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...

Examples

example 2

Renaturation of recombinant collagen .alpha. 1(III) propeptides.

[0086] As an example, recombinant human PIIICP4.1 was renatured, the production of which was described in Burchardt, 1998, and in the patents: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1). Furthermore, recombinant human PIIINP 4.5.2 was renatured, the production of which was described in the patent: Monoclonal antibody and assay for detecting PIIINP (WO 99 / 61477A2). Finally, recombinant murine PIIINP, the production of which was described in Kauschke, 1999, was renatured using the method described below.

[0087] However, the method is not limited to the renaturation of these exemplary propeptides, but is suitable for the renaturation of other procollagen propeptides and similar compounds.

[0088] Method.

[0089] Used Buffers.

[0090] A) Dialysis Buffer.

[0091] 300 mM Trizma-Base, pH 7.4.

[0092] 400 mM L-Arginine.

[0093] 10 mM EDTA.

[0094] 0.4 mM Pefabloc SC.

[0095] 1 mM Glutathione reduce...

example 3

Measurement of the Elimination Kinetics of Renatured PIIICP in the Anaesthetized Rat.

[0107] Materials and Methods.

[0108] PIIICP-Plate-ELISA: The determination of PIIICP concentrations in biological samples was carried out with a sandwich ELISA assay. Two monoclonal anti-PIIICP antibodies were used (Burchardt, 1998).

[0109] As a catching antibody, antibody 48B14 (Burchardt, 1998) was immobilized on an ELISA plate at a concentration of 5 .mu.g / ml. After blocking free unspecific binding sites on the plate by incubation with a 3% (v / v) BSA solution, biological samples or buffered solutions with known PIIICP concentrations were added together with a known concentration of a FITC-labelled secondary antibody (48D19) (Burchardt, 1998) for 30 minutes to the immobilized secondary antibody. Remaining free PIIICP antigen and free secondary antibody were subsequently removed by washing steps and the amount of bound, labelled secondary antibody was determined. The aforementioned antibodies can be ...

example 4

Demonstration of the Biological Efficacy of PIIICP and PIIINP.

[0115] The biological efficacy of the compounds can be demonstrated in cell culture assays and in vivo. For example, after addition of the inhibitors to human cell lines, a drop in the concentration of free .alpha. 1(III) propeptide in the supernatant can be measured because the peptide is released by the enzymatic activity of PCP. To measure PIIICP concentrations in the supernatant a recently established assay can be used (Burchardt, 1997, and patent application: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1)).

[0116] In this patent the biological efficacy of PIIICP4.1 (production described in Burchardt, 1998, and in the patents: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1), purification and renaturation described above in this patent as an example); of PIIINP4.5.2 (production described in the patent: Monoclonal antibody and assay for ...

[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...

Examples

example 2

Renaturation of recombinant collagen .alpha. 1(III) propeptides.

[0086] As an example, recombinant human PIIICP4.1 was renatured, the production of which was described in Burchardt, 1998, and in the patents: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1). Furthermore, recombinant human PIIINP 4.5.2 was renatured, the production of which was described in the patent: Monoclonal antibody and assay for detecting PIIINP (WO 99 / 61477A2). Finally, recombinant murine PIIINP, the production of which was described in Kauschke, 1999, was renatured using the method described below.

[0087] However, the method is not limited to the renaturation of these exemplary propeptides, but is suitable for the renaturation of other procollagen propeptides and similar compounds.

[0088] Method.

[0089] Used Buffers.

[0090] A) Dialysis Buffer.

[0091] 300 mM Trizma-Base, pH 7.4.

[0092] 400 mM L-Arginine.

[0093] 10 mM EDTA.

[0094] 0.4 mM Pefabloc SC.

[0095] 1 mM Glutathione reduce...

example 3

Measurement of the Elimination Kinetics of Renatured PIIICP in the Anaesthetized Rat.

[0107] Materials and Methods.

[0108] PIIICP-Plate-ELISA: The determination of PIIICP concentrations in biological samples was carried out with a sandwich ELISA assay. Two monoclonal anti-PIIICP antibodies were used (Burchardt, 1998).

[0109] As a catching antibody, antibody 48B14 (Burchardt, 1998) was immobilized on an ELISA plate at a concentration of 5 .mu.g / ml. After blocking free unspecific binding sites on the plate by incubation with a 3% (v / v) BSA solution, biological samples or buffered solutions with known PIIICP concentrations were added together with a known concentration of a FITC-labelled secondary antibody (48D19) (Burchardt, 1998) for 30 minutes to the immobilized secondary antibody. Remaining free PIIICP antigen and free secondary antibody were subsequently removed by washing steps and the amount of bound, labelled secondary antibody was determined. The aforementioned antibodies can be ...

example 4

Demonstration of the Biological Efficacy of PIIICP and PIIINP.

[0115] The biological efficacy of the compounds can be demonstrated in cell culture assays and in vivo. For example, after addition of the inhibitors to human cell lines, a drop in the concentration of free .alpha. 1(III) propeptide in the supernatant can be measured because the peptide is released by the enzymatic activity of PCP. To measure PIIICP concentrations in the supernatant a recently established assay can be used (Burchardt, 1997, and patent application: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1)).

[0116] In this patent the biological efficacy of PIIICP4.1 (production described in Burchardt, 1998, and in the patents: An immunoassay for procollagen-III-C-terminal propeptide (WO 99 / 24835A2 and EP00988964A1), purification and renaturation described above in this patent as an example); of PIIINP4.5.2 (production described in the patent: Monoclonal antibody and assay for ...

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