EP0918537A1

EP0918537A1 - Use of proteolytic enzymes for influencing cytokines

Info

Publication number: EP0918537A1
Application number: EP98934985A
Authority: EP
Inventors: Gerhard Stauder; Karl Ransberger
Original assignee: Mucos Pharma GmbH and Co
Current assignee: Mucos Pharma GmbH and Co
Priority date: 1997-06-20
Filing date: 1998-06-19
Publication date: 1999-06-02
Also published as: WO1998058663A1; DE19726255C2; DE19726255A1

Abstract

The invention relates to the use of at least one proteolytic enzyme for influencing cytokines. The proteolytic enzymes of choice are trypsin, bromelain and papain. Rutoside may also be used.

Description

Influence of cytokines by proteolytic enzymes

The present invention relates to a method for influencing cytokines by proteolytic enzymes and the use of proteolytic enzymes for influencing cytokines.

The cytokines are important metabolic substances in triggering various disease states. Cytokines are small proteins with molecular weights of 8,000 to 30,000, with each cytokine having its own amino acid sequence and cell surface receptors. They are made from a variety of different cell types and act on almost every tissue and organ system. The names given to the various cytokines do not follow a logical system, but rather correspond to their biological properties. IL-1 and TNF (tumor necrosis factor) are the primary pro-inflammatory cytokines, and moreover, these two cytokines act in a synergistic manner in inducing inflammation, shock and death.

In the past 20 years, more than 100 different surface molecules have been described on leukocytes in human blood. Their identification, characterization and functional description has been accelerated in particular by the technique of producing monoclonal antibodies. Defined surface molecules are generally recorded in a so-called CD nomenclature ("cluster of differentiation").

Their function or their physiological ligand is known for some of the surface molecules. For the interleukin-2 receptor (CD2S) e.g. interleukin-2 is the natural ligand. Other cytokines are also ligands of the surface molecules and are therefore influenced by them.

It has also been described for various surface molecules of leukocytes and endothelial cells that their antigen density is regulated by immune mediators. The expression density of the inflammatory marker HLA-DR on monocytes and the adhesion molecule CD54 (ICAM-1) on endothelial cells.

Certain infections or cancers result in a shift in various subpopulations of lymphocytes, granulocytes and monocytes in homeostasis. Normally the organism is able to restore the distributional equilibria of the subpopulations in the process of recovery.

In chronic diseases, especially autoimmune diseases and the associated permanent burden on the immune system (e.g. due to immune complexes and in connection with chronic persistent viral infections such as AIDS), there is a shift in the course of the disease, which the organism can no longer compensate for, both the balance of the cell populations and also the expression density of some specific antigens. In general, one speaks of an uncontrolled activation of the immune system.

According to the current state of knowledge, the assumption is confirmed that the modulation of cell surface molecules of the immune system could represent a special field of "medication".

The present invention was therefore based on the technical problem of specifying a method for influencing cytokines.

This object is achieved by the invention specified in claim 1. Advantageous further developments are specified in the subclaims.

According to claim 1, the use of at least one proteolytic enzyme and optionally rutoside is provided for influencing cytokines. The immunoglobulins modulated by the proteolytic enzyme or enzymes are distinguished by the fact that their binding capacity for the complement component C1q is reduced. This applies to natural C1 q-binding immunoglobulins (subclasses IgG1, IgG2, IgG3, IgM and partly IgA).

The changed C1 q binding capacity is - without being bound by any theory - 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 enzyme-induced 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. Domain.

Treatment of neutrophils with proteolytic enzymes significantly reduced expression e.g. of the receptor III for the Fc region of IgG and apparently also influences the number of Fcγ-R-Il on the cell surface. The binding of IgG-covered erytrocytes to neutrophils is increased; their ability to perform IgG-mediated functions, e.g. increases the antibody-dependent cellular cytotoxicity and chemiluminescence induced by IgE. These reactions are completely dependent on Fcγ-R-Il.

In a preferred embodiment, trypsin, bromelain, papain and optionally rutin or a combination thereof is used as the proteolytic enzyme.

The enzymes used according to the invention can be isolated inexpensively from the following raw materials.

Bromelain is a proteolytically active enzyme from the pineapple juice and can also be isolated from ripe fruit. Papain is a proteolytic enzyme obtained from the milk sap of the immature, meaty fruits of the Carica Papaya melon tree. Pure papain is a crystalline polypeptide with an MG. from 23350 consisting of a chain of 212 amino acid residues with 4 disulfide bridges; Sequence and spatial structure are known. Papain is used in many different ways: due to its protein-splitting properties as "meat tenderizer" or "short salt", for clarifying beer, for bread and hard biscuit production, in leather preparation, in the textile industry for degassing silk and for preventing wool matting, in the tobacco industry for quality improvement, for the recovery of silver from used photographic material, furthermore in bacteriology for the extraction of peptone. In medicine, papain is already used to support enzymatic digestion, for enzymatic wound cleaning and as an additive to denture cleaning agents. For special purposes, papain preparations are also available bound to plastic polymers or agarose. Papain has also been used as a catalyst for the synthesis of oligopeptides.

Trypsin is a proteolytic enzyme that is also formed in the pancreas and is already used therapeutically in conjunction with other enzymes. It belongs to the serine proteinases. Crystalline trypsin has a MW of approx. 23300, is soluble in water but not in alcohol, has an optimum activity at pH 7-9 and cleaves peptide chains specifically on the carboxy side of the basic amino acid residues L-lysine and L-arginine. The spatial structure of the 223 amino acid trypsin is known.

A particularly good effectiveness is shown when using a combination of the enzymes bromelain, papain and / or trypsin. In addition to the remarkable and unexpected effect of these enzymes, the combined use of the enzymes mentioned has the further advantage that even with long-term use there are no harmful side effects.

Rutoside can also be added to the medication. Rutoside is a glycoside that belongs to the flavonoids. The combined use of 20 to 100 mg bromelain, 40 to 120 mg papain and 10 to 50 mg trypsin per dose unit is particularly effective.

A combination of 90 mg bromelain, 120 mg papain and 100 mg rutoside x 3H ₂ O is very particularly preferred.

In another preferred embodiment, a combination of 48 mg trypsin, 90 mg bromelain and 100 mg rutoside x 3H ₂ 0 is used.

In a further preferred embodiment, 10 to 100 mg, particularly preferably 100 mg of rutoside x 3 H ₂ 0 are used per dose unit.

The medicament can furthermore contain all customary auxiliaries and / or carriers.

Auxiliaries and carriers include e.g. Lactose, magnesium stearate, stearic acid, talc, methacrylic acid, copolymer type A, Shellack, Makrogol 6000, di-butyl phthalate, vanillin, titanium dioxide, white clay, polyindone, yellow wax and camauba wax.

The invention further relates to the additional use of α ₂ -Macroglobulin (α ₂ -M). α ₂ -M is one of the most important inhibitors of proteinases. ₂ -M reacts with a variety of endopeptidases. The reaction of α ₂ -M with the respective proteinase usually takes place in such a way that α ₂ -M is subject to a change in conformation after it has come into contact with the proteinase and then holds it in the molecule. The enzyme inhibition is based on steric hindrance, although the active center of the enzyme remains functional. By influencing proteinase, α ₂ -M contributes to immunomodulation by influencing cytokines. The invention further relates to a method for influencing cytokines, the cytokines being brought into contact with one or more proteolytic enzymes and optionally rutoside.

The following examples are intended to illustrate the invention in detail.

example 1

material and methods

1. Material

The RPMI 1640 cell culture medium was used with the following additives: NaHC03 ⁽ biochrom, 2 g / 1); L-glutamine (biochrom, 2 mM), Na pyruvate (biochrom,

1mM), NaN ₃ (Sigma, 0.01%).

If the cells were stimulated, either phythaemagglutinin M (biochrome, 5 μg / ml) or γ-interferon (100 U / ml) was used. The cells were stimulated over 1-3 days with the addition of 10% fetal calf serum (biochrom).

Freshly isolated, human, peripheral, mononuclear blood cells (citrate blood) were used as targets for the enzymes to be examined. After the usual isolation using Ficoll, the cells were washed several times and used fresh in the experiments. Neutrophil granulocytes were also isolated from fresh citrate blood. Here, the lymphocytes / monocytes were separated using a 2-stage Ficoll gradient.

2. Monoclonal antibodies used

Monoclonal antibodies were used for the specific recognition of the surface structures of the leukocytes. These recognize on the corresponding each have a defined epitope, which occurs only once in the structure of the antigens examined by us. Overview 1 shows the surface markers examined, the monoclonal antibodies and the analyzed target cells.

Overview 1: Analyzed surface markers, monoclonal antibodies and target cells used in enzyme treatment

¹ phycoerythrin, red fluorescence;

² fluorescein isothiocyanate, green fluorescence;

³ Becton Dickinson, Heidelberg;

⁴ Ortho Diagnostics

3. Incubation conditions

The freshly isolated and prepared cells were incubated with the enzymes bromelain, papain and trypsin (pharmaceutical ingredients from Mucos) with the concentrations given in the legends of the tables and figures. When the three enzymes were mixed, the mixing ratio corresponded to 22.7: 15.5: 11.9 (bromelain: papain: trypsin, based on 40 μg / ml, “BPT”). Three enzyme concentrations (40, 10, 2.5 μg / ml) were examined. The incubation took place in serum-free medium at 37 ° C.

The proteases were set up immediately before the incubations. 0.01% sodium acid was contained in the cell culture media. This addition prevents the cells from during the incubation process or the washing process. would express receptor molecules again. After washing out the cell culture medium, the cells can be reactivated (not demonstrated).

After appropriate incubation of the cells with the respective enzymes, washing out of the enzymes and labeling with monoclonal antibodies (according to the manufacturers), the analysis of the surface markers was carried out immediately.

4. Analytical flow cytometry

All studies on the modulation of cell surface molecules were carried out by means of analytical flow cytometry (FACSCAN, Becton Dickinson, Heidelberg) using device-specific software (Lysis I). With appropriate setting of the device and the carrying of references, i.e. Cells that were treated without enzymes but in the same procedure were measured.

A corresponding fluorescence-conjugated isotype control was carried out for each subclass of the monoclonal antibodies. This is mouse immunoglobulin, with which the capacity of the non-specific binding of the target cells is determined by flow cytometry.

10,000 cells were measured per histogram. The respective cell population was separated with a so-called "electronic gate", in which there were at least 3 500 cells.

5. Presentation of the results

The evaluation and analysis of the data was carried out independently of the measurement process on the flow cytometer using device-specific software. The histograms of the controls, ie the untreated cell samples, were compared with the histograms of the enzyme-treated cells. The raw representation comprises an optically impressive, but relatively confusing histogram, in which various individual measurements are shown superimposed on one another. Here, in particular, the effect of the enzymes can be made optically transparent compared to the reference. For these investigations, it is not the percentage of a subpopulation of the total leukocyte population that is the measured variable, but rather the relative receptor density, which is shown as the fluorescence intensity.

From these histograms, which are exemplary and have an illustrative character, data can be derived which reflect the relative fluorescence intensity of the individual measurement. This is a measure of the relative receptor or surface molecule density in a measured cell population.

The bar graphs show the media of relative fluorescence in a logarithmic representation. Compared to the reference, the reduction in the density of the respective surface molecule as a function of the enzyme concentration can be represented.

The tables contain the data from independent experiments. The donor numbers are only valid for the respective table and cannot be transferred to other tables. If, for example, a value of 40% is given in the table, this means that in this antigen 40% of all surface molecules are changed by the enzyme in such a way that the specific monoclonal antibody no longer recognizes its epitope. If no reduction is observed, the value "0" appears in the tables. The percentage given thus expresses the enzyme performance against the individual antigens. Values up to 20% are not considered relevant in individual cases.

To evaluate the effect of the enzymes on the individual antigens, it is favorable to choose a suitable comparison scale. With a few exceptions, all tests were carried out under standardized incubation conditions. carried out. This means that the half-effect concentration known from previous research reports can be given for these experimental conditions.

To calculate the half-effect concentration, the data were evaluated using non-linear regression. The median of the fluorescence intensity versus the enzyme concentration used and the reference are correlated with one another, and from this the amount of enzyme can be calculated which leads to a 50% reduction in the relative fluorescence intensity or in the structure of the changed receptor density.

Figure I: CD2 modulation by proteases; The median of the relative fluorescence intensity is given; Target cells: lymphocytes. The positive control (reference) are untreated cells that were incubated with the monoclonal antibody specific for CD2. The negative controls are untreated cells incubated with the antibody isotype. The incubation time with the enzymes was 60 min. The data from donor 1 (see Tab. 1) are shown as the mean of double determinations and standard deviation.

Figure 2: CD4 (epitope Leu3a) modulation by proteases; The median of the relative fluorescence intensity is given; Target cells: lymphocytes. The positive control (reference) are untreated cells that were incubated with the monoclonal antibody specific for CD4. The negative controls are untreated cells incubated with the antibody isotype. The incubation time with the enzymes was 60 min. The data from donor 2 (see Tab. 2) are shown as the mean of double determinations and standard deviation.

Figure 3: CD4 (Epitope OKT4 and Leu3a) modulation by trypsin; The median of the relative fluorescence intensity is given; Target cells: lymphocytes. The positive control (reference) are untreated cells that were incubated with the monoclonal antibody specific for CD4. The negative controls are untreated cells incubated with the antibody isotype. The incubation on time with the enzymes was 60 min. The data from a donor (see Table 3) are shown as the mean of double determinations and standard deviation.

Figure 4: CD25 modulation by proteases; The median of the relative fluorescence intensity is given; Target cells: PHA blasts. The positive control (reference) are untreated cells that were incubated with the monoclonal antibody specific for CD25. The negative controls are untreated cells incubated with the antibody isotype. The incubation time with the enzymes was 60 min. The data from donor 1 are shown as the mean of duplicate determinations and standard deviation.

Figure 5: Reduction of the Leu3a antigen density by trypsin. Freshly isolated human peripheral, mononuclear blood cells were incubated with trypsin in the serum-free medium, then washed and labeled with the monoclonal antibody anti-Leu3a. The reduction in the relative fluorescence density of CD4 in the lymphocyte population is the measure of enzyme activity. The half-effect concentration of trypsin compared to the epitope Leu3a of CD4 is calculated using the fitted curve.

Results

Table 1: CD2 modulation by proteases; Target cells: lymphocytes. The percentage reduction in the median of the relative fluorescence intensity, which is a measure of the enzyme performance, is given. The incubation time with the enzymes was 60 min. The results of three experiments with cells from three different donors are shown.

Enzyme No. 40 μg / ml 10 μg / ml 2.5 μg / ml

Bromelain 1 11, 1 17.9 17.4

2 6.1 12.2 14.3

3 0 0 0 Papain 1 22.4 21, 4 34.7 2 5.4 9.5 17.0 3 0 0 0

Trypsin 1 75.7 73.6 73.0 2 83.7 21, 1 0.7 3 96.3 85.4 50.9

BPT 1 71, 9 54.7 38.2 2 74.1 8.8 18.4 3 94.8 72.6 25.2

Table 2: CD4 (epitope Leu3a) modulation by proteases; Target cells: lymphocytes. The percentage reduction in the median of the relative fluorescence intensity, which is a measure of the enzyme performance, is given. The incubation time with the enzymes was 60 min. The results of two experiments with cells from two different donors are shown.

Enzyme No. 40 μg / ml 10 μg / ml 2.5 μg / ml

Bromelain 1 24.6 0 0 2 16.2 0 0

Papain 1 2.5 3.2 0 2 0 0 5.7

Trypsin 1 99.3 93.0 64.1 2 99.4 96.2 57.5

BPT 1 98.3 49.3 23.0 2 99.4 79.2 20.2

Table 3: Modulation of the CD4 epitopes Leu3a and OKT4 by trypsin; Target cells: lymphocytes. The percentage reduction in the median of the relative fluorescence intensity, which is a measure of the enzyme performance, is given. The incubation time with the enzymes was 60 min. The data from a donor are shown. Enzyme epitope 40 μg / ml 20 μg / ml 10 μg / ml

Trypsin Leu3a 98.5 96.7 87.1 OCT4 6.5 6.3 19.5 Epitope 5 μg / ml 2.5 μg / ml 1, 25 μg / ml

Trypsin Leu3a 65.2 41, 9 28.8 OCT4 13.3 10.8 9.3

Table 4: CD25 modulation by proteases; Target cells: lymphocytes, monocytes, NK cells. The percentage reduction in the median of the relative fluorescence intensity, which is a measure of the enzyme performance, is given. The incubation time with the enzymes was 60 min. The results of two experiments with cells from two different donors are shown. The cells were mitogen stimulated for 3 days prior to the experiment.

Enzyme No. 40 μg / ml 10 μg / ml 2.5 μg / ml

Bromelain 1 90.7 80.1 59.7 2 62.6 43.6 0

Papain 1 92.2 81.0 60.7 2 62.6 43.6 0

Trypsin 1 91, 2 88.2 71, 0 2 78.9 73.9 52.8

BPT 1 92.1 89.7 50.9 2 79.4 44.1 12.2

Table 5: Calculated half-effect concentration (μg / ml) of bromelain, papain, trypsin and their combination for the 50% reduction in the density of cellular surface molecules. The enzyme incubation was 1 hour. Enzymes CD2 CD4 ³ CD25 ⁴

Bromelain half-effect conc. n w n w 18.4

Area ² 14-23 Papain Semi Effect Conc. ¹ nwnw 12.4

Area ² 11-14 Trypsin Semi Effect Conc. ¹ 1, 5 2.5 <2.5

Range ² 1-2 2.3-3.1 BPT ⁵ half-effect conc. ¹ 9.3 16.4 16.0

Area ² 7.5-14 15.5-18 13-19

¹ calculated from averages of 2 or 3 independent experiments

² minimum and maximum value, 95% confidence interval = 1 δ, estimated

³ Modulation of CD4 epitope Leu3a

^{4 present} on stimulated cells

⁵ mixture of proteases in the ratio 22.7: 15.5: 11, 9 - bromelain: papain: trypsin nw: not effective in the system under investigation (max. 40 μg enzyme / ml, 1 h incubation)

Claims

claims

1. Use of at least one proteolytic enzyme and optionally rutoside to influence cytokines.

2. Use according to claim 1, characterized in that trypsin, bromelain or papain or a combination of one or more of these enzymes is used as the proteolytic enzyme.

3. Use according to claim 1 or 2, characterized in that rutoside is additionally used.

4. Use according to one or more of claims 1 to 3, characterized in that 20 to 100 mg bromelain, 40 to 120 mg papain and 10 to 50 mg trypsin are used per dose unit.

5. Use according to one or more of claims 1 to 4, characterized in that 90 mg bromelain, 120 mg papain and 100 mg rutoside x 3 H ₂ 0 are used per dose unit.

6. Use according to one or more of claims 1 to 3, characterized in that 90 mg bromelain, 48 mg trypsin and 100 mg rutoside x 3 H ₂ 0 are used per dose unit.

7. Use according to one or more of the preceding claims, characterized in that α ₂ macroglobulin is additionally used.

8. A method for influencing cytokines, the cytokines being brought into contact with one or more proteolytic enzymes and optionally rutoside.