WO1996000579A1

WO1996000579A1 - Aqueous cell extracts and aqueous suspensions of cell wall fractions from mycobacteria

Info

Publication number: WO1996000579A1
Application number: PCT/EP1995/002524
Authority: WO
Inventors: Hans-Georg Laves; Anne Thomsen
Original assignee: Laves-Arzneimittel Gmbh
Priority date: 1994-06-30
Filing date: 1995-06-29
Publication date: 1996-01-11
Also published as: JP3031718B2; DE4422859A1; DE4422859C2; AU2888495A; EP0804211A1; CA2194138A1; CZ285731B6; CZ375396A3; PL317892A1; NO965593L; JPH09507857A; NO965593D0

Abstract

The invention relates to aqueous extracts of not usually human-pathogenic mycobacteria, in which the extracts give an increase in the cytokinene IL-1 and IL-6 content in the ratio of 1:6 or 1:1.25 respectively according to the j.Immunol. 120:2027-2032 cyto-test, with a change in concentration of 1 to 4 to 1 to 16.

Description

Aqueous cell extracts and aqueous suspensions of cell wall fractions at Mvkohacterien.

The invention relates to aqueous extracts and aqueous suspensions of Zeil wall fractions from mycobacteria which are generally not human-pathogenic.

It is known that Mykobacterium tuberculosis in the variety hominis and Mykobacterium leprae are human pathogenic, but there are also a number of mycobacteria, which are usually only animal pathogenic; for example, Mycobacterium tuberculosis var. bovis is pathogenic for many mammals and Mycobacterium avium is a pathogen for birds. However, it has been found that, in rare cases, infections with mycobacteria, which are only animal-pathogenic, can be observed in people with a severely attacked immune system. It is also known that both the proteins and the various lipoid fractions of the mycobacteria have an antigenic character, while the polysaccharides probably only function as haptens in the sense of semi-antigens. It is furthermore known that tubercle bacteria which penetrate into a host organism which has already been in contact with mycobacteria in the sense of an infection which has already passed through trigger an allergy, that is to say an immunologically caused specifically altered reactivity. The state of allergy to tubercle bacteria is checked by intracutaneous injection of tuberculin; Tuberculin consists of dissolved decay products from tubercle bacteria. The tuberculin sample can therefore be used to determine whether a host organism is allergic to tubercle bacilli or not. An allergic organism reacts much faster to a natural infection and is therefore more likely to contain it than a non-allergic organism. This fact is used to correct a malfunction or a delayed response of the immune system by an unspecific modulation of the immune functions.

In the body's immune response, cytokines such as IL-1 and IL-6 play a central role in the regulation, proliferation and differentiation of immune cells. An unspecific modulation of the immune function essentially arises from the activation of monocytes or macrophages, which then release cytokines to an increased extent. Interleukin research is still at the experimental stage, but it is now known that interleukin 1 causes an increase in PHA-induced lymphocyte proliferation, fibroplast proliferation and an increase in the activity of natural killer cells in the presence of IFN. The same applies essentially to interleukin 6, which also causes an increase in the activity of natural killer cells and a T cell proliferation. Experimentally, the interleukins are therefore used in immunotherapy for the treatment of tumors and for the substitution of T cell defects. Interleukin 2 is used successfully, for example, in metastatic renal cancer and the cytokines interferon-δ and interferon-α 2B have been successful in rheumatoid arthritis and hairy cell leukemia. Recently, the granulocyte colony-stimulating factor G-CSF has also been used in the clinic for leukopenia in patients with previous myelosuppressive chemotherapy. In addition to the isolated cytokines, substances are also used therapeutically which stimulate or modulate the immune system non-specifically by activating monocytes or macrophages, which then release cytokines such as IL-1 and IL-6.

There is therefore still a strong need for substances which are able to generate non-specific immunomodulation, since a whole range of diseases or their accompanying symptoms can be therapeutically influenced by an increased release of cytokines and interferons.

According to the invention, aqueous extracts or aqueous suspensions of cell wall fractions of mycobacteria which are generally not human-pathogenic are therefore proposed with the features of claims 1 and 2.

Mycobacteria, which are generally not human pathogenic, can be used, inter alia, as M. tuberculosis var. Bovis, M. avium, M. kansasii, M. marinum, M. scrofulaceum, M. intercellulare, M. xenopi, M. fortuitum and M. chelonae ssp.chelone. These strains are known as such and can be obtained, for example, from the comprehensive London strain collection of the Center for Public Health Laboratory, National Collection of Type Cultures. This institute also provides information about the culture conditions of the respective species or subspecies. The extracts or suspensions prepared according to the invention have proven to be effective immunomodulators in the first clinical trials and can be used, for example, as supportive therapy for infectious diseases, neoplastic diseases, bronchial asthma, allergies of unknown origin and rheumatoid arthritis.

The invention is explained in more detail below with the aid of the examples:

Example 1 :

Production of the extract or the cell wall fractions

For the preparation of the aqueous extract or the cell wall fraction, 250 ml of liquid medium from 5 g / liter sodium chloride, 14 g / liter peptone, 7 g / liter Na2HP0 ₄ x 2H ₂ 0, 2 g / liter KH2PO4, 3% glycerol (85% ) inoculated with a bacterial loop with a mycobacterial culture and incubated for 4 weeks at 35 ° C. 4.5 g of moist bacteria are filtered through a sterile suction filter with a glass frit and then triturated homogeneously. After adding 100 ml of sterilized isotonic sodium chloride solution, the bacterial count is 9.7 x 10 ^ CFU per ml.

The resuspended bacterial cells are centrifuged at 12,000 RPM for 20 minutes. The pellet is mixed dry with glass beads, again taken up in sodium chloride solution and broken with a Vibrogen cell mill at 4 ° C. for 20 minutes. After suction filtering over a glass frit and adding 3 drops of an anti-foaming agent, centrifugation is carried out again (18,000 RPM, 20 minutes). The supernatant obtained is used to produce the mycobacteria extract and the pellet to produce the cell wall fraction.

In the further processing to the extract, 43 ml of the supernatant are autoclaved at 121 ° C for 20 minutes and stored at 4 ° C. This is followed by filtration using a depth filter (EKS) and sterile filtration (sterile filter with a pore size of 0.54 μm). Finally, the filtered extract is kept in a water bath at 80 ° C. for 2 hours.

To obtain the cell wall fraction, the pellet is resuspended in 27 ml isotonic sodium chloride solution. Example 2:

Attempts to quantify cytokine induction

These attempts refer to a number of publications; the numbers in brackets refer to the attached list of references.

Cell extract and cell wall fraction show a significant induction of the cytokines interleukin-1 (IL-1) and interleukin-6 (IL-6) in a dose-dependent reaction. Cytokines are vital messenger substances that are found between cells, e.g. mediate the cells of the immune system.

IL-1 and IL-6 are pleiotropic cytokines that are produced by different cells and play a central role in immune defense and hematopoiesis. They cause proliferation and differentiation of T and B cells (immunomodulating effect), the regulation of acute phase proteins by the liver in the course of an inflammatory process (anti-inflammatory effect) and the stimulation of colony-forming cells for megakaryocytes and megakaryocytes themselves (regulation of hematopoiesis ). IL-1 also induces the synthesis of other lymphokines such as IL-4, IL-5 and IL-6.

Isolation of mononuclear cells:

Heparinized blood from healthy donors was isolated by gradient centrifugation over Ficoll, washed several times and incubated in serum-free RPMI 1640 (1) with L-glutamine and antibiotics. The various stimuli were incubated with the cells for 24 hours for cytokine production (2). The supernatants were removed, provided with protective protein, stored at -20 ° C. and then examined in the corresponding cytokine test.

Isolation of v. Scular endothelial and smooth muscle cells: Endothelial and smooth muscle cells (EC and SMC) were isolated from pieces of the saphenous vein that could not be used in bypass surgery. EC were isolated by collagenase treatment (3) and SMC by a balancing technique (4). The cells were incubated in M199, ECGF and heparin or DMEM with 10% FCS, L-glutamine and antibiotics (5, 6, 7). The various stimuli were incubated with the cells for 24 hours for cytokine production. The supernatants were removed, provided with protective protein, stored at -20 ° C. and then examined in the corresponding cytokine test. 7TD1 assass for the detection of IL-6:

The above-mentioned supernatants of mono-cellular and vascular cells were diluted in 96-well culture plates in 8 to 12 steps. 2,000 cells / well of the IL-6-dependent murine B cell line 7TD1 (8) were added to these dilution series, similarly as described for B9 cells. The cultures were incubated for 72 hours, radioactive thymidine was added for a further 6 hours and the proliferation of the cells was determined by measuring the built-in radioactivity. The activity of the samples was determined by comparing the samples with a defined standard using probit analysis (9).

Fibroblast Assav for Detection of Interlenkin-1:

Fibroblasts were cultivated in 96 well plates. After 24 hours, the medium was changed and samples were added to the fibroblast cultures in dilution series. After 72 hours, the cultures were provided with radioactive thymidine and, after a further 24 hours, evaluated as described for 7TD1 cells.

Results;

Depending on the dose, the extract induces IL-1 (pg / ml) as follows:

Concentration 1: 4: 206 pg / ml; 1: 16: 1,250 pg / ml; 1:64: 947 pg / ml; 1: 1.024: 625 pg / ml

The extract induces IL-6 (pg / ml) depending on the dose as follows:

Concentration 1: 4: 95.082 pg / ml; 1: 16: 1 18,447 pg / ml; 1:64: 76,326 pg / ml; 1: 1,024: 51,961 pg / ml

The cell wall fraction induced dose-dependent IL-1 (pg / ml) as follows:

Concentration 1: 4: 103 pg / ml; 1:16: 1,649 pg / ml; 1:64: 1,539 pg / ml; 1: 1.024: 54 pg / ml

The cell wall fraction induced dose-dependent IL-6 (pg / ml) as follows:

Concentration 1: 4: 2013 pg / ml; 1: 16: 41 .71 1 pg / ml; 1:64: 35,374 pg / ml; 1: 1.024: 19,331 pg / ml literature

1. Loppno, H., H. Brade, I. Dürrbaum, C. A. Dinarello, S. usumoto, E. T. Rietschel and H.-D. Flad. 1989.

IL1 induction capacity of defined lipopolysaccharide partial structures. J. Immunol. 142: 3229-3238.

2. Loppnow, H., H. Brade, E. T. Rietschel and H.-D. Flad. 1994. Induction of cytokines in mononuclear and vascular cells by endctoxin and other bacterial products. In: Virginia L. Clark anc Patrik M. Bavoil (eds.), Methods in Enzy ology; Bacterial pathogenesis, Part B; Interaction of pathogenic bacteria with host cells. Academic Press, Orlando. In press.

3. Jaffe, E.A., R.L. Nachmann, C.G. Becker and C.R. Minick. 1973. Culture of human endothelial cells derived fro umbilical veins. J. Clin. Invest. 52: 2745-2756.

4. Ross, R. and B. Kariya. 1980. Morphogenesis of vascular smooth muscle in atherosclerosis and cell structure. In: "Handbook of Physiology" The Cardiovascular System, Section 2.

(D.F. Bohr, A.P. Somlyo and H.Y. Sparks, eds.), American

Physiological Society, Bethesda. p. 66-91.

5. Loppnow, H. and P. Libby. 1989. Adult human vascular endothelial cells express the IL6 gene differentially in response to LPS or IL1. Cell. Immunol. 122: 493-503.

6. Loppnow, H. and P. Libby. 1990. Proliferating or Interleukin 1-activated Human Vascular Smooth Muscle Cells Secrete Copious Interleukin 6th J. Clin. Invest. 85:

731 - 738. ^"

7. Loppnow, H. and P. Libby. 1992. Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways. Exp. Cell Res. 198: 283-290.

8. Van Snick, J., S. Cayphas, A. Vink, C. Uyttenhove, PG Coulie, MR Rubira and RJ Simpson. 1986. Purification and NH ₂ terminal amino acid sequence of a T-cell derived lymphokine with growth factor activity for B-cell hybridomas. Proc. Natl. Acad. Be. USA 83: 9679-9683.

9. Gillis, S., M.M. Ferm, W. Ou and K.A. Smith. 1978.

T-cell growth factor: parameters for production and a quantitative microassay for activity. J. Immunol. 120: 2027-2032.

RSATZBL _Ä TT (REGEL26)

Claims

claims

1 . Aqueous extract from mycobacteria, which are generally not pathogenic to humans, characterized in that the extract in the cytokine test according to J. Immunol. 120: 2027 - 2032 when the concentration changes from 1: 4 to 1:16, the cytokines IL-1 and IL-6 are increased in a ratio of 1: 6 and 1: 1, 25, respectively.

2. Aqueous suspension of cell wall fractions of generally non-human pathogenic mycobacteria, characterized in that these fractions in the cytokine test according to J. Immunol. 120: 2027 - 2032 with a change in concentration from 1: 4 to 1:16 IL-1 and IL-6 induce cytokines in a ratio of 1:16 and 1:20, respectively.

3. A process for the preparation of an aqueous extract or an aqueous suspension of cell wall fractions of mycobacteria which are generally not pathogenic to humans, characterized in that the mycobacteria are incubated on a peptone agar at 35 ° C. for 4 weeks, filtered off from the nutrient medium, resuspended in isotonic saline solution and then Centrifuged for 20 minutes at 12,000 RPM, the sediment was taken up in saline and ground in a cell mill at 4 ° Celsius and centrifuged again for 20 minutes at 18,000 RPM and the supernatant was separated, autoclaved and sterile filtered and then kept at 80 ° Celsius for a period of 2 hours becomes.