DE4130221A1 - Use of proteolytic enzymes e.g. papain or trypsin - Google Patents

Abstract

Diseases the origin of which involves proteins (A) contg. at least one CH2 domain are treated with a compsn. contg. at least one proteolytic enzyme (I). More specifically (I) is papain and/or trypsin, and (A) is a member of the immunoglobulin superfamily, esp. an immunoglobulin.

Description

The present invention relates to the use of at least one proteolytic enzyme for the manufacture of a medicament for the disease, the development of which involves at least one protein containing at least one C _H 2 domain.

The immune system is a biological functional unit with highly specific reactions on both humoral like at the cellular level. Disorders in this complex Systems are at the origin of numerous diseases causally involved. This is how defects lead to development the immune system to so-called immune deficiency diseases, caused by an increased susceptibility to infections and Tumors are marked, such as the Agammaglobulinemia. Excessive willingness to react can cause allergic reactions or autoimmune diseases trigger, and a disturbed immunological Monitoring function of the organism can Tumor formation and the spread of pathogenic Favor microorganisms in the body.

Antibodies are essential molecules for the humoral response. The heavy and light protein chains that form the antibody are each divided into different functional domains. The variable domain of the heavy chain V _{H is} involved in the binding of the antigen, and the subsequent domains C _H 1, C _H 2 and C _H 3 are responsible for the so-called effector functions of an antibody, such as the binding of complement proteins. The C _H 2 domain in particular is involved in the initial step of complement activation.

However, the presence of a C _H 2 domain is not limited to immunoglobulins, but the C _H 2 structure is also very similar, for example, in T cell receptor proteins, cell adhesion molecules and the class I and II proteins encoded in the main histocompatibility complex. Numerous proteins with a structure similar to the domain structure of the immunoglobulins are combined to form the proteins of the so-called immunoglobulin superfamily. An overview of proteins containing a C _H 2 structure is given in AF Williams and AN Barcley, "The Immunoglobulin Superfamily Domains for Cell Surface Recognition", Ann. Rev. Immunol., 1988, 6, 381-405.

In the following, the term “C _H 2 structure” or “C _H 2 domain” means the region of a protein molecule that has a structure similar to the C _H 2 domain of IgG. The term C2 domain or C2 set is also used in the literature for such a structure.

As explained above for the immunoglobulins, the C _H 2 structure also frequently has an effector function in the other members of the immunoglobulin superfamily, which can be involved in the development of a wide variety of clinical pictures. Table 1 shows a list of the C _H 2 structure of associated clinical pictures. This table lists clinical pictures in whose formation different proteins containing at least one C _H 2 structure are involved.

Table 1

Occurrence of C _H 2 -related protein structures in various clinical pictures

Table 2 lists the immunological reaction partners for a C _H 2 structure from various proteins and the pathoimmunological or physiological subsequent reactions which can be caused by the interaction of the C _H 2 structure with the immunological reaction partner.

Table 2

Immunological reaction partner of structures containing C _H 2

It is an object of the present invention to provide a medicament with which a disease in the development of which at least one protein containing at least one C _H 2 domain is involved can be effectively treated.

This object is achieved by a Medicament, for the manufacture of which at least one proteolytic enzyme is used.

It has surprisingly been found that proteolytic enzymes modulate the C _H 2 structure in proteins extremely selectively, so that the C _H 2 structure loses its effector function, but the other humoral or cellular functions are retained.

According to the invention, particularly suitable proteolytic enzymes are trypsin, papain, α-chymotrypsin and pancreatin. All papain and trypsin are particularly suitable, one being Combination of papain and trypsin turned out to be special proves effective.

Papain is a proteolytic enzyme that is derived from the Milky juice of the immature, fleshy fruits of the Melon tree Carica papaia according to usual methods can be produced.

Trypsin and α-chymotrypsin are proteolytic enzymes, which can be produced from pancreas using conventional methods.

Pancreatin is made from pig or bovine pancreas won.  

The proteolytic enzymes are particularly suitable for Treatment of a disease, the emergence of which at least a protein belonging to the immunoglobulin superfamily, is involved, and especially if an immunoglobulin is involved.

The immunoglobulins modulated by the proteolytic enzyme (s) are distinguished by the fact that their binding capacity for the complement component C1q is reduced. This applies to natural C1q-binding immunoglobulins (subclasses IgG ₁ , IgG ₂ , IgG ₃ , IgM and partly IgA).

The altered C1q binding capacity is presumably caused by a change in the conformation of the C _H 2 domain due to the action of the enzyme. The conformational change can either be caused directly by an enzymatically caused change in the C _H 2 domain, or the proteolytic enzymes cause a structural change in the regions adjacent to the C _H 2 domain, and these changes influence the conformation of the C _H 2- Domain.

For the manufacture of a medication the following amounts of enzyme per tablet are particularly suitable:

Trypsin is preferably in an amount of 10 to 30 mg, particularly preferably in an amount of 24 mg use. For α-chymotrypsin, 1 up to 10 mg, in particular 1 mg, used. For papain are 40 to 100 mg are particularly suitable, 60 mg are whole particularly suitable. For pancreatin there are 50 to 200 mg preferably suitable, 100 mg being particularly suitable are. Preferably the drug is the usual  Auxiliaries and carriers added. If necessary, that can Drug additional proteolytic enzymes contain.

The following examples illustrate the invention.

The F (ab ′) ₂ fragment of human immunoglobulin G was adsorptively bound to a microtiter plate with 96 wells and increased adsorption (available from Nunc) as a solid phase. To prevent adsorptive binding of other molecules to the solid phase, the active plastic surface was then covered with a 0.1% bovine serum albumin solution.

A rabbit antibody of class G against human IgG F (ab ′) ₂ , which then together form an immune complex, was then bound to the human F (ab ′) ₂ fragment fixed to the solid phase, which functions as an antigen. This immune complex was exposed to the various solutions with proteolytic enzyme containing different concentrations of enzyme for periods of different lengths.

The change in the C1q binding ability of the immune complex due to the enzyme action was determined by the quantitative determination of bound, human C1q. The bound C1q was detected with an anti-human C1q class G antibody from sheep, the sheep antibody being coupled to alkaline phosphatase, the conversion of p-nitrophenyl phosphate at 405 nm was determined photometrically. The quantification of the immune complex-bound amount of C1q was carried out using a calibration curve. In Figs. 1 to 4 some results of the test series are plotted.

Fig. 1 shows a result obtained with papain, in which the immune complex was incubated for 30 min at 37 ° C and various enzyme concentrations. The reference concentration of the C1q was 2000 ng / ml. Four parallel tests were carried out for each measured value. The mean values of the determined C1q concentration are shown as diamond-shaped symbols with the associated standard deviation. The standard deviation could no longer be represented graphically for symbols without error bars.

In FIG. 2, the values obtained for enzyme amounts between 10 and 100 ng / ml are plotted reciprocally to the representation in FIG. 1. This diagram shows the papain concentration that leads to halving the C1q binding capacity (half-effect concentration).

In FIGS. 3 and 4 the results obtained with trypsin in a manner analogous to Fig. 1 and 2.. The incubation time with trypsin was 120 min at 37 ° C. Each measured value represents the result of 4 parallel tests.

Table 3 contains a compilation of the Enzyme concentrations required to halve the C1q binding ability in the experiments described above were needed.  

Table 3

The minimum and maximum values found are in brackets specified.

As control attempts to ensure that the effect caused by trypsin or papain on the binding ability of immune complexes for C1q is not due to unspecific proteolytic degradation of proteins from the incubation batch, experiments were carried out in which the addition of the proteolytic enzymes to the incubation batch only after it had taken place C1q was bound to the immune complex. In these incubation approaches, no changes in the amount of C1q bound to the immune complex with enzyme amounts of the half-value concentration and the same incubation period were observed. This result clearly leads to the conclusion that the proteolytic enzymes used selectively modulate the binding domain for C1q, ie the C _H 2 domain, in such a way that their C1q binding capacity is reduced. The proteolytic enzymes are therefore particularly suitable for the treatment of inflammation reactions mediated with complement.

The modulation of the C _H 2 structure by proteolytic enzymes could also be observed for the membrane-bound proteins CD4 on T lymphocytes or monocytes and CD54 on monocytes. In the cases of the CD4 and CD54 molecules, the action of trypsin leads to a reduction in the receptor epitope density on the cells. Trypsin has a similar effect on the receptor protein CD31 of B cells.

The above-described use of proteolytic enzymes according to the invention thus makes it possible to effectively treat a wide spectrum of the most diverse diseases in whose formation at least one protein with a C _H 2 domain is involved, such as, for example, complement-mediated inflammatory reactions.

Claims (7)

1. Use of at least one proteolytic enzyme for the manufacture of a medicament for the treatment of a disease, the development of which involves at least one protein containing at least one C _H 2 domain. 2. Use according to claim 1, characterized in that as a proteolytic enzyme papain and / or trypsin is used. 3. Use according to claim 2, characterized in that Trypsin is used as the proteolytic enzyme. 4. Use according to claim 2, characterized in that papain is used as the proteolytic enzyme. 5. Use according to one of claims 1 to 4, characterized characterized that a protein of Immunoglobulin superfamily at the onset of the disease is involved. 6. Use according to claim 5, characterized in that an immunoglobulin at the onset of the disease is involved. 7. Use according to claim 6, characterized in that Complement-mediated inflammation can be treated.