to the use of an N-terminal procollagen (III) propeptide and / or a C-terminal procollagen (III) propeptide, which was prepared by the method according to the invention, for the treatment or prevention of fibrotic diseases.

In a preferred embodiment, the fibrotic diseases are selected from systemic or local scleroderma, liver fibrosis of various origins, such as alcoholic cirrhosis of the liver, biliary cirrhosis, hepatitis viral or other genesis, the so-called "veno-occlusive disease", idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, acute pulmonary fibrosis, the "acute respiratory distress syndrome" (ARDS), perimuscular fibrosis, pericentral fibrosis, dermatofibromas, kidney fibrosis, diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scarring, joint adhesions, arthrosis, corneal fibrosis, myelofibular fibroids muscular fibrosis, Duchenne muscular dystrophy, esophageal strictures, Ormond's disease, Crohn's disease, ulcerative colitis, atherosclerotic changes, pulmonary hypertension, angiopathies of the arteries and veins and aneurysms of the large vessels or are caused or initiated by surgical scar revisions, plastic surgery, glaucoma, cataract fibrosis, the scarring of the cornea graft versus host disease ", surgical interventions on tendons, nerve pinching syndromes, Dupuytren's contracture, adhesions as a result of gynecological interventions, pelvic adhesions, infertility, peridural fibrosis, diseases of the thyroid or parathyroid glands, due to metastatic bone infection or due to multiple myeloma, or due to multiple myeloma ,

The tables show:

Table 1 shows the body weight at the end of the experiment, relative liver weight, GIDH serum activities, collagen type III and type I and TGFß mRNA concentrations after chronic application of various procollagen αι (III) propeptides in the mouse CCI ₄ model. Differences in mRNA concentration according to TaqMan analysis are given as -Δct- values in comparison to the intact control. (Mean ± SEM)

Table 2 shows the body weight at the end of the experiment, relative liver weight, GIDH serum activities, collagen type III and type I and TGFβ mRNA concentrations after chronic application of different concentrations of the PIIICP4.1 protein in the mouse CCI ₄ model. Differences in mRNA concentration according to TaqMan analysis are as -Δct values compared to the intact control. (Mean ± SEM)

Table 1

Table 2

The figures show:

Figure 1 (I) shows the amino acid sequences in the region of the human C-terminal procollagen (III) propeptide (PIIICP). The N-terminal end of the prosequence is formed by the procollagen-C-proteinase interface (see arrow). The actual propeptide sequence is printed in bold. The sequence section that is in the vicinity of the procollagen C proteinase interface is underlined. Figure 1 (II) shows the Amino acid sequences in the area of human N-terminal procollagen (III) propeptide (PIIINP). The N-terminal end of the prosequence is adjacent to the presequence. The procollagen-N-proteinase interface is indicated by an arrow. The actual propeptide sequence is printed in bold. The section of the sequence which is in the vicinity of the procollagen-N-proteinase interface is underlined.

FIG. 2 shows the PlllCP serum concentration curve in the anesthetized rat model. The terminal half-life was approx. 80 min, the terminal volume of distribution was approx. 7.4 l / kg and the elimination rate was approx. 6.3 l / (h * kg). See Example 3 in the specification for details.

Figure 3 shows (A) total collagen area and (B) CTGF mRNA concentration after chronic application of various procollagen αι (III) propeptides in the mouse CCI ₄ model (mean ± SEM).

FIG. 4 shows (A) total collagen area fraction and (B) CTGF mRNA concentration after chronic application of different concentrations of the PIIICP4.1 protein in the mouse CCI ₄ model (mean ± SEM). See Example 4 in the specification for details.

FIG. 5 shows immunohistochemical studies on the localization of the PIIICP antigen on representative sections from rat liver. The anti-PIIICP monoclonal antibody 48D19 was used. (A) intact control after seven days infusion of a buffer control solution (240-fold); (B) CCI ₄ -induced liver fibrosis after seven days of PlIICP protein infusion (400-fold); (C) CCI-induced liver fibrosis after a seven-day infusion of a buffer control solution (fibrosis control, 320-fold).

The examples illustrate the invention.

In the examples, "v / v" means volume percent and "w / v" means weight percent. Example 1: Purification of PIIICP from Inclusion Bodies in E. coli

PIIICP was produced in E. coli in the form of inclusion bodies (Burchardt 1998). The cell paste of three 200 ml cultures in each case was resuspended in 24 ml 50 mM Tris-HCl (pH 8.0), 1 M NaCl, frozen overnight at -20 ° C. and centrifuged after thawing (5,000 g, 10 min ). 4.8 ml of the same buffer were added to the precipitate, and 4.8 ml of 25 mM Tris-HCl (pH 8.0), 5 mg / ml lysozyme and 4.8 ml of a 0.5 M EDTA solution (pH 8 , 0) added. After 1 h of incubation at 37 ° C, the bacterial cells were disrupted using ultrasound. 12 ml of 10% Triton-X-100, 1 mM EDTA, dissolved in 50 mM Tris-HCl (pH 8.0), were then added and, after 30 minutes of incubation, centrifuged off (5,000 g, 10 minutes). The insoluble fraction was dissolved in 24 ml of 5% Triton-X-100, dissolved in 50 mM Tris-HCl (pH 8.0), sonicated, incubated again at 37 ° C. and centrifuged again (5,000 g, 10 min). The tritone extraction to remove membrane lipids was repeated once. The pellet was then dissolved in 24 ml of 1 M urea, 1 mM EDTA, dissolved in 50 mM Tris-HCl (pH 8.0), with ultrasound and centrifuged again after 30 min of incubation (5,000 g, 10 min). The pellet consisted of almost pure PIIICP in the form of inclusion bodies. The protein could be dissolved in 6 M urea, dissolved in 50 mM Tris-HCl (pH 8.0). If the sample was centrifuged at 15,800 g for 30 min, the inclusion body protein remained in the supernatant.

Example 2: Renaturation of recombinant collagen αidlO propeptides

For example, recombinant human PIIICP4.1, the genetic engineering of which was described in Burchardt, 1998, and in the patent applications: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1) was renatured. Furthermore, recombinant human PIIINP 4.5.2. renatured, the genetic engineering of which is described in the patent application: Monoclonal antibody and assay for detecting PIIINP (WO09961477A2). Finally, recombinant murine PIIINP, the genetic engineering of which is described in Kauschke, 1999, was also renatured using the method described below.

However, the method is in no way limited to the renaturation of these propeptides mentioned by way of example, but is suitable for the renaturation of other procollagen propeptides and similar compounds.

method

Buffers used:

Dialysis buffer: 300 mM Trizma base, pH 7.4

400 mM L-arginine

10mM EDTA

0.4 mM Pefabloc SC

1 mM glutathione reduced

0.1 mM glutathione oxidized

Limited dilution buffer: 50 mM Trizma base, pH 7.4

800 mM L-arginine

10 mM NaCl

10mM EDTA

0.4 mM Pefabloc SC

1 mM glutathione reduced

0.1 mM glutathione oxidized The collagen α-ι (III) propeptides can be dissolved in a 6 M urea buffer. The buffer conditions of the proteins were changed abruptly by a limited dilution step. A redox system in the target buffer favored the formation of disulfide bridges. For this purpose, the protein solution was dripped into an excess of ice-cold limited dilution buffer with gentle shaking. This batch was stored at 4 ° C overnight.

All dialysis steps took place at 4 ° C. Each step was dialyzed against 100-fold buffer volume for at least three hours. The limited dilution approach was transferred to a tube of suitable pore size and dialyzed against the dialysis buffer. In the next dialysis steps the L-arginine concentration was reduced to 100 mM, then a buffer without L-arginine was used. The fourth buffer also lacked the glutathione redox system. The final buffer was selected according to the intended use. For in v / Vo experiments it consisted, for example, of 10 mM Trizma base, pH 7.4, 145 mM NaCl. The dialysate was then stored at 4 ° C.

Example 3: Measurement of the elimination kinetics of renatured PIIICP in the anesthetized rat

Materials and methods

PIIICP plate ELISA: The determination of PIIICP concentrations in biological samples was carried out in a sandwich ELISA assay. Two monoclonal anti-PIIICP antibodies were used (Burchardt 1998).

Antibody 48B14 (Burchardt, 1998) was immobilized on the test plate in a concentration of 5 μg / ml as a capture antibody. After blocking free binding sites on the plate by incubation with a 3% (v / v) BSA solution in PBS biological samples or buffer solutions with known PIIICP concentrations together with a known concentration of FITC-labeled secondary antibody (48D19) (Burchardt, 1998) were added to the immobilized primary antibody for 30 min. Unbound PIIICP antigen and still unbound secondary antibody were then removed by washing steps and the amount of bound, labeled secondary antibody was determined. The above antibodies can be replaced with other anti-PIIICP antibody pairs that can be prepared by conventional methods (Harlow and Lane, 1988).

The measured in vivo concentrations in human serum were for the most part below the detection limit (below 0.5 ng / ml, see patent application: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1)). These results are almost an order of magnitude below the physiological PIIINP concentrations in human serum.

Animal experiment: Fasting Sprague-Dawley rats weighing approx. 300 g were anesthetized with an intraperitoneal trapanal dose of 100 mg / kg body weight.

Liquids or substance dilutions were administered via a catheter in the jugular vein. The blood pressure was measured and the blood was drawn using a catheter in the aorta femoralis. A tracheal incision was made to facilitate spontaneous breathing. During the experiment, the animals received infrared heat radiation. After opening, the animals received a bolus of 5 ml of physiological saline per kg body weight to compensate for the loss of blood. Following a fifteen-minute recovery phase, the substance dilutions in physiological buffer solution (10 mM Trizma base, pH 7.4, 145 mM NaCl) were administered by a two-hour infusion of renatured PIIICP4.1 (Burchardt 1998) with a flow rate of 100 μl per kg body weight and minute (PIIICP4.1 concentration in the infusion solution approx. 150 μg / ml). Blood samples were taken at 2, 60, 115, 150, 180, and 225 minutes after the start of the infusion in parallel with a recording of the respective blood pressure. 400 μl of blood were taken at each point in time, 20 μl of heparin (250 IU / ml) were added immediately, centrifuged off sharply and the plasma was stored at -20 ° C. until the test. After the end of the experiment, the experimental animals were killed by adding KCI solution. After the PIIICP concentration determinations in the samples, the pharmacokinetic parameters were determined.

Results

The terminal half-life was determined to be approximately 80 min. In the terminal curve area, 6.1% of the total area was still below the curve. The volume of distribution in the terminal phase was approx. 7.4 l / kg, the elimination rate was determined to be approx. 6.3 l / h / kg) (see FIG. 2).

Example 4: Demonstration of the biological effectiveness of PIIICP and PIIINP

The biological effectiveness of the substances can be demonstrated in cell culture assays and in vivo. After application of the inhibitors, e.g. in human cell lines the drop in the concentration of free α-ι (III) propeptide in the supernatants can be measured because this peptide is released by the activity of the PCP. A recently established assay can be used to measure the PIIICP concentrations in the supernatant (Burchardt, 1997, and patent applications: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1)).

This patent application exemplifies the biological activity of PIIICP4.1 (preparation described in Burchardt, 1998, and in the patents: An immunoassay for procollagen III C-terminal propeptide (WO09924835A2 and EP00988964A1); Recovery and renaturation described above in this patent application as an example), PIIINP4.5.2 (preparation described in the patent: Monocional antibody and assay for detecting PIIINP (WO09961477A2); renaturation described above in this patent application as an example) and of murine recombinant PIIINP (production described in Kauschke, 1999; renaturation described above in this patent application as an example) in the model of CCI-induced liver fibrosis in the mouse.

For PIIICP, the antifibrotic effect of 3 different doses of this compound is described in this animal model as an example.

Depending on the organ manifestation or the type of fibrotic damage, animal models for other fibrosis manifestations, e.g. in the heart, kidneys, lungs, skin or other organs.

In the example of CCI ₄ -induced liver fibrosis in the mouse, the reduction of the collagen deposition of PIIICP and PIIINP by quantitative morphometry is described. It can also be done, for example, by determining the hydroxyproline content (Gerling, 1996) of the fibrotic organs or by quantitative morphometry (Kauschke 1999). The example also describes the organ-protective effect of PIIICP and PIIINP by reducing cell damage, which is determined by measuring the activities of intracellular marker enzymes (eg GIDH). The organ-protective effect can also be measured in another way, for example by measuring the inhibition of the extent of apoptosis and necrosis or the like. Materials and methods

Animal experiments: The experimental animals were administered 0.2 ml of a mixture of CCI and mineral oil in a ratio of 1: 8 twice a week per 100 g body weight over the entire experimental period. The substance was administered by daily intraperitoneal application of 0.5 ml of a substance dilution. In the fibrous and intact controls, buffer control solution without procollagen propeptides was applied. The animals had free access to standard food and water throughout the experiment. The body weight of the animals was measured at the beginning and at the end of the experiment. At the end of the test, the wet weight of the liver was determined, the liver portioned for the subsequent examinations and immediately snap-frozen in liquid nitrogen. Storage until use was at -80 ° C. Plasma samples were also taken from the test animals to determine clinico-chemical parameters.

Total collagen staining with Sirius Red / Fast Green: 14 μm frozen sections were dried overnight. After a ten-minute treatment with a 10% (v / v) formaldehyde solution, the sections were distilled twice with H ₂ 0 dist. washed. The Picrosirius red staining was carried out for 30 minutes at room temperature in a 0.1% (w / v) Sirius Red solution in saturated picric acid (additionally per 400 ml one PBS tablet and 10 ml glacial acetic acid). The cuts were again twice with H ₂ 0 dest. washed and then colored for 30 min in a 0.1% (w / v) Fast Green solution in saturated picric acid (additionally per 400 ml a PBS tablet and 10 ml glacial acetic acid). The decolorization and dewatering steps were carried out by a sequence of washing steps: ten seconds H ₂ 0 dest, ten seconds 70% (v / v) ethanol, one minute each 80% (v / v) and 90% (v / v) ethanol, three times two minutes each of pure ethanol. In front After the final covering in Leica CV Mount, the sections were washed three times for 5 minutes each in xylene.

Total RNA preparation from tissue: About 20 mg of tissue was ground in liquid nitrogen and transferred to an Eppendorf reaction vessel. The tissue powder was mixed with 600 ul buffer RLT (with 0.1% (v / v) ß-mercaptoethanol), mixed homogeneously and applied to a QIAshredder column. The column was centrifuged at 18,000 g and 4 ° C for 2 min. The retained solids were discarded and the flow continued to be used. The following processing was carried out using the RNeasy Total RNA Kit (Qiagen, Hilden) according to the manufacturer's instructions. Elution was carried out with 35 μl RNase-free H ₂ 0. The RNA content of each preparation was determined photometrically immediately following an aliquot, and a further sample was checked for the integrity of the RNA bands obtained using an RNA-formaldehyde agarose gel , Storage until use was at -80 ° C.

cDNA synthesis from total RNA: The synthesis of the cDNA from the prepared total RNA samples was carried out in all cases using the SuperScript II preamplification system according to the manufacturer's instructions (Gibco BRL). All work steps were carried out on ice. In all cases, 1 μg of total RNA and master mixes were used. Prior to reverse transcription, any genomic DNA contamination in all samples was removed by DNase I digestion. For this purpose, 1 μg of total RNA was made up to a volume of 8 μl with RNase-free water, 1 μl of DNase I (Superscript Kit) and 1 μl of 10-fold buffer were added, and the digestion was carried out for 10 min at room temperature. The DNase I digestion was stopped by adding 1 μl of 25 mM EDTA solution and subsequent incubation (10 min at 65 ° C.). The entire approach was used in cDNA synthesis. Following the cDNA synthesis, the batches were made up to a total volume of 100 μl Fill up with DNase-free water and store at -80 ° C until use. In all cDNA syntheses, random checks were carried out for genomic DNA contamination without the addition of reverse transcriptase.

mRNA concentration determination with a TaqMan PCR analysis: The

Specific mRNA concentrations were determined by a TaqMan analysis. During the PCR reaction, specific hybridizing fluorescence-labeled probes are cleaved by exonuclease activity of the Taq polymerase and the resulting fluorescence signal is measured in real time. Results are given in the form of the number of cycles at which the fluorescence released exceeds a predetermined threshold value for the first time. The parameter et (threshold cycle) specifies the arithmetically determined number of cycles at which the measured fluorescence signal for the first time delivers a specific signal value above the threshold value (threshold). Lower ct values indicate that there was a higher specific mRNA concentration in the original batch. The maximum possible product doubling with each cycle in the PCR reaction. By choosing suitable primers and probes, and by the small size of the amplified sequence, approximately a product doubling can be assumed for each PCR cycle during the exponential phase of the reaction. As a result, the differences in the mRNA concentrations of the transcript in question in the original sample can be calculated from the measured differences in the ct units after normalization. A standardized difference of 3 ct units means that if a product doubling is valid with each PCR cycle, the mRNA concentrations of this transcript differ from the comparison samples by a factor of 8 (= 2 ³ ).

When considering the mRNA concentrations, all measured mRNA concentrations were normalized to the respective HPRT mRNA concentration. For the HPRT mRNA concentration it could be shown that this is within the scope of a Fibrosis does not change and, as a result, HPRT can serve as a standard, ie as a reference level.

All primers and probes were selected with regard to their position on the gene and the amplification product to be expected so that in the context of the TaqMan PCR reaction a doubling of the product concentration was to be expected in each case per cycle. These assumptions were checked and confirmed in advance by control experiments. The primers and the 6-FAM-labeled probes were all present in concentrations of 100 μM. In order to avoid variability between approaches, master mixes were used in all cases and at least double determinations for each approach. A single transcript was examined for all samples collected on a plate. In all investigations, controls without a template or without a previous reverse transcription reaction were carried out. All work took place under ice cooling. The master mix contained 12.5 μl of the TaqMan Universal PCR Master Mix (Röche), 7.5 μl of a primer-probe mix (1 μM per primer and 0.5 μM probe in DNase / RNase-free water) as well as 3 , 75 μl DNase / RNase-free water. 2.5 μl cDNA were placed in 96-well plates with optical lids and mixed with 22.5 μl of the master mix per batch. The plate was centrifuged for 1 min at 500 g and 4 ° C before the PCR reaction. The program of the TaqMan PCR reaction included a heating phase of 2 min at 50 ° C, a ten-minute denaturation at 95 ° C and then 40 cycles with a denaturation at 95 ° C for 15 seconds and a combined one-minute annealing / expansion reaction 60 ° C. During the cycles, the fluorescence of the released probes was automatically measured at the time of denaturation. The evaluation was carried out with the ABI PRISM Sequence Detection Software. The baseline was set at the average of cycles 3 to 15, the threshold was 0.04. Results

Effects of infusion of procollagen (III) propeptides in the chronic ip experiment in the mouse CCU model: To investigate the effects of the recombinantly produced procollagen -I (III) propeptides on the formation of an experimentally induced liver fibrosis, groups of 10 mice each only treated with CCI ₄ (fibrosis control) or additionally with protein solutions with concentrations of 500 μg / ml. In addition, one group was treated only with a buffer control solution (intact control). Human PIIICP4.1 (preparation described in Burchardt, 1998, and in the patent applications: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1); recovery and renaturation described above in this patent application as an example) were used as therapeutic recombinant proteins. , human complete PIIINP 4.5.2 (preparation described in the patent: Monocional antibody and assay for detecting PIIINP (WO09961477A2); renaturation described above in this patent application as an example) and murine PIIINP with 18 amino acid residues of the prosequence (referred to as M1, preparation described in Kauschke , 1999; renaturation described above in this patent application as an example).

At the end of treatment, the PIIICP dissolved in the infusion solution was not degraded in all cases. This was demonstrated with an SDS-PAGE gel.

In all animals, the total collagen area in liver sections, the GIDH activity in the serum and the mRNA concentrations of selected transcripts were measured morphometrically using a TaqMan analysis. The proteins were well tolerated by the animals in the concentration used during the period examined. There were no pathological abnormalities, and there were no frequent deaths.

The morphometically determined total collagen area, which is determined by automated Evaluation of the Sirius red-stained areas showed the highest values in the fibrosis control group (Kauschke, 1999). The total collagen areas in the treatment groups were significantly reduced in all cases (FIG. 3A). With PIIICP4.1 to 71% of the fibrosis control (p <0.04), with PIIINP 4.5.2 to 63% of the fibrosis control (p <0.01) and with M1 to 68% of the fibrosis control (p <0.02).

When looking at the mRNA concentrations in the liver, CTGF showed a significant reduction in concentration in all treatment groups compared to fibrosis control (FIG. 3B). The differences compared to the fibrosis control were +1.9 Δct units (PIIICP4.1, p <0.001), +1.5 Δct units (PIIINP 4.5.2, p <0.01) and +1, 2 Δct units (M1, p <0.02).

The GIDH serum activity was also reduced in all treatment groups compared to fibrosis control (Tabellel) (Kauschke, 1999). Serum activities were 17% (PIIICP4.1) and 11% (PIIINP 4.5.2, M1) lower than in the fibrosis control. Due to individual fluctuations, however, the deviations never reached the level of significance (p <0.05).

All animals with CCI ₄ application showed a reduction in body weight (Table 1). The treatment groups showed no differences from the fibrosis control.

The relative liver weight was above the intact controls in all CCI ₄ groups. The treated animals tended to have a slightly increased relative liver weight compared to the fibrosis control. The absolute liver weights between the groups were not significantly different (Table 1).

The mRNA concentrations of the transcripts collagen type III and type I, TGFßi (Table 1) as well as lysyl oxidase, MMP-1, PAI-1 and tenascin (data not shown) showed none significant differences between the fibrosis control group and the treatment groups with the procollagen αι (III) propeptide applications.

More precise dose-effect studies of the in wVo effects after PIIICP4.1 infusion: More precise studies of the in wVo effects of the recombinantly produced PIIICP4.1 protein (production described in Burchardt, 1998, and in the patent applications: An immunoassay for procollagen-III -C-terminal propeptides (WO09924835A2 and EP00988964A1); recovery and renaturation described above in this patent application as an example) on the formation of an experimentally induced liver fibrosis were carried out in the mouse CCI ₄ model.

Groups of 8 mice were treated with CCI (fibrosis control) or additionally with PIIICP4.1 protein solutions with concentrations of 50 μg / ml PIIICP4.1, 150 μg / ml and 500 μg / ml over a period of 14 days , In addition, one group was treated with buffer control solution only (intact control).

At the end of the treatment, the PIIICP dissolved in the application solutions was not degraded in all cases. This was shown with an SDS-PAGE gel analysis.

The total collagen area in liver sections, the GIDH activity in the serum and the mRNA concentrations of selected transcripts were measured morphometrically in all animals using the TaqMan analysis.

Overall, the protein was well tolerated by the animals in all concentrations used during the treatment period. There were no pathological abnormalities, and there were no frequent deaths.

All animals with a CCI application showed in comparison to the untreated Control animals had a lower body weight at the end of the experiment (see Table 2). In comparison to the fibrosis control, the PIIICP groups showed no significant differences. The tendency was that the animals with PIIICP application weighed somewhat more, regardless of the concentration obtained.

The relative liver weight was above the values of the untreated intact control in all CCI-treated groups. There were no significant differences when comparing the individual treatment groups with the fibrosis control (Table 2).

When considering the morphometrically determined total collagen deposition, which was carried out via an automated evaluation of the Sirius red-stained areas, significant differences between the individual groups of animals were shown (FIG. 4A). After chronic treatment with i.p.-applied renatured PIIICP4.1 solution, a reduced total collagen area was measured in all treatment groups. In the treatment group with the lowest PIIICP dose (50 μg / ml), the total collagen area was significantly reduced by 32% compared to the fibrosis control (p <0.05). After treatment with 150 μg / ml PIIICP4.1, the significant reduction compared to the fibrosis control was 44% (p <0.02). The treatment group with the highest protein concentration (500 μg / ml PIIICP4.1) showed significant fluctuations in the total collagen area measured within the treatment group and a reduction in the total collagen area by 31%. In comparison to the fibrosis control, the level of significance (p <0.05) was not reached.

The clinical parameter of GIDH activity as a measure of cell damage determined in parallel in the serum was significantly reduced in the groups additionally treated with PIIICP4.1 protein solution compared to the fibrosis control (Table 2). After chronic treatment with 50 μg / ml PIIICP4.1, GIDH serum activity showed 68% of the activity of the fibrosis control, after 150 μg / ml PIIICP4.1 in 60% of the fibrosis control and after 500 μg / ml PIIICP4.1 in 67% the fibrosis control. Only after treatment with 150 μg / ml PIIICP4.1 was the reduction compared to the fibrosis control significant (P <0.05). In the other two treatment groups, the significance level was not reached due to the fluctuations in the results from animal to animal.

After normalization to HPRT, all transcripts (type I collagen, type III collagen, tenascin, PAI-1, MMP-1, lysyloxidase and CTGF) showed differences between the intact control animals (without CCI ₄ application) and the fibrosis controls. All of the transcripts examined were genes which are involved in the synthesis of the extracellular matrix. As a result, the mRNA concentrations in the fibrosis controls were on average 6-10 times higher than in the intact controls.

Significant differences between the animals treated with PIIICP4.1 in addition to the CCI ₄ damage and the fibrosis control were measured in the transcripts examined for the CTGF transcript (FIG. 4B). At 50 μg / ml PIIICP4.1 the difference was +0.74 Δct units (p <0.04), at 150 μg / ml at +0.75 Δct units (p <0.05) and 500 μg / ml PIIICP4.1 at +1.41 Δct units (p <0.02).

Example 5: Immunohistochemical detection of PIIICP on liver sections

material and methods

Model of liver fibrosis in the biliary ligated rat: In fasted female Sprague-Dawley rats, the main biliary duct was isolated after barbiturate anesthesia after medial opening of the upper abdomen. The bile duct system was closed using an inserted catheter by applying approximately 0.1 ml of Ethibloc per animal. The bile duct was then ligated distally and proximally and severed. Intact control animals were also operated on, but the bile duct system was not blocked. The PIIICP4.1 application (preparation described in Burchardt, 1998, and in the patent applications: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1); recovery and renaturation described above in this patent application as Examples 1 and 2) was carried out by means of a permanent venous infusion using an implanted indwelling catheter.

The femoral vein was implanted parallel to the bile duct occlusion. For this purpose, the skin in the area of the right groin was opened and the vena femoralis isolated atraumatically and fixed by two ligatures. After the incision, a venous catheter was inserted into the heart and fixed. The catheter was then guided subcutaneously to the neck using a trocar and the surgical wound was closed with a skin suture. The venous catheter was connected via a collar to a rotation adapter connected to an infusion pump by a protection spiral. To prevent infection, 0.1 ml of Tardomyocel was applied subcutaneously postoperatively.

The start of the PIIICP4.1 infusion with a protein concentration of 300 μg / ml took place 24 hours after the operation. Buffer control infusions were given to intact and fibrosis controls. The infusion was carried out at a flow rate of 0.2 ml per hour over a period of seven days. The infusion solution was cooled (approx. 7 ° C) during the entire test period.

The rats spent the duration of the experiment in a round cage with free access to standard food and water. The body weight of the animals was measured at the beginning and at the end. At the end of the experiment, the liver was portioned for the subsequent examinations and immediately snap frozen in liquid nitrogen and stored at -80 ° C. until use. Tissue fixation for immunohistochemistry: The tissues for immunohistochemical examinations were fixed in 3.6% (v / v) formaldehyde solution for 24 h. After washing out with distilled water, the water was removed in an ascending row with ethanol and the tissues embedded in 52 ° C. paraffin.

Immunohistochemistry: 5 μm paraffin sections were dewaxed. For this purpose, the sections were suksessive with xylene, with pure ethanol and then over a concentration series of an ethanol-water mixture (90% (v / v), 80% (v / v), 70% (v / v)) in pure water transferred. After five minutes of treatment with 3.6% (v / v) H2O2, the mixture was washed with distilled H ₂ 0. It was then washed with PBS for five minutes. Blocking was carried out for 20 minutes in a solution of 5% (w / v) skim milk powder and 1% (w / v) BSA in PBS. After washing twice for three min each of PBS, the primary antibody (mouse anti-PIIICP antibody 48D19) was added in a concentration of 4 μg / ml in PBS and incubated for one hour at room temperature. After a further two washing steps, the ExtrAvidin Peroxidase Staining Kit (Sigma Aldrich) was used. The sections were incubated with biotinylated anti-mouse IgG's at a 1:15 dilution in PBS with 1% (w / v) BSA for 20 min at room temperature. After two washing steps, a 1:15 dilution of the ExtrAvidin peroxidase was treated at room temperature for 20 min. This dilution was made with PBS with the addition of 1% (w / v) BSA. Following two washes, the tissue sections were developed using the DAB system (1 tablet per 5 ml of water) (Sigma Aldrich) for 10 minutes. Remains of the staining solution were distilled with H ₂ 0. away. The counter-staining of the cell nuclei was carried out with hemalum. The tissue sections were placed for 1 min in an acidic Mayer haemun solution (1: 4 dilution with dist. H ₂ 0), the dye was then washed with dist. H ₂ 0 washed out. The sections were blued by treating them with tap water for five minutes. After a washing step in H ₂ 0 dist. the cuts were drained. With the aid of aqueous ethanol solutions of increasing concentration, the water was removed (70% (v / v), 80% (v / v), 90% (v / v), finally three times pure ethanol). The dewatered tissue sections were washed three times in xylene for 5 minutes each and covered with the Leica CV Mount synthetic resin.

Results

FIG. 5 shows immunohistochemical investigations with the anti-PIIICP antibody 48D19 on representative sections from rat liver. Liver fibrosis was induced by bile duct ligation. A PIMCP protein or buffer control solution was then infused through a permanent catheter for seven days.

In the intact control, i.e. Sham surgery and infusion of a buffer control solution showed hardly any immunohistochemical staining with the PIIICP antibody (FIG. 5A).

The fibrosis control showed severe damage to the liver (FIG. 5C). There was a massive proliferation of the bile ducts in the damaged livers. In the area of the sinusoids, individual cells that had proven positive on smooth muscle cell actin (α-SMA) were strongly stained. They were transformed hepatic star cells that were selectively stained. The conductivity of the sinusoids was clearly limited. Staining of the hepatocytes could not be determined.

Sections from animals with bile duct ligation and PlllCP infusion solution also showed the strong intracellular staining of hepatic star cells (FIG. 5B). In contrast to the fibrosis control, these cells showed additional intracellular staining in the form of granules. These intracellular stains were also found in hepatocytes with access to the sinusoids. As far as can be seen, there was no specific immune response in the cell nucleus. The extent of the fibrosis was less compared to the fibrosis control. literature

Afdhal NH, Keaveny AP, Cohen SB, Nunes DP, Maldonado N, O'Brien M, Stone

PJ (1997) Urinar assays for desmosine and hydroxylysylpyridinoline in the detection of cirrhosis. J Hepatol. 27: 993-1002.

Ala-Kokko L, Kontusaari S, Baldwin CL, Kuivaniemi H, Prockop DJ (1989) Structure of cDNA clones coding for the entire preproalphal (III) chain of human type III collagen. Differences from type I procollagen structure and conservation of codon preferences. Biochemical Journal 260: 509-516

Amenta PS, Gay S, Vaheri A, Martinez-Hernandes A (1986) The extracellular matrix is an integrated unit: Ultrastructural localization of collagen types I, III, IV, VI, fibronectin, and laminin in human term placenta. Collagen Related Research 6: 125-152

Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D

(1997) Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 26:

643-649.

Brooks DG, Steinert C, Gerl M, Knolle J, Neubauer HP, Günzler V (1993) A radioimmunoassay for the N-terminal propeptide of rat procollagen type III. Matrix 13: 381-387

Bulleid NJ, Wilson R, Lees JF (1996) Type-Ill procollagen assembly in semi-intact cells: Chain association, nucleation and triple-helix folding do not require formation of inter-chain disulfide bonds but triple-helix nucleation does require hydroxylation. Biochemical Journal 317: 195-202 Burchardt ER et al. Monocional antibody and assay for detecting PIIINP. WO09961477A2

Burchardt ER et al. An immunoassay for procollagen-III-C-terminal propeptide. WO09924835A2 and EP00988964A1

Burchardt ER, Schröder W, Heke M, Kohlmeyer J, Neumann R, Kroll W (1997) Expression cloning of C-terminal procollagen (III) propeptide and its use in a novel serum assay to monitor liver fibrogenesis. Hepatology 26: 487A

Burchardt ER, Heke M, Kauschke SG, Harjes P, Kohlmeyer J, Kroll W, Schauer M, Schroeder W, Voelker M (1998) Epitope-specific monocional antibodies against human C-terminal procollagen α-ι (lll) -propeptide. Matrix Biology 17: 673-677

Byers PH, Duvic M, Atkinson M, Robinow M, Smith LT, Krane SM, Greally MT, Ludman M, Matalon R, Pauker S, Quanbeck D, Schwarze U (1997) Ehiers-Danlos syndrome type VI IA and VI IB result from splice-junction mutations or genomic deletions that involve exon 6 in the COL1A1 and COL1A2 genes of type I collagen. American Journal of Medical Genetics 72: 94-105

Davis BH, Madri JA (1987) Type I and type III procollagen peptides during hepatic fibrogenesis. An immunohistochemical and ELISA serum study in the CCI ₄ rat model. American Journal of Physiology 126: 137-147

Dion AS, Myers JC (1987) COOH-terminal propeptides of the major human procollagens. Journal of Molecular Biology 193: 127-143

Fessler Ll, Fessler JH (1979) Characterization of type III procollagen from chick embryo blood vessels. Journal of Biological Chemistry 254: 233-239 Fouser L, Sage EH, Clark J, Bornstein P (1991) Feedback regulation of collagen gene expression: A trojan horse approach. Proceedings of the National Academy of Sciences USA 88: 10158-10162

Gerling B, Becker M. Waldschmidt J, Rehmann M, Schuppan D (1996) Elevated serum amino terminal procollagen type-III-peptide parallels collagen accumulation in rats with secondary biliary fibrosis. Hepatology 25: 79-84

Harlow and Lane (1988) Antibodies, A Laboratory Manual. CSH Press, Cold Spring Harbor, NY, USA.

Janitz M, Heiser V, Boettcher U, Landt O, Lauster R (1998) Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1. Journal of Molecular Medicine 76: 141-146

Jensen LT, Henriksen JH, Risteli J, Olesen HP, Nielsen MD, Lorenzen I (1989) Fate of circulating amino-terminal propeptide of type III procollagen in conscious pigs. American Journal of Physiology 265: R139-R145

Katayama K, Seyer JM, Raghow R, Kang AH (1991) Regulation of extracellular matrix production by chemically synthesized subfragments of type I collagen carboxy propeptide. Biochemistry 30: 7097-7104

Katayama K, Armendariz-Borunda J, Raghow R, Kang AH, Seyer JM (1993) A pentapeptide from type I procollagen promotes K, Seyer JM, Raghow R extracellular matrix production. Journal of Biological Chemistry 268: 9941-9944

Kauschke SG, Knorr A, Heke M, Kohlmeyer J, Schauer M, Theiss G, Waehler R, Burchardt ER (1999) Two assays for measuring fibrosis: RT-PCR of collagen alphal type III is an early predictor of subsequent collagen deposition while a novel N-terminal procollagen (III) propeptide assay reflects manifest fibrosis in CCI ₄ -treated rats. Analytical Biochemistry 275: 131-140

Keene DR, Engvall E, Glanville RW (1988) Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network. Journal of Cell Biology 107: 1995-2006

Kessler E, Takahara K, Biniaminov L, Brusel M, Greenspan D (1996) Bone morphogenetic protein-1: The type I procollagen C-proteinase. Science 271: 360-362

Kühn K, Wiestner M, Krieg T, Müller PK (1982) Structure and function of the amino terminal propeptide of type I and III collagen. Connective Tissue Research 10: 43-50

Lee ST, Kessler E, Greenspan DS (1990) Analysis of site-directed mutations in human pro-alpha2 (l) collagen which block cleavage by the C-proteinase. Journal of Biological Chemistry 265: 21992-21996

Lees JF, Bulleid NJ (1994) The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen. Journal of Biological Chemistry 269: 24354-24360.

Loidl HR, Brinker JM, May M, Pihlajaniemi T, Morrow S, Rosenbloom J, Myers C (1984) Molecular cloning and carboxyl-propeptide analysis of human type III procollagen. Nucleic Acids Research 12: 9383-9384

Ma X, Svegliati-Baroni G, Poniachik J, Baraona E, Lieber CS (1997) Collagen synthesis by Iiver stellate cells is released from its normal feedback regulation by acetaldehyde- induced modification of the carboxyl-terminal propeptide of procollagen. Alcoholism: Clinical and Experimental Research 21: 1204-1211 Melkko J, Hellevik T, Risteli L, Risteli J, Smedsrod B (1994) Clearance of NH ₂ -terminal propeptides of types I and III procollagen is a physiological function of the scavanger receptor in liver endothelial cells. Journal of Experimental Medicine 179: 405-412.

Paglia L, Wilczek J, de Leon LD, Martin GR, Hörlein D, Müller P (1979) Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides. Biochemistry 18: 5030-5034.

Schuppan D (1991) Connective tissue polypeptides in serum as parameters to monitor antifibrotic treatment in hepatic fibrogenesis. Journal of Hepatology 13: S17-S25.

Smedsrod B (1988) Aminoterminal propeptide of type III procollagen is cleared from the circulation by receptor-mediated endocytosis in liver endothelial cells. Collagen Related Research 8: 375-388

Wu CH, Donovan CB, Wu GY (1986) Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides, Journal of Biological Chemistry 261: 10482-10484

Zafarullah K, Brown EM, Kuivaniemi H, Tromp G, Sieron AL, Fertala A, Prockop DJ (1997) Synthesis and conformational properties of a recombinant C-propeptide of human type III procollagen. Matrix Biology 16: 201-209

Claims

claims

1. Containing drugs

(a) a (poly) peptide comprising the N-terminal procollagen (III) propeptide and / or the C-terminal procollagen (III) propeptide or

(b) a fragment or derivative thereof having essentially the antifibrotic activity of the (poly) peptide (a) and / or

(c) a peptide comprising the recognition sequence of type III procollagen C protease and / or a peptide comprising the recognition sequence of type III procollagen N proteinase in combination with a pharmaceutically acceptable carrier or diluent.

2. Medicament according to claim 1, wherein the (poly) peptide or fragment or derivative and / or the peptide is derived from human procollagen (III).

3. Medicament according to claim 1 or 2, wherein the (poly) peptide comprising the recognition sequence comprises 10 to 15 amino acids N-terminal from the cleavage site.

4. Medicament according to one of claims 1 to 3, wherein the (poly) peptide comprising the recognition sequence comprises 10 to 15 amino acids C-terminal from the cleavage site.

5. Medicament according to one of claims 1 to 4, wherein the (poly) peptide is a fusion protein.

6. Medicament according to claim 5, wherein the (poly) peptide contains a His tag.

7. Medicament according to claim 6, wherein the His tag is 6 His tag and is attached N-terminal.

8. Use of a (poly) peptide or fragment or derivative thereof, as defined in one of the preceding claims, for the manufacture of a medicament or medical device for the treatment or prevention of fibrotic diseases.

9. Use according to claim 8, wherein the fibrotic diseases are selected from systemic or local scleroderma, liver fibrosis of different origins, such as alcoholic liver cirrhosis, biliary cirrhosis, hepatitis viral or other genesis, the so-called "veno-occlusive disease", idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, acute pulmonary fibrosis, the "acute respiratory distress syndrome" (ARDS), perimuscular fibrosis, pericentral fibrosis, dermatofibroma, renal fibrosis, diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scarring, arthroplasty, arthibiosis, arterial fibrosis, arthibrosis, articular fibroids, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthroplasty, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthropathy, arthroplasty cystic fibrosis, muscular fibrosis, Duchenne muscular dystrophy, esophageal strictures, Ormond's disease, Crohn's disease, ulcerative colitis, atherosclerotic changes, pulmonary hypertension, angiopathies of the arteries and veins and aneurysms of the large vessels or are caused or initiated by surgical scar revisions, plastic surgery, glaucoma, cataract fibrosis, scarring of the cornea, the so-called "graft versus host disease", surgical procedures on tendons, nerve trapping syndromes, Dupuytren's contracture, adhesions due to gynecological infectious disease, pelvic adhesions , peridural fibrosis, diseases of the thyroid or parathyroid glands, due to metastatic bone infestation, multiple myeloma or restenosis.

10. Process for the preparation of renatured N-terminal procollagen (III) propeptide and / or C-terminal procollagen (III) propeptide, wherein one

(a) dissolving the inclusion body (s) containing the propeptide (s) recombinantly prepared by conventional methods with E. coli in a 0.5 to 8 M denaturing buffer;

(b) the buffer according to (a) is added dropwise to a limit dilution buffer which buffers around a neutral pH, contains L-arginine in a final concentration of 200 to 1000 mM and a disulfide bridge-reducing coupled redox system until a volume ratio of the denaturing buffer to limit dilution buffer reached at most 1: 3;

(c) dialyzing the buffer mixture according to (b) for at least 2 hours against a physiological buffer containing L-arginine in a final concentration of 50 to 200 mM and a disulfide bridge-reducing coupled redox system;

(d) dialyzing the buffer mixture according to (c) for at least 2 hours against a physiological buffer containing a disulfide bridge-reducing coupled redox system; and

(e) dialyzing the buffer mixture according to (d) for at least 2 hours against a physiological buffer.

11. The method according to claim 10, wherein a chelator is added to the buffer in at least one of steps (b) to (d).

12. The method of claim 10 or 11, wherein the redox system reduces glutathione - glutathione is oxidized.

13. The method according to any one of claims 10 to 12, wherein a further protease inhibitor is added to the buffer in at least one of steps (b) to (d).

14. The method according to any one of claims 10 to 13, wherein in one of steps (c) and / or (d) a salt is added to the buffer in a final concentration of about 10 mM.

15. The method according to any one of claims 10 to 14, wherein the denaturing buffer in step (a) contains urea in a final concentration of 0.5 to 8 M.

16. The method according to any one of claims 10 to 15, wherein Trizma base is used as buffering agent.

17. The method according to claim 10, wherein the dialysis steps are carried out at about 4 ° C.

18. The method according to any one of claims 10 to 17, wherein in steps (c) to (e) dialyzed against at least 100 times the dialysis buffer volume.

19. A process for the preparation of a medicament, wherein the renatured N-terminal procollagen (III) propeptide and / or C-terminal procollagen (III) propeptide prepared by the process according to one of claims 10 to 18 is concentrated or lyophilized by customary methods and mixed with a pharmaceutically acceptable carrier or with a pharmaceutically acceptable diluent.

20. Use of an N-terminal procollagen (III) propeptide and / or a C-terminal procollagen (III) propeptide, which was prepared by the method according to any one of claims 10 to 18, for the treatment or prevention of fibrotic diseases.

21. Use according to claim 20, wherein the fibrotic diseases are selected from systemic or local scleroderma, liver fibrosis of different origins, such as alcoholic cirrhosis of the liver, biliary cirrhosis, hepatitis viral or other genesis, the so-called "veno-occlusive disease", idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, acute pulmonary fibrosis, "acute respiratory distress syndrome" (ARDS), perimuscular fibrosis , pericentral fibrosis, dermatofibroma, renal fibrosis, diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scarring, joint adhesions, arthrosis, myelofibrosis, scarring of the cornea, cystic fibrosis, muscular fibrosis, morbus, morbus , Ulcerative colitis, atherosclerotic changes, pulmonary hypertension, angiopathies of the arteries and veins and aneurysms of the large vessels or are caused or initiated by surgical scar revisions, plastic surgery, glaucoma, cataract fibrosis, scarring gene of the cornea, the so-called "graft versus host disease", surgical interventions on tendons, nerve pinching syndromes, Dupuytren's contracture, adhesions due to gynecological interventions, pelvic adhesions, infertility, peridural fibrosis, diseases of the thyroid gland or parathyroid gland, due to metastatic disorders multiple myeloma or restenosis.