DE4130221C2 - Use of proteolytic enzymes for the manufacture of a medicament for the treatment of autoimmune diseases, in whose formation proteins with a C¶H¶2 domain are involved - Google Patents

Description

The present invention relates to the use of at least one proteolytic enzyme for the manufacture of a medicament for the treatment of autoimmune diseases, the formation 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 immunodeficiency 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 restricted to immunoglobulins, but the C _H 2 structure is also very similarly present, for example, in T cell receptor proteins, cell adhesion molecules and the proteins of class I and II 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 various 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

References 1-13:

• 1) G. S. Hahn, Three-Dimensional Structural Analysis of Human Immunoglobulin, G: Localization of Fc Actrive Sites, in: Cellular and Humoral Defense against Disease, 6th ann. Meet. Long Beach, Clin. Physiol Biochem., S. Karger AG, Basel (1983), pp. 117-144.
• 2) T.A. Springer; Adhesion receptors of the immune system, Nature 346 (1990), pp. 425-434.
• 3) D.E. Staunton; S.D. Marlin and C. Stratowa et al., Primary Structure of ICAM-1 Demonstrates Interaction between Members of the Immunoglobulin and Integrin Supergene Families, Cell 52 (1988), pp. 925-933.
• 4) A.F. Williams and A.N. Barclay; The Immunoglobulin Superfamily domains for Cell Surface Recognition, Ann.Rev.Immunol. 6 (1988), pp. 381-405.
• 5) D.L. Simmons; C. Walter; C. Power and R. Pigott; Molecular Cloning of CD31, A Putative Intercellular Adhesion Molecule Closely Related to Carcinoembryonic Antigen, J. Exp. Med. 171 (1990), pp. 2147-52.
• 6) D.P. Stites and A.I. Terr, Cardiac and Vascular Diseases, in: Basic and Clinical Immunology, 7th ed., Cape. 39, Appleton & Lange, East Norwalk (1991), Pp. 492-505.
• 7) D.P. Stites and A.I. Terr, Rheumatic Diseases, in: Basic and Clinical Immunology, 7th ed., Chap. 36, Appleton & Lange, East Norwalk (1991), pp. 438-463.  
• 8) D.P. Stites and A.I. Terr, Renal Diseases, in: Basic and Clinical Immunology, 7th ed., chap. 41, Appleton & Lange, East Norwalk (1991), pp. 526-538.
• 9) D.P. Stites and A.I. Terr, Neurologic Diseases, in: Basic and Clinical Immunology, 7th ed., Chap. 43, Appleton & Lange, East Norwalk (1991), pp. 552-563.
• 10) M.H. Wansbrough-Jones et al .; Complement activation and spleen function in erythema multiforme associated with Herpes simplex virus reactivation, J. Clin. Lab. Immunol. (1986) No. 21, pp. 83-85.
• 11) C.Y. Lin et al .; Nephrotic syndrome associated with Varicella infection, Pediartics 75 (1985) No. 6, p. 1127 ff.
• 12) H. Greiling and A.M. Gressner, Clinical Textbook Chemistry and Pathobiochemistry, 2nd ed., Schattauer, Stuttgart (1989), p. 1017.
• 13) P.J. Späth et al .; Complement levels and circulating immune complexes in a controlled, longitudinal, multicentre study on effects of intravenous immunoglobulin in adults with AIDS-related complex / WaLter-Reed 5, Vox Sang (1990) No. 59, pp. 51-58.

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

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

According to the invention, this object is achieved by a Medicament for the manufacture of which at least one proteolytic enzyme is used, being used as a proteolytic Enzyme papain and / or trypsin is used.

It was surprisingly 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.

Proteolytic enzymes suitable according to the invention are Papain and trypsin, being one 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 is a proteolytic enzyme that can be produced from pancreas using conventional methods.  

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

Those modulated by the proteolytic enzyme (s) Immunoglobulins are characterized in that their Binding ability for the complement component C1q is lowered. This applies to natural C1q-binding Immunoglobulins (subclasses IgG₁, IgG₂, IgG₃, IgM and partly IgA).

The changed C1q binding capacity is probably 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 bring about 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 papain are 40 to 100 mg are particularly suitable, 60 mg are whole particularly suitable. 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.

On a microtiter plate with 96 wells and higher Adsorption (available from Nunc) as a solid phase the F (ab ′) ₂ fragment of human immunoglobulin G adsorptively bound. For an adsorptive bond further Preventing molecules from entering the solid phase became the active one Plastic surface with a 0.1% bovine serum albumin solution then layered.

To the human F (ab ′) ₂ fragment fixed to the solid phase, that acts as an antigen then became a Rabbit antibody of class G against human IgG F (ab ′) ₂ bound, which then together form an immune complex. This immune complex was created using the various solutions proteolytic enzyme, the different Concentrations of enzyme contained different lengths exposed to continuous periods.

The change in the C1q binding capacity 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 2,000 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. In the case of symbols without an error bar, the standard deviation could no longer be represented graphically.

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 tests 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 the 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 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 case 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 use of proteolytic enzymes according to the invention explained above thus allows autoimmune diseases, in the development of which at least one protein with a C _H 2 domain is involved, to be treated effectively.

Claims (1)

Use of at least one proteolytic enzyme for the treatment of autoimmune diseases, the development of which involves proteins with a C _H 2 domain, characterized in that papain and / or trypsin is used as the proteolytic enzyme.