Procollagen (III) Propeptides and Related Substances for Treating Fibrotic Diseases

Abstract

A medicament for treating or preventing fibrotic diseases contains an antifibrotic substance that is a (poly) peptide having antifibrotic activity and comprising at least one of an N-terminal procollagen (III) propeptide and a C-terminal procollagen (III) propeptide, or a fragment of the (poly) peptide defined having antifibrotic activity or a derivative of the (poly) peptide having antifibrotic activity. The antifibrotic substance is combined with a pharmaceutically tolerable carrier or dilutant.

Description

[0001] This application is a continuaton of International Application PCT / EP01 / 12663 with an international filing date of Oct. 31, 2001, not published in the English language under PCT Article 21(2), and now abandoned.BACKGROUND OF INVENTION[0002] This invention relates to the use of procollagen (III) propeptides and related substances for treating fibrotic diseases and 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.[0003] Collagen Biosynthesis.[0004] The collagens of types I and III are synthesized as prepropeptides and are extensively modified posttranslationally. Among the intracellular modifications are glycosylations, enzymatic hydroxylation rea...

AI Technical Summary

Benefits of technology

[0127] All primers and probes were so chosen, with respect to their localization on the gene and to the expected amplification product, that a doubling of the concentration of the product in each cycle was to be expected in the course of a TaqMan PCR reaction. These assumptions were checked by control experiments before and verified. The primers and the 6-FAM-labeled probes were all present at concentrations of 100 .mu.M. To prevent variability between the different incubations master mixes were used in all cases and every incubation was carried out at least in duplicate. Determinations of every single transcript were carried out for all samples on the same plate. In all experiments, control experiments without template or without previous reverse transcription were carried out. All work was performed on ice. The master mix contained 12.5 .mu.l of the TaqMan Universal Master Mix (Roche), 7.5 .mu.l of the primer-probe-mix (1 .mu.M with respect to each primer and 0.5 .mu.M probe in DNase / RNase-free water) as well as 3.75 .mu.l DNase / RNase-free water. Per determination, 2.5 .mu.l cDNA solution were pipetted into 96 well plates with optical lids and mixed with 22.5 .mu.l of the master mix. The plates were centrifuged for 1 minute at 500 g and 4.degree. C. The program of the TaqMan PR reaction encompassed a heating phase of 2 minutes at 50.degree. C., a 10 minute denaturing step at 95.degree. C. as well as 40 cycles with a denaturing step at 95.degree. C. for 15 seconds and a combined one minute annealing / expansion step at 60.degree. C. Within the cycles, the fluorescence of the liberated fluorescent probe was measured automatically at the time point of the denaturation step. The evaluation was carried out with the ABI PRISM Sequence Detection Software. The baseline was set at the mean of cycles 3 and 15, the threshold was 0.04.

Problems solved by technology

It has been observed, however, that the region around these cysteine residues is critical for the correct formation of intramolecular disulfide bridges because a Cys.fwdarw.Ser mutation in that region leads to impaired intramolecular disulfide bridge formation.

Consequently, PINP cannot be cleaved off.

The recombinant protein could only be produced in small quantities for analytical purposes, however.

For chronic in vivo applications, these proteins were less suitable because of the potential immunogenicity of the His tag and because the biological half-life of the recombinant proteins may be decreased for this reason.

Their solubility was too low for most therapeutic applications.

For chronic in vivo applications, these proteins were also less suitable because of the potential immunogenicity of the His tag and because the biological half-life of the recombinant proteins may be decreased for this reason.

The solubility of PIIINP in aqueous solutions was too low for most therapeutic applications.

In many of the aforementioned diseases, a successful therapy has not been established so far.

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