DE102009034779A1 - Synthetic analogues of bacterial lipopeptides and their application for the therapy and prophylaxis of allergic diseases - Google Patents

Abstract

The present invention relates to new lipopepitides derived from a spore germination protein of the species Bacillus cereus with immunostimulatory activity, processes for the preparation of synthetic analogues, their use as active ingredients for the therapy and prophylaxis of numerous allergic diseases, preferably allergic asthma, and formulations of corresponding pharmaceutical agents. The previous approaches to treating allergic diseases are very time-consuming and are often accompanied by numerous side effects. Often, only a short-term relief of the symptoms of the disease can be achieved. A reliable way to effectively reduce the allergy risk is not yet known. The described analogues of bacterial lipopeptides have a good immunostimulatory effect. By activating the TLR receptors 1, 2 and 6, they contribute to the maturation of the immune system and thus to a reduction in the risk of allergies. In the animal model, a significant reduction in sensitivity to allergenic substances with good tolerability was demonstrated. The compounds are to be used for the therapy and prophylaxis of allergic diseases.

Description

The The present invention relates to novel spore germination protein the species Bacillus cereus derived lipopeptides with immunostimulatory Effect, process for the preparation of synthetic analogues, their Use as active ingredients for the therapy and prophylaxis of numerous allergic diseases, preferably allergic asthma, as well as formulations of appropriate pharmaceutical agents.

for the first time is the immune stimulation by lipoproteins from bacterial spores and by synthetic analogues thereof at the molecular level demonstrated.

State of the art

When Allergy becomes an overactive defense reaction of the immune system to certain, under normal circumstances harmless stimuli from the environment (allergens). The allergic Reaction manifests itself in typical, by inflammatory Processes triggered symptoms. It comes to mild to serious damage to various organs of the body. Depending on the disease form can be different Be affected individually or in combination. While allergic symptoms on the mucous membranes typically More likely to occur acutely, diseases such as bronchial asthma or atopic dermatitis take a chronic course. In the Usually, allergic persons develop hypersensitivities during the course of the disease against a growing number of substances.

The Mechanisms in the development of allergies correspond to those at the pathophysiological pathogens that control pathogens Operations. After the immunological mechanisms of action are differentiated subtypes of allergy. This is understood Today in the narrower sense of allergy often only the type I allergy. This form is due to an inappropriate reaction of the humoral Immune system, its actual purpose in the defense against parasites exists, causes and is due to the formation of allergen-specific Immunoglobulin E antibodies (IgE).

Small amounts of an allergen are sufficient to trigger sensitization. In the process, the allergens that have entered the body are taken up by dendritic cells and processed. The thus activated dendritic cells present via their MHC-II surface receptors fragments of allergens to naive T-cells induced by CD4 costimulation and under the influence, inter alia, of the cytokine interleukin-4 (IL-4) to activated T _H 2 cells tires. At the same time, naive B cells come into contact with the allergen. If these activated B cells then encounter the allergen-specifically activated T _H 2 cells, a T cell B cell interaction ensues, as a result of which the B cell becomes the IgE-secreting plasma cell and begins to to form allergen-specific IgE in large quantities.

The formed IgE antibodies are for the most part bound on the cell surfaces of mast cells and basophils Granulocytes. At the same time, they also become antigen-presenting Cells are bound and thus enhance the presentation the allergens to the immune system. The IgE concentration in the serum is on the other hand comparatively low.

The Sensitization phase results in one through which increased Number of allergen-specific IgE antibodies, increased allergy readiness, but runs without Disease symptoms. However, if there is a second contact with the allergen against which the sensitization has occurred, this becomes by the IgE antibodies on the mast cells and basophils Granulocytes bound. The following release of histamines, Cytokines, leukotrienes and the like a. partly toxic compounds the activated leukocytes leads to further activation of the immune system and attractant of inflammatory Eosinophils. Within a short time can be an increased vascular Permeability, the relaxation of smooth muscle as well as the stimulation of the cutaneous nerve endings are observed the typical allergic symptoms of hay fever, conjunctivitis, allergic asthma, food allergies or even anaphylactic Cause shock.

The Binding of allergens to the IgE antibodies on the mast cells accelerates simultaneously, with the participation of the cytokines IL-4 and the expression of CD40 ligands, the production of other allergen-specific IgE antibodies by the plasma B cells.

In addition to this immediate reaction, chronic symptoms can occur in most allergies, especially in advanced stages. These are due to the activation of allergen-specific T cells antigen-presenting dendritic cells as well as IL-5 mediated chemotactic recruitment and immigration of eosinophils, basophils and monocytes to the site of the allergic reaction. At the same time, further, particularly long-lived mast cells are activated. This chronic course is independent of the current presence of the allergen and contributes to the maintenance of the inflammatory immune response.

To the causes of allergic diseases, v. a. the steady increase the number of those affected, are so far not conclusive Explanations have been provided. However, a number of factors are moved into the closer focus. Undeniably there it is a genetic predisposition to an increased risk of allergy. Also the changed environmental conditions with increasing Pollution, as well as climate change with the so Associated shifts in flora and fauna come as a cause into consideration. An important role is also the changed Food and eating habits awarded. For example find increasingly artificial additives in food and care products use. Novel medicines often unknown side effects are allowed.

An essential step in the development of allergies is the imprinting of the immune system, in particular the maturation or differentiation of the T _H 1 and T _H 2 cells. IL-4 producing T _H 2 cells are necessary for the formation of the allergy-mediating IgE antibodies, whereas T _H 1 cells producing interferon-γ inhibit the formation of IgE antibodies. For a functioning immune defense, both cell types are needed in a balanced ratio.

It is believed that the immune system is more T _H 2 -containing during pregnancy and is directed in the first few years of life by contact with microbial components, especially bacteria, in the direction of an efficient cell-based T _H 1 response less associated with allergic reactions.

By the changed urban living environment, life in small families and the use of medically effective and disinfecting substances even in normal detergents, has the burden of bacterial and parasitic pathogens, however, decreased sharply. That is why a large number of scientists see the Causes of the observed increase in allergic diseases in the lack of activation of the immune system in infancy ("hygiene hypothesis").

The Possibilities for the prevention of new sensitisations as well as for the therapy of already pronounced allergies limited. In addition to the symptomatic therapy of allergy patients On the one hand, the avoidance of the allergen is recommended as far as possible.

In the context of hyposensitization, however, the direct confrontation with attenuated doses of the allergen in question takes place ( GB Pajno et al. 2001. Clinical and Experimental Allergy 31: 1392-1397 ; L. Jacobsen et al. 2007. Allergy 62 (8): 943-948 ; C. Möller et al. 2002. J Allergy Clin Immunol 109 (2): 251-256 ; A. Bufe et al. 2009. J Allergy Clin Immunol 123 (1): 167-173 ). However, this immunotherapeutic approach has different success. Especially with the frequently occurring bronchial asthma, this form of therapy is rather ineffective.

Other approaches, building on the "hygiene hypothesis", support the T _H 1 / T _H 2 shift by confronting immunomodulatory, disease-independent substances.

In epidemiological studies, for example, the influence of a rural environment, especially contact with animals, has been demonstrated to reduce the risk of developing allergic asthma and hay fever in children. A prominent study on this topic is the ALEX study ( J. Riedler et al. 2001. Lancet 358: 1129-1133 ).

In WO 2001/049319 describes the preparation of an aqueous suspension of microorganisms from stable air and their use for the prophylactic treatment of children with an increased risk of allergies or first signs of an allergy syndrome. WO 2006/029685 describes an aqueous extract of barn dust. In the mouse model inhalation of this extract showed a clear reduction in the symptoms of allergic asthma and suppression of the T _H 2-dominated immune response ( M. Peters et al. 2006. Thorax 61: 134-139 ). Which substances ultimately trigger the protective effect from the large number of potentially immunostimulatory components of bacteria, viruses, fungi, plants and animals, however, is still unknown.

While these two papers assume a limited number of treatments, other studies point to the need for lifelong exposure in the rural environment to enable effective prophylaxis ( J. Douwes et al. 2008. Eur Respir J 32: 603-611 ).

in the Track this relative non-specific microbial burden some other treatment approaches targeted stimulation the immune response via the toll-like receptors (TLR).

TLR are structures of the innate immune system. As membrane proteins bound to the cells of the immune system, they serve to recognize functional structures that occur exclusively on or in pathogens. Each of the 13 (known for mammalia) receptors recognizes a very specific pattern of a pathogen class. Thus, TLR 1 is described in conjunction with TLR 2 as a receptor for cell wall components of bacteria. TLR 2 detects gram-positive bacteria, such as B. mycobacteria. In conjunction with TLR 6, this receptor responds to lipopeptides of mycoplasmas and zymosan from yeasts and fungi. Other TLRs detect viral components in the cells (TLR 3, 7, 9), other bacterial components such as. B. Lipopolysaccharides from the cell membrane of Gram-negative bacteria (TLR 4) or flagellin (TLR 5) ( H. Heine et al. 2003. Int Arch Allergy Immunol 130: 180-192 ).

about a biochemical signaling cascade occurs in the TLR carrier cells a corresponding intracellular mechanism, usually the Expression of certain, the antigen-specific defense of pathogens serving Genes, triggered. As a result, in the first stage initiates the non-specific immune response, which later with the formation of specific antibodies, the reaction the acquired immune system follows.

dendritic Cells that play an important role in the development of allergies as a rule, all types of TLR carry. At her Activation they express the corresponding MHC complexes and costimulatory enzymes. In the lymph nodes they stimulate the Maturation of antigen-specific T cells.

A targeted stimulation of certain TLR should therefore be appropriate support the maturation of the immune system later to induce an adequate response of the organism to foreign substances.

As such an approach z. B. the treatment and prophylaxis of allergies with specific, derived from non-human pathogenic microorganisms DNA sections, preferably oligonucleotides containing at least one non-methylated CpG sequence described. Often, these agents are combined with other ingredients such as allergens, adjuvants and viral components. Mycobacteria strains are very often described as bacterial components in this therapeutic approach. The short DNA sequences derived therefrom are recognized by the Toll-like receptor 9 of the dendritic cells and stimulate the T _H 1 immune response with simultaneous inhibition of the allergy-correlated T _H 2 reaction ( US 2006/0217328 . US 2006/0251677 . WO 2008/014979 . WO 2007/095976 ).

Three of these are currently in clinical trials. The company Cytos used in CYT003-QbG10 a composition of a virus-like particles, an immunostimulatory DNA sequence of Mycobacteria species, an unspecified TLR ligand and optionally an antigen, preferably an allergen such. Pollen extract, for the treatment of allergies and asthma ( WO 2005/004907 , Cytos press release dated 13.01.2009). Dynavax is using TOLAMBA ^® and AIC to test two preparations of a short synthetic DNA sequence conjugated to the major allergen of ragweed for the treatment of ragweed hay fever ( WO 2006/096497 , Dynavax press releases dated 06.03.2006 and 04.10.2006) and allergic asthma ( WO 2008/073661 ).

Against the background of the hygiene hypothesis and to support the T _H 1 / T _H 2 shift and thus for the prophylaxis and therapy of allergies, TLR 1, 2 and 6 are also suitable. The cytokines expressed upon activation are more similar to the pattern necessary for the differentiation of T _H 1 lymphocyte cells. In addition, TLR 2 is also found in the epithelia of the trachea and the alveoli, which qualifies this receptor especially for the treatment of allergic asthma ( C. Zuany-Amorim 2002. Nat Rev Drug Discov 1: 797-807 ).

Whole microorganisms, parts of microorganisms or derived lipoproteins or lipopeptides are often used to stimulate the TLR 2 receptor ( EP 1387167 . WO 2004/092210 . US 2008/0241139 ). Non-pathogenic bacterial strains such as Borrelia spp, Escherichia spp, Heliobacter spp, Leptospira spp, Listeria spp, Mycobacterium spp, Mycoplasma spp, Pseu are preferred for humans domonas spp, Salmonella spp, Staphylococcus spp, Streptococcus spp, particularly preferably Bacillus thuringiensis, Borrelia burgdorfei, Mycobacterium tuberculosis, Mycoplasma fermentans, Treponema pallidium and Escherichia coli. Lipopolysaccharides are also described for a selective immune stimulation of the TLR receptors for the treatment of allergies ( WO 2008/059035 . US 2008/0119145 ).

WO 2004/074435 claims the targeted influencing of the differentiation process of T-helper cells via the TLR of dendritic cells. By applying a TLR agonist, in combination with synthetic peptides, nanoparticles, virus-like particles or T-cell or B-cell epitopes in bound / unbound form, the treatment of immune deficiencies and allergies is to be made possible. As TLR agonists microbes such as viruses, bacteria, parasites and parts or molecular components thereof are used.

TLR can also be activated by synthetic compounds. Thus, the TLR 1 / TLR 2 heterodimer also reacts to triacylated synthetic lipopeptides such as the Pam ₃ Cys derivatives ( EP 0210412 ). TLR 6 / TLR 2 heterodimers are sensitive to diacylated synthetic lipopeptides such as the Pam ₂ Cys derivatives ( EP 0519327 ).

Based on these findings, various drugs and vaccines containing acylated lipopeptides have already been developed for use in immunomodulatory therapies. Typically, for this purpose, the lipopeptides, preferably Pam ₃ Cys and Pam ₂ Cys derivatives as TLR 2 targeting substance, combined with an antigenic component for B lymphocytes or for cytotoxic T cells and other excipients to induce synergistic effects ( WO 2007/103322 . WO 2006/104389 . US 7,387,271 ). Also, liposomes containing lipopeptides, preferably Pam ₃ Cys, in addition to antigens and possibly cytostatics are claimed for the treatment of various diseases and allergies ( WO 2005/063201 . WO 2005/063288 ).

Also described are conjugates of Pam ₃ Cys derivatives and antigens as vaccines. So in WO 2007/078879 a combination of a membrane protein of the influenza virus and a Pam ₃ Cys derivative, in WO 2007/079448 a linear tandem molecule of a B cell epitope, a T cell epitope and Pam ₃ Cys or in WO 2004/014956 and WO 2004/014957 a molecule of a B-cell or CTL epitope and a T helper cell epitope bridged by a Pam ₃ Cys lipopeptide.

In contrast to the previously described methods for stimulating the TLR receptors, the suppression of the TLR 2-mediated immune reaction and the associated inflammatory reaction are proposed for the treatment of already pronounced allergic diseases ( WO 2005/039504 . WO 2007/047396 . WO 2005/115430 . WO 2005/067959 ). In WO 2007/098371 the relief of inflammatory reactions via the TLR 5 is achieved by means of flagellin preparations. WO 2007/118901 describes the targeted suppression of the T _H 2-correlated immune response by a tracheal cytotoxin or a muramyl-tetrapeptide with the addition of Pam ₃ Cys for the treatment of allergies, asthma and other T _H 2-correlated diseases.

Also for the treatment of allergic diseases at the level of allergen-specific antibodies Pam ₃ Cys conjugates are used. WO 1996/12740 describes a synthetic linear conjugate of a lipopeptide immunostimulator, a bacterial T-helper cell epitope and an immunogenic peptide capable of binding to membrane-bound IgE.

Pharmaceutical agents having an oligopeptide covalently coupled to a lipid, preferably a Pam ₂ Cys or Pam ₃ Cys derivative, without further additions of specific antigens or B cell or T cell epitopes, become very little described. In most cases, Pam ₃ Cys is simply given as a stimulant adjuvant in a mixture of antigens, epitopes and microorganisms or constituents of microorganisms. The covalent coupling, if any, is then usually with specific immunization agents via activation of the adaptive immune system.

The closest to this construction still come in WO 1996/22067 described conjugates with a library of short fragments of a CTL epitope covalently linked to a Pam ₃ Cys derivative and described in WO 2007/078879 described covalent compounds from a membrane protein of the influenza virus and a Pam ₃ Cys derivative, for use as vaccines. From Mühlradt et al. For example, a diacylated dihydroxypropylcysteine peptide having a Mycoplasma fermentans-derived peptide (MALP-2) is claimed for various applications in immunological medicine ( WO 1998/027110 . WO 1999/059610 . WO 2002/028887 . WO 2003/084568 . WO 2004/108145 ). US 5,688,771 describes the coupling of peptidoglycans with a lipophilic alkyl chain as an immunomodulator.

applications such compounds for the therapy and prophylaxis of allergic Diseases via the targeted modulation of TLR receptors have not been described yet.

task

Allergies are a major health problem with a multifaceted clinical picture. In the industrialized countries, a clear increase in the incidence of allergic diseases has been observed in recent years. Today, more than 20% of the population in Western industrialized countries suffer from allergic diseases (World Health Organization 2002. Prevention of Allergy and Allergic Asthma). There are 80 million allergic persons in Europe alone (GA ² LEN-Global Allergy and Asthma European Network, www.ga2len.net, 2008). By 2025, approximately 400 million people worldwide will suffer from allergic asthma. Of particular concern is the increase in the proportion of children affected.

Usually be three approaches to the treatment of allergic diseases pursued: i) prevention of allergen contact as far as possible, (ii) the prescription of medications which relieve the symptoms of the disease and iii) conventional immunotherapy, also called desensitization designated.

The drug-related symptom treatment with cortisone preparations and histamine inhibitors allows only a short-term improvement of the condition, which is a repeated application for the patients, often even several times a day, means. In addition, the conventional agents often with considerable Afflicted side effects. The desensitization on the other hand, is very time consuming. 3-5 years of treatment with up to 80 allergen injections are for the patients quite uncomfortable, so that only a few allergic people use this form of therapy use.

Also there are no effective ways of prophylaxis, already at risk for an allergy to be effective to lower. Thus, there is a great need for new effective ones Prevention and treatment strategies.

task The present invention is therefore novel pharmaceutical Active ingredients for the prevention and treatment of allergic diseases, preferably from allergic asthma, to provide which the mentioned disadvantages of conventional means bypass. It is based on the "hygiene hypothesis" Way of activating and maturing the innate immune system a completely new approach.

The Task is solved by a selection of first described, from the structure of a germination protein from the spores of the species Bacillus cereus derived lipopeptides, with immunostimulatory Effect and their synthetic analogues having the general structure, as set forth in claim 1. Preferred embodiments These compounds are claimed in claims 2 to 13. Claims 14 and 15 relate to a manufacturing process for the compounds according to the invention, claims 16 and 17 the use of the invention Compounds and claims 18 to 29 pharmaceutical Preparations containing the inventive Compounds, their preparation and use. The wording of all Claims are hereby incorporated by reference Description made.

The essential feature of the present invention consists in a Series from a spore germination protein of the species Bacillus cereus-derived lipopeptides and their synthetic analogues, with regard to the immunomodulatory effect of these compounds to take special account of stereoselectivity is. With the present invention is the first time the immunostimulatory Effect of bacilli spores detected at the molecular level. The derived peptides or sequence motifs are so far none immunologically active epitope has been assigned.

The The invention further includes the use of the immunostimulatory Effect of these compounds to simulate a natural Environment for the purpose of maturing the immune system and building on it their use for the therapy and prophylaxis of many allergic Diseases of humans and animals, preferably allergic Asthma.

Detailed description

In genetic studies, various astro-susceptible modulating TLRs have been identified. This establishes a link between the microbiological environment and the innate im munsystem postulated in asthmatics.

in the Framework of the search for new drugs for prophylaxis and therapy of allergic diseases, most preferably allergic Asthma, therefore, became the immunostimulating effect of various synthetic analogues of naturally occurring lipoproteins examined as possible TLR agonists.

lipoproteins are components of Gram-negative bacteria, Gram-positive bacteria and mycoplasma. They represent between 1% and 3.5% the genome of bacteria. Bacterial lipoproteins have no Sequence homology. They are, however, by the unusual Amino acid S- [2,3-di (hydroxy) -propyl] - [R] -cysteine at the N-terminus, to which two or three fatty acids are bound.

Lipoproteins and their synthetic analogues are potent immune modulators of the early innate immune response. As adjuvants, they also support the effective response of the adaptive immune system. They are usually recognized by the Toll-like receptors TLR 1, TLR 2 and TLR 6 via the formation of homo- or heterodimers. Via an intercellular signal cascade, the elements of innate immunity, such as macrophages, polymorphic nuclear cells and dendritic cells, are activated, which form the first antibodies. In the further course begins the specific antibody formation. The involved cytokines are z. Tumor necrosis factor alpha (TNF-α) and various interleukins (IL-1α, IL-1β, IL-6, IL-8, IL-12). By this cytokine pattern, which is more of a T _H 1-dominated immune response, immunodeviation away from the T _H 2 expression is observed in most experimental models.

The DOLOP database (DOLOP-A Database of Bacterial Lipoproteins. http://www.mrc-lmb.cam.ac.uk/genomes/dolop ) contains a collection of previously described bacterial lipoproteins. The amino acid sequences of proteins are based largely on predictions based on the enlightened genome of the species. Few recorded lipoproteins are also based on a complete structure elucidation.

Out This database was initially made of lipoproteins of frequent occurring bacterial species selected. Starting from the hygiene hypothesis and the previously published work on the preventive antiallergic efficacy of dust extracts, the focus was doing so in normal concentrations for the healthy People non-pathogenic microorganisms that are particularly common occur in animal stables.

These Pathogens whose spores and decay products are usually considered Airborne particles are present and are inhaled into the bronchi and the lung tissue was taken. Learning through repeated contact the immune system, between the ubiquitous microbial Load and non-pathogenic substances. This Maturation process may possibly be an allergic dysregulation prevent the immune response.

Of the selected lipoproteins, peptides containing the N-terminal last 15 amino acids and later other analogues were initially synthesized. To simulate the lipid components, the peptides were covalently attached at their N-terminal end to S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] -N-palmitoyl- [R] -cysteine (Pam ₃ Cys). coupled. Pam ₃ Cys is representative of various lipid components, as previous studies have shown that replacing palmitic acid with similarly sized fatty acids does not cause any relevant activity changes. It was also shown in the investigations that the peptides bound to the diacylated lipoamino acid (Pam ₂ Cys) have a comparable biological activity.

In one assay, the ability of these synthetic analogs to stimulate innate immunity receptors was determined as a function of concentration. The immunostimulatory activity of the lipopeptides was demonstrated by IL-8 secretion by THP-1 cells, a human monocytic leukemia cell line. The THP-1 cells were incubated with different concentrations of the respective lipopeptides. Subsequently, the IL-8 concentration in the supernatants was determined by ELISA (procedure according to Example B). Tab. 1: Selection of the tested frequently occurring bacterial lipopeptides.

Most of the lipopeptides tested, as well as the synthetic Pam ₃ Cys derivative, achieve their half-maximal activity in the IL-8 assay in the concentration range of 10 to 100 nM ( - ).

It has surprisingly been found that the lipopeptide sbLP11 (SEQ ID NO 1) derived from the germination protein GerD of the spores from Bacillus cereus ATCC 10987 has an up to 1000 times increased stimulatory activity compared with the other synthetic lipopeptides tested, ie its half maximal activity in the IL-8 assay already achieved in the concentration range of 10 to 50 pM ( . ).

So far could still have an immune-stimulating effect of bacterial spores never be detected at the molecular level. In addition, the arbitrarily selected section of the lipoprotein not previously known as an immunogenic epitope.

Also in a collection of lipopeptides associated with the germination proteins of spores of various Bacillus species, the lipopeptides derived from the Bacillus cereus clones (SEQ ID NO 1, SEQ ID NO 2) surprisingly exhibited significantly higher stimulation activity of IL-8 secretion as other germination proteins ( . ). Tab. 2: Selection of the tested lipopeptides derived from the germination proteins of bacterial spores:

In order to verify the exceptional immunological activity of the identified lipopeptide sGerD from Bacillus cereus (SEQ ID NO 1) and the transferability of this effect to other synthetic analogues, further investigations of the structure-activity relationship were made. Table 3: Synthetic analogues to study the structure-activity relationship of sGerD.

The IL-8-stimulating activity of the 13 amino acid-containing lipopeptide variant (SEQ ID NO 3) corresponded to that of the sGerD with 15 amino acids (SEQ ID NO 1). Further shortening of the lipopeptide resulted in a marked reduction of the immunomodulatory activity ( . ).

In the alanine scan with the sGerD-derived tridecapeptide sequence (SEQ ID NO 3) as the basis, the replacement of the amino acids at positions 3, 4, and 5 of the amino acid sequence following Pam ₃ Cys markedly reduced cytokine secretion ( . ).

Obviously represents the subject EKE with the sequence of oppositely loaded Amino acids, in addition to the characteristic lipoamino acid with 2 or 3 fatty acids, another key structural feature for the immunostimulating action of the lipopeptide.

Further variations showed that lipopeptides with sterically undemanding small amino acids such as alanine or glycine are exceptionally active at position 1 of the amino acid sequence following Pam ₃ Cys. Obviously, these amino acids improve the interaction with the binding pocket of the receptor.

Based on these findings, further structural analogues of the previously investigated lipopeptides were derived and characterized with regard to their immunostimulatory effect. The found structural moti They were consistently implemented in order to further increase the solubility and absorbability of the compounds and thus their bioavailability.

Overall, the lipopeptides with the structural motifs derived in this way have an enhanced action ( ). The half-maximal activity in the IL-8 assay is below 0.1 nM for all compounds. The two lipopeptides Pam ₃ Cys-AEEKEAEEK (SEQ ID NO 4) and Pam ₃ Cys-GEEKEEK (SEQ ID NO 5) proved to be particularly effective with an amino acid sequence which was greatly altered compared to the sGerD original sequence.

Another study concerned the effect of palmitic acid substituents. Significantly increased stimulatory activities were detected for both the Pam ₃ Cys derivative and the Pam ₂ Cys derivative of the sGerD lipopeptide truncated to 13 amino acids (SEQ ID NO 3) ( ). The coupling of the third fatty acid residue via an amide bond to the N-terminus of the 2,3-dihydroxypropyl cysteine thus gives no decisive advantage over the free N-terminus lipopeptide in the immunological activity of sGerD.

In contrast, the immunomodulatory activity of sGerD is substantially dependent on the existence of the 2,3-dihydroxypropyl cysteine esterified at both OH groups with a fatty acid residue ( ). The non-acylated and the mono-N-palmitoylated peptides have no IL-8-stimulating activities. This applies both to the 15 amino acid-containing original sequence of sGerD (SEQ ID NO 1) and to the variant truncated to 13 amino acids (SEQ ID NO 3).

Farther gave a study on the influence of stereoisomerism of the invention Lipopeptides have a significantly higher immunomodulatory effect the compounds containing the RR isomer of the dihydroxypropylcysteine parent contain.

outgoing from these investigations could surprisingly be found be that both the derived lipopeptides sGerD from Bacillus cereus (SEQ ID NO 1, SEQ ID NO 2) as well as other specifically modified Analogs that no longer correspond to the original sequence, preferably the compounds having the sequences according to SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, an exceptionally high immunostimulatory Have activity. This show the lipopeptides, the contain the RR isomer of the dihydroxypropyl cysteine main body, a very special activity.

This is surprising even more so than that so far the immunostimulatory effect of Bacillus spores were not detected at the molecular level.

at the microorganism Bacillus cereus is a Gram-positive Bacteria, naturally in the ground, so also in the dust occurs. The ubiquitous amounts are for healthy people and animals are not pathogenic. In very high concentrations the bacterium can be used as food poisoner, v. a. in rice and Milk, act.

There are isolated references in the literature to vaccines against this bacterium. Adjuvants must always be used in order to achieve a sufficient immunostimulatory effect of this bacterium or its parts ( WO 2004/030608 . WO 2008/054892 . US 2003/0190322 ). A patent claims the use of Bacillus cereus spores as a transport system for immunogenic polynucleotide sequences ( WO 2006/087576 ).

The independent immunomodulatory effect of Bacillus cereus spores per se, certain parts thereof or individual lipopeptides from these spores is not described yet. Also was the use of selected short amino acid sequences of these Lipopeptides that are not specific to immunity Epitope can be assigned, so far not considered drawn.

In the present invention, for the first time, a peptide as long as 25 amino acids derived from the amino acid sequence at the N-terminal end of the total 25 kDalton germination protein GerD of Bacillus cereus spores was selected and covalently attached to a di- or triacylated dihydroxypropylcysteine (Pam ₂ Cys or Pam ₃ Cys) coupled to achieve a non-specific immunostimulatory effect in humans and animals via one of the Toll-like receptors TLR 1, TLR 2 or TLR 6. This stimulation should contribute to the maturation of the immune system and minimize the risk of developing a manifested allergic disease.

sowie deren Verwendung als Immunstimulanzien zur Therapie und Prophylaxe zahlreicher allergischer Erkrankungen, vorzugsweise von allergischem Asthma.The present invention therefore relates to compounds of the formula

as well as their use as immunostimulants for the therapy and prophylaxis of numerous allergic diseases, preferably of allergic asthma.

R 1 and R 2, which may be identical or different, represent a saturated or unsaturated acyl radical having 7 to 25 C atoms,
R3 is a saturated or unsaturated acyl radical having 7 to 25 carbon atoms or H and
R4 for a physiologically acceptable peptide consisting of 1 to 25 amino acid residues and not immunogenic per se in the treated species.

Preferred embodiments relate to lipopeptides according to the invention having two or three saturated C ₁₆ -acyl substituents for R 1, R 2 and, where appropriate, R 3 and a peptide corresponding to the following formulas (II) - (IX).

SEQ ID NO 1

SEQ ID NO 3

SEQ ID NO 4

SEQ ID NO 5

The Definition of the compounds of the formulas (I) to (IX) all pharmaceutically acceptable salts, stereoisomers, stereoisomer mixtures and prodrug compounds of the invention Lipopeptides.

additionally for the detection of the immunostimulatory activity of the invention Lipopeptides via the in vitro stimulation of IL-8 secretion were their complete allergy-protective effect as well as the physiological compatibility in the standardized animal model allergic asthma.

In the immunological and cell biological functional studies (Examples C and D) could be shown that by the inhalation of the lipopeptides according to the invention both the asthma-related symptoms, the inflammatory response and the allergenic sensitization to almost the level of non-sensitized mice (corresponding values M. Peters et al. 2006. Thorax 61: 134-139 described) could be pushed back. The T _H 1-stimulated immune response was only slightly enhanced. Restrictive side effects were not observed in the mouse model.

A particular effectiveness showed the inventive Lipopeptides directly on the lung tissue. The concentration the allergen-specific antibody was in the lung fluid significantly higher than in serum. This puts the inhalation as particularly effective application form of the invention Close to lipopeptides.

These surprising Insights into the outstanding immunomodulatory Activity of the lipopeptides according to the invention and their effectiveness in the therapy and prophylaxis of allergic Diseases, preferably allergic asthma completely new possibilities for their application as active ingredients for the treatment and vaccination against allergies.

The Spectrum of activity of the compounds of the invention includes due to the non-specific immunization approach via the TLR receptors the full breadth of the known allergic Diseases, especially allergies and hypersensitivities against environmental substances and foods that are affected by symptoms the mucous membranes (allergic rhinitis, swelling of the buccal mucosa, Conjunctivitis), the respiratory tract (bronchial asthma), the skin (atopic Dermatitis, atopic dermatitis, contact dermatitis, urticaria), in the gastrointestinal tract (Vomiting, diarrhea) or as an acute emergency (anaphylactic) Shock).

Especially the compounds of the invention are effective in the treatment of respiratory allergic reactions, in particular allergic asthma and allergic rhinitis which may occur a. through house dust and pollens are caused.

typical Medical indications include prophylaxis, for example Individuals with clear genetic disposition and treatment of humans and animals at first symptoms of an emerging one Hypersensitiviät.

One Another object of the invention is the preparation of synthetic Lipopeptides according to formula (I), which according to the invention as Immunostimulants for the therapy and prophylaxis of allergic diseases can be used.

A possible route is the convergent synthesis of the compounds according to the invention, wherein the unusual amino acid Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteine (Fmoc-Dhc-OH) in solution or in solid phase synthesis with a side chain protected peptide synthesized on a synthetic resin. This precursor can be bisacylated on the hydroxy groups. After Fmoc cleavage of additional side-chain protecting groups, the target compound is separated from the resin. In the synthesis of the Pam ₃ Cys derivative, the acylation of the liberated amino group takes place before the cleavage.

at Another production variant is the synthesis of the invention Compounds with the building block Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2R) -propyl] - [R] -cysteine (Fmoc-RR-Dhc-OH). This will be the destination connection as a stereochemically defined derivative obtained from L-amino acids, from the modified amino acid S- [2,3-di (hydroxy) - [2R] -propyl] - [R] -cysteine and composed of two or three acyl radicals.

One Another object of the invention is the use of the invention synthetic lipopeptides according to formula (I) for the preparation of pharmaceutical Preparations for the treatment and prevention of Diseases of humans and animals associated with allergies stand. These immunomodulatory compositions containing at least one inventive synthetic Lipopeptide according to formula (I), further contain at least one or no immunological effect enhancing adjuvant, at least one or no non-toxic, inert, pharmaceutically suitable carrier and at least one or no non-toxic, inert, pharmaceutically suitable auxiliary and / or additive.

Pharmaceutically suitable materials are those in the field of pharmacy and food technology as well as substances known to be usable in adjoining areas, in particular those listed in relevant pharmacopoeias whose properties do not preclude physiological application.

The Pharmaceutical compositions of the invention containing at least one synthetic according to the invention Lipopeptide according to formula (I), for example as tablets, Dragees, capsules, pills, granules, suppositories, patches, injectables, Solutions, suspensions, emulsions, pastes, ointments, gels, creams, Lotions, powders or sprays, preferably as a suspension or solution for intranasal inhalation.

The Preparation of the pharmaceutical according to the invention Preparations are carried out in the usual way according to the person skilled in the art known methods.

The The invention also relates to methods of treatment and prevention of diseases in humans and animals associated with allergies stand. For this one gives to a person or an animal, which or that requires such treatment, an immunostimulatory acting amount of a pharmaceutical according to the invention Preparation containing at least one inventive synthetic lipopeptide of formula (I). The active substance or the Pharmaceutical preparation according to the invention local or systemic, oral or parenteral (especially mucosal, transdermally, by injection), preferably intrapulmonary or intranasal.

For details, to particularly preferred embodiments and others Features and advantages of the invention will become apparent from the following Description of embodiments in connection with the subclaims, wherein the above and to be explained below individual features in each case and in any combination with each other are claimed.

The Embodiments are purely illustrative and restrictive the scope of the invention is not a.

pictures

Immunostimulatory activity of synthetic bacterial derived lipopeptides as measured by IL-8 secretion of THP-1 cells as a function of concentration (sbLP 1-7).

Immunostimulatory activity of synthetic bacterial-derived lipopeptides as measured by IL-8 secretion of THP-1 cells as a function of concentration (sbLP 8-14).

Immunostimulatory activity of synthetic bacterial derived lipopeptides as measured by IL-8 secretion of THP-1 cells as a function of concentration (sbLP 15-21).

Immunostimulatory activity of the synthetic bacterial derived from the Bacillus cereus spore germination protein GerD lipopeptides sGerD (sbLP 11) compared to Pam ₃ Cys-SK ₄ , as measured by the IL-8 secretion of THP-1 cells depending on the concentration.

Immunostimulatory activity of synthetic lipopeptides derived from the germination proteins of various Bacillus species as measured by IL-8 secretion of THP-1 cells at a concentration of 1 nM.

Immunostimulatory activity of synthetic lipopeptides derived from the germinating proteins GerD of different Bacillus species as measured by IL-8 secretion of THP-1 cells as a function of concentration.

Immunostimulatory activity of truncated structural analogues of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells at a concentration of 1 nM.

Immunostimulatory activity of truncated structural analogues of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells as a function of concentration.

Immune-stimulating activity of alanine-modified structure analogues of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells at egg concentration of 1 nM.

Immunostimulatory activity of alanine-modified structure analogues of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells as a function of concentration.

Immunostimulatory activity of correspondingly derived structural analogues of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells as a function of concentration.

Immunostimulatory activity of the Pam ₂ Cys and Pam ₃ Cys structural analogs of Bacillus cereus-derived lipopeptide sGerD as measured by IL-8 secretion of THP-1 cells at a concentration of 1 nM.

Immunostimulatory activity of 2,3-dihydroxypropylcysteine-free structure analogues of Bacillus cereus-derived lipopeptide sGerD, measured by IL-8 secretion of THP-1 cells at 10 μM and 1 nM, respectively.

Treatment protocol for the asthma and sensitization model in the BALB / c laboratory mouse.

Respiratory hyperreactivity of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Modulation of eosinophilic granulocytes in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Modulation of lymphocytes in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Modulation of neutrophil granulocytes in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Number of macrophages in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

IgE concentration in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Serum IgE concentration of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

IgG1 concentration in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

IgG1 concentration in serum of sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

IgG2a concentration in the bronchoalveolar lavage of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

IgG2a concentration in the serum of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

Modulation of interleukin-5 production by the spleen cells of the sensitized laboratory mice after treatment with different doses of the lipopeptide LPM compared to the control groups.

embodiments

Example A - Preparation of Synthetic Bacterial Lipopeptides Pam ₂ Cys-AQEKEAKSELDYD and Pam ₃ Cys-AQEKEAKSELDYD

In This embodiment uses the general methodology for the preparation of the lipopeptides according to the invention described with the methods of solid phase synthesis. structural modifications can be avoided by omitting individual synthesis steps or adding from the skilled synthetic chemist known Steps are made.

Becomes in the synthesis of the extraordinary amino acid S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteine (Dhc) the racemic Synthesis building block uses RS-2,3-Epoxy-1-propanol, receives a diastereomeric mixture of the target compound. By use of the enantiomer R-2,3-epoxy-1-propanol becomes immunomodulatory more active RR stereoisomer obtained from Dhc.

The Synthesis of the peptides is carried out according to classical literature Methods in solution or in an automated process at the solid phase. The process steps for the synthesis on solid phase are for almost all amino acid sequences equal. For this reason, in the example, only the principal Synthesis process described, without being peculiar to individual peptides enter into.

a) Synthesis of the peptide AQEKEAKSELDYD with automated solid-phase synthesis

The Peptide AQEKEAKSELDYD is synthesized on a synthesis robot from a Fmoc-L-Asp loaded synthetic resin by the Fmoc peptide synthesis method built up. This is the loaded trityl chloride-polystyrene resin (8 mg, 7 μmol) weighed into a reactor. The amino acids are dissolved in a 1-hydroxybenzotriazole solution (0.5 M in N, N-dimethylformamide) to 0.5 M solutions. Subsequently, the filled reactors and Vials placed on the intended positions of the synthesis robot.

In the first step, the cleavage of the Fmoc protective group takes place according to the program sequence shown in Tab. Tab. 4: Cycle for cleavage of the Fmoc protective group. synthesis step reagents Volumes [μL] Duration [min] Start resin swell N, N-dimethylformamide 800 2 1. deblocking 30% piperidine in N, N-dimethylformamide 200 5 2. Washing N, N-dimethylformamide 300 1 3. Deblocking 30% piperidine in N, N-dimethylformamide 200 10 4. Wash, 7x N, N-dimethylformamide 300 1

Subsequently the corresponding peptide is built up on the resin.

This is done by the stepwise linking of the respective Amino acids. Immediately after each coupling, the N-Fmoc protecting group becomes the just-coupled amino acid cleaved to the binding the next amino acid at the released To enable amino group.

The program sequence of the synthesizer robot is shown in the following table. Tab. 5: Cycle for the synthesis of the peptides. synthesis step reagents Volumes [μL] Duration [min] 1st clutch 3 MN, N'-diisopropylcarbodiimide in N, N-dimethylformamide, amino acid solution 25 100 120 2. Wash, 3 × N, N-dimethylformamide 200 1 3. Deblocking 30% piperidine in N, N-dimethylformamide 200 5 4. Wash, 3x N, N-dimethylformamide 300 1 5. Deblocking 30% piperidine in N, N-dimethylformamide 200 10 6. Wash, 7x N, N-dimethylformamide 300 1 7. Continue with step 1.

b) Synthesis of the N, N'-bis (fluorenylmethoxycarbonyl) - [R] -cystin bis-tert-butyl ester (Fmoc-Cys-OtBu) ₂

L-cystine-bis-tert-butyl ester (5.5 g, 13 mmol) and Fmoc-N-hydroxysuccinimidyl ester (7.04 g, 21 mmol) are dissolved in tetrahydrofuran (15 mL). While stirring add N-ethylmorpholine (3.83 g, 4.2 mL, 30 mmol) in tetrahydrofuran (7.5 mL).

The Reaction solution is stirred for 3 h at room temperature and then with the help of the rotary evaporator until evaporated to dryness. The residue is taken up in ethyl acetate (100 mL) and in a separating funnel with potassium bisulfate solution (5%, 3 x 80 mL) and water (3 x 80 mL). After drying with sodium sulfate, the organic phase is Rotary evaporator evaporated and the residue as an oil receive.

This residue is dissolved in dichloromethane / methanol (1: 4, 385 ml) by means of an ultrasonic bath and recrystallized (18 h, -20 ° C.). The resulting colorless precipitate is filtered through a suction filter, washed with tert-butyl alcohol / 2-propanol (1: 1, 2 × 100 mL) and dried in vacuo.
Yield (Fmoc-Cys-OtBu) ₂ : 7.48 g (72%); [M + H] ⁺ : 797

c) Synthesis of the Nα -fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) (2RS) -propyl] - [R] -cysteine tert -butyl ester (Fmoc-RS-Dhc-OtBu)

The (Fmoc-Cys-OtBu) ₂ (4.96 g, 6.2 mmol) is dissolved in dichloromethane (39 mL). Subsequently, zinc (2.84 g, 43.4 mmol) and a solution (20.7 mL) of methanol, hydrochloric acid (32%) and sulfuric acid (98%) (100: 7: 1) are added with vigorous stirring.

To 15 min reaction time at room temperature becomes RS-2,3-epoxy-1-propanol Add (45.9 g, 41.3 mL, 62 mmol) to the reaction mixture. The whole is further stirred in an oil bath (5 h, 40 ° C). Subsequently, the solution is on a rotary evaporator evaporated to half volume, with potassium bisulfate solution (5%, 5.2 mL) and stored at -2 ° C for 16 h.

The product is extracted with dichloromethane (3 x 50 mL). After drying with sodium sulfate, the organic phase is evaporated on a rotary evaporator to dryness.
Yield (Fmoc-RS-Dhc-OtBu): 5.67 g (96%); [M + H] ⁺ : 474

In analogously, the RR stereoisomer of Fmoc-Dhc-OtBu is under Use of R-2,3-epoxy-1-propanol obtained.

d) Synthesis of Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteine (Fmoc-RS-Dhc-OH)

Fmoc RS-bile duct-OtBu (5.67 g, 12 mmol) is dissolved in trifluoroacetic acid (99%, 142 mL) and the reaction mixture for 1 h at room temperature touched.

The solution is then evaporated on a rotary evaporator, treated with N-hexane (50 mL) and diethyl ether (30 mL) and evaporated again on a rotary evaporator. The viscous, oily residue is then dissolved in tert-butyl alcohol / water (80%, 100 mL) and lyophilized (64 h).
Yield (Fmoc-RS-Dhc-OH): 3.03 g (60%); [M + H] ⁺ : 418

e) Synthesis of Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD

One loaded with the side-chain protected peptide AQEKEAKSELDYD Synthetic resin (5.22 μmol) is reacted with N, N-dimethylformamide (100 μL) and allowed to swell for 5 min in a reactor.

To filtering off the N, N-dimethylformamide supernatant N, N-diisopropylethylamine (3M in N, N-dimethylformamide; 12.2 μL; 36.6 μmol; 7 equivalents), Nα -fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteine (15 mg in 100 ul of a 0.5 M 1-hydroxybenzotriazole solution in N, N-dimethylformamide; 36.6 μmol; 7 equivalents) and Diisopropylcarbodiimide solution (3 M in N, N-dimethylformamide; 12.2 μL; 36.6 μmol; 7 equivalents) to the resin Pipette.

The Resin suspension is then added for 2 h 37 ° C incubated in the heating block. Thereafter, the resin with N, N-dimethylformamide (3 x 600 μL) and dichloromethane (3 x 600 μL) washed.

f) Esterification of Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD with palmitic acid

The with Nα -fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD loaded resin (5.22 μmol) is treated with N, N-dimethylformamide (100 μL) and allowed to swell for 5 min in a reactor.

To filtering off the N, N-dimethylformamide supernatant N, N-diisopropylethylamine (3M in N, N-dimethylformamide; 24.4 μL; 73.2 μmol; 14 equivalents), palmitic acid (1 M in N, N-dimethylformamide / dichloromethane (1: 1; 104 μL; 104 μmol; 20 equivalents) and diisopropylcarbodiimide (3M in N, N-dimethylformamide; 24.4 μL; 73.2 μmol; 14 equivalents) to the resin Pipette. After 5 min, 4-dimethylaminopyridine (1M in N, N-dimethylformamide; 30 μL; 30 μmol; 5.7 equivalents).

The Resin suspension is then added for 2 h 37 ° C incubated in the heating block. Thereafter, the resin with N, N-dimethylformamide (3 x 600 μL) and dichloromethane (3 x 600 μL) washed. The esterification will once again be under the same Test conditions repeated.

g) Cleavage of the Fmoc and the side chain protecting groups

The with Nα -fluorenylmethoxycarbonyl-S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD loaded resin (5.22 μmol) is treated with dichloromethane (100 μL) added and allowed to swell for 5 min in a reactor.

To the filtration of the dichloromethane supernatant is piperidine (30% in N, N-dimethylformamide, 200 μL) pipetted to the resin and leave the mixture at room temperature for 10 minutes. After that will the resin with N, N-dimethylformamide (3 × 600 μL) washed. Then again piperidine (30% in N, N-dimethylformamide; 200 μL) to the resin and pipetted Leave the suspension at room temperature for 20 minutes. After that it will be Resin with N, N-dimethylformamide (3 x 600 μL), dichloromethane (3 x 600 μL) and diethyl ether (4 x 600 μL) washed.

The Completeness of the reaction is determined by the ninhydrin test controlled.

h) Cleavage of the lipopeptide S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD from the synthesis resin

The Cleavage solution trifluoroacetic acid / triisopropylsilane / water (92.5: 5: 2.5; 300 μL) is added to the resin (2.6 μmol). Pipetted and leave the suspension for 2 h at room temperature. Subsequently The cleavage solution is compressed with compressed air into a test tube filtered. Subsequently, another 200 μL cleavage solution pipetted to the resin and the suspension again for 30 min at room temperature leave.

After re-filtration of the cleavage solution with compressed air, the resin is washed with dichloromethane (200 μL). The filtrate and washings are combined and evaporated on a rotary evaporator. The residue is digested with diethyl ether (3 × 300 μL) and then centrifuged. The sediment is taken up in tert-butyl alcohol / water (4: 1, 2 ml) and freeze-dried (18 h).
Yield (Pam ₂ - (2RS) -Hc-AQEKEAKSELDYD): 5.6 mg (98%);
M: 2180; [M + 2H] ²⁺ : 1091; [M + 3H] ³⁺ : 728

i) Acylation of S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD with palmitic acid

The with S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [R] -cysteinyl-AQEKEAKSELDYD loaded resin (5 mg, 2.6 μmol) is treated with N, N-dimethylformamide (100 μL) and allowed to swell for 5 min in a reactor.

To filtering off the N, N-dimethylformamide supernatant N, N-diisopropylethylamine (3M in N, N-dimethylformamide; 12.2 μL; 36.6 μmol; 14 equivalents), palmitic acid (1 M in N, N-dimethylformamide / dichloromethane (1: 1, 36.6 μL, 36.6 μmol; 14 equivalents) and diisopropylcarbodiimide (3M in N, N-dimethylformamide; 12.2 μL; 36.6 μmol; 14 equivalents) to the resin Pipette.

The Resin suspension is then added for 2 h 37 ° C incubated in the heating block. Thereafter, the resin with N, N-dimethylformamide (3 x 600 μL) and dichloromethane (3 x 600 μL) washed.

The cleavage of the product from the resin is carried out according to the method described under h).
Yield (Pam ₃ - (2RS) -Hc-AQEKEAKSELDYD): 5.6 mg (89%);
M: 2418; [M + 2H] ²⁺ : 1210; [M + 3H] ³⁺ : 807

Example B - Determination of immunostimulating Activity of synthetic bacterial lipopeptides IL-8 secretion of THP-1 cells

The immunostimulatory activity of lipopeptides was characterized by the IL-8 secretion of THP-1 cells, a human monocytic leukemia cell line (DSMZ Braunschweig, No. ACC 15). The THP-1 cells were cultured at 37 ° C in RPMI 1640 cell culture medium with 10% fetal bovine serum (PAN Biotech) in a 5% CO ₂ enriched atmosphere.

To carry out the determinations, 180 μL (5 × 10 ⁴ cells) were added to the wells of a microtiter plate. After 1 h incubation at 37 ° C, the cell suspensions were mixed with the lipopeptide solutions.

To was initially a 1 mM lipopeptide stock solution made in DMSO. This was 1:20 with tert-butyl alcohol / water (4: 1, v / v) and then 1: 500 with the cell culture medium further diluted. The final dilution to the final concentration desired in the test was carried out by the choice of volume added to the cell suspension of this 100 nM lipopeptide solution. As a control, a lipopeptide-free Solution containing the same amounts of DMSO and tert-butyl alcohol contains, carried.

To 5 h incubation of the lipopeptides with the cell suspension were the Cell culture supernatants removed and until measurement frozen at -80 ° C.

The determination of the IL-8 concentration was carried out in a sandwich ELISA according to the principle of a non-competitive assay. The ELISA was performed in microtiter plates with a maximum volume of 300 μL per well at room temperature (Nunc-Immuno ^™ 96 MicroWell ^™ Plates / MaxiSorp ^™ ). Per plate, two IL-8 solutions of known concentration (Recombinant Human Interleukin-8 Standard / Sigma / Product No. I 1645) were included as an internal standard.

a) coating the microtiter plate with human anti-IL-8 antibody

The Microtiter plates were washed with an antibody to human Coated IL-8. This so-called "coating" is based on a nonspecific hydrophobic, noncovalent, interaction between the support material of the microtiter plate and the Antibody.

A solution of the primary antibody (human anti-IL-8 antibody / Conc. 0.5 mg / mL / BD Biosciences Pharmingen / Product No. 554716) with PBS buffer (pH 7.2 / Gibco ^™ Invitrogen Corporation / Product No. 14200-067) to 1 μg / mL. Into each well of the microtiter plate was added 100 μL of this solution pi pipette by. The microtiter plate was then capped and incubated for 16 h at room temperature.

b) washing step

To the incubation was the remaining free antibody removed by washing.

To The plate was washed 3 times with 250 μL washing buffer (50 mM Tris (pH 8.0) + 0.2% Tween 20 in ultrapure water) each well, washing the wash solutions after each 30 s residence time by decantation and knocking the plate were removed.

c) blocking step

Around later unwanted adsorption of constituents to avoid from the test substance, possibly uncoated surfaces were in the microtiter plates with BSA solution (Bovine Albumin / Sigma / Product No. A7906) saturated.

To 200 μL of the blocking buffer (4% BSA solution in 9 parts ultrapure water and one part PBS buffer pH 7.2) in each Well the microtiter plate pipetted and the plates for 60 min at Room temperature incubated.

d) washing step

The excess Blocking buffer was removed according to step b).

e) Addition of cell culture supernatants

Subsequently the cell culture supernatants were coated in the Microtiter plate, in whose wells the immobilized antibodies specifically bind the interleukin-8 from the antigen-containing sample, given.

To 50 μL of blocking buffer were presented per well, add 50 μL each of the sample to be tested and the whole Incubated for 90 min at room temperature. Each sample was in triplicate examined.

f) washing step

To the antigen-antibody reaction became unbound sample material according to step b) removed from the microtiter plate.

g) Incubation with secondary antibody

Subsequently was a biotinylated secondary antibody in given the wells that oppose another determinant of interleukin-8 is directed and binds to them.

The Solution of the secondary antibody (biotin mouse anti-human IL-8 antibody / conc. 0.5 mg / mL / BD Biosciences Pharmingen / Product no. 554718) was added with blocking buffer diluted to a concentration of 1 μg / mL. Per Well, 100 μL of this solution were pipetted and The microtiter plate is incubated for 60 min at room temperature.

h) washing step

To the reaction was the excess of antibodies according to step b) removed from the microtiter plate.

i) Incubation with streptavidin-horseradish peroxidase

Last the horseradish peroxidase conjugated streptavidin was added, that to the biotin molecules of the secondary antibody binds.

For this purpose, the streptavidin-HRP solution (streptavidin-HRP (horseradish peroxidase) / conc. 1.25 mg / mL / PIERCE ENDOGEN / Product-No. 21126) with blocking buffer to 0.25 μg / mL. Per 100 μL of this solution were pipetted into the wells and the whole incubated for 60 min at room temperature.

j) washing step

To the reaction became the excess enzyme accordingly Step b) removed.

k) adding the substrate solution 3,3 ', 5,5'-tetramethylbenzidine

The as a substrate added 3,3 ', 5,5'-tetramethylbenzidine is of the bound to the biotinylated secondary antibodies Peroxidase reacted to a blue end product.

To 100 μL of the substrate solution (3,3 ', 5,5'-tetramethylbenzidine / Sigma / product no. T0440) per well and the microtiter plate for 5 min at room temperature incubated in the dark. The reaction was by addition of 1 M sulfuric acid completed. This resulted in a color change from blue to blue yellow.

l) detection

The The optical density of the solution was measured at 450 nm using a microtiter plate reader Spectra MAX 340 (Molecular Devices). The IL-8 concentration was determined from the calibration curve.

Example C - Detection of allergy-protective Potency of the Lipopeptides of the Invention in the Standardized Animal Model - Methods

The complete allergy-protective potency, activity and effect of the lipopeptides of the present invention through immunological and cell biological functional investigations determined in the standardized animal model of the mouse in vivo.

in the The following are the methods of sensitization and induction of allergic asthma in mice, for treatment with the lipopeptide solutions and for the analysis of allergic and inflammatory reactions.

The treatment protocol with the timing of the model experiment is in shown graphically.

a) Sensitization and induction of allergic asthma in mice with ovalbumin (OVA)

The Laboratory animals, seven-week-old female mice of the inbred strain BALB / c (Charles River, Sulzfeld), were 14 days to the surrounding area used. They had access to water and water at will Food.

Mice were sensitized by intraperitoneal injection of a mixture of 20 μg ovalbumin (Grade V / Sigma) and 2.2 mg aluminum hydroxide (ImjectAlum / Pierce) twice in a total volume of 200 μL PBS buffer. Aluminum hydroxide was used as an adjuvant because it is known to elicit T _H 2-stimulated immune responses caused by the production of cytokines IL-4, IL-5 and IL-13 and antibodies of the isotype IgE and IgG1 (corresponding to IgG4 in humans).

Around subsequently an allergic reaction in the lungs of the Mice, so the acute allergic asthma The mice were raised on day 28 and day 38 each for 20 min with an aerosol of a 1% ovalbumin solution treated. For this, the mice were placed in an 11 L Plexiglas chamber set to which a Pari-Boy Turbo Aerosol Generator Pari was connected.

• 1) verstärkte Reaktion gegen Methacholin (bronchiale Hyperreaktivität)
• 2) verstärkte Infiltration von eosinophilen Granulozyten und Lymphozyten in das Atemwegslumen
• 3) Produktion von Ovalbumin-spezifischen Antikörpern der Isotypen IgE und IgG1
• 4) Produktion von Interleukin-5 nach in vitro Restimulierung der Milz-Zell-Lymphozyten mit Ovalbumin

After the second application of the OVA aerosol, the mice could be diagnosed with the symptoms of acute allergic asthma for a few days:

• 1) enhanced reaction against methacholine (bronchial hyperreactivity)
• 2) Increased infiltration of eosinophilic granulocytes and lymphocytes into the airway lumen
• 3) Production of ovalbumin-specific antibodies of the isotypes IgE and IgG1
• 4) Production of interleukin-5 after in vitro restimulation of the spleen cell lymphocytes with ovalbumin

b) Measurement of bronchial hyperreactivity

One crucial diagnostic parameter to an allergic asthmatic Diagnose disease is bronchial hyperreactivity. This is a hypersensitivity of the smooth Musculature of the bronchi against nonspecific irritation. For diagnosis in humans, low concentrations of histamine are applied. Subsequently, the airway resistance is measured. asthmatic Individuals show a histamine concentration dependent stronger bronchoconstriction and thus a higher Airway resistance as healthy persons.

to Provocation in mice is used instead of histamine Methacholine, as the bronchi of mice are not on histamine react. The mice are transformed into a whole body plethysmograph (Buxco Electronics), through whose chambers a continuous flow of air flows, set. From the changes in pressure due to respiration the so-called Penh value is calculated for each respiratory cycle, which correlates with the airway resistance. In hyperreactive mice the airway resistance increases with the methacholine concentration, which is expressed by the increase of the Penh value.

The Measurement of bronchial hyperreactivity was 24 hours after the last OVA aerosol exposure. For the measurement the mice were after a habituation period of 10 min in plethysmograph for 7 min with PBS aerosol in contact brought. Then took place in each case for 7 min exposure to ascending methacholine concentrations (6 mg / mL, 12 mg / mL, 25 mg / mL, 50 mg / mL / Sigma). The Penh value was continuously measured and averaged over periods of 30 s. The highest Penh mean of a concentration level was then plotted against the methacholine concentration. The area under this dose-response curve is the greater the more the mice react to methacholine, it correlates with the hyper-responsiveness of the animals.

c) examination of the cellular Composition of bronchoalveolar lavage (BAL)

The in healthy volunteers or untreated mice in lung tissue and leukocytes found in the lumen of the respiratory tract are almost exclusively Macrophages. Other leukocyte types are usually undetectable. In connection with allergic asthma it does not come out yet quite clear causes for massive infiltration of the respiratory tract by eosinophilic granulocytes and to a lesser extent Lymphocytes and neutrophils.

Around the composition of the leukocyte infiltrate in the respiratory tract The mice were examined three days after the last one OVA aerosol exposure killed. The trachea was uncovered and the lumen of the airways via a 24G venous indwelling cannula washed twice with 1 ml each of isotonic buffer (bronchoalveolar Lavage). The total number of leukocytes that look that way the lungs were flushed out by Neubauer Counting chamber counted microscopically.

Subsequently The leucocytes were treated with a Zyto centrifuge Shandon centrifuged on slides and with the HAEME quick-dyeing kit the company Labor + Technik Eberhard Lehmann, as in the product supplement described, stained. After staining 300 cells on the slide were counted microscopically and according to the common criteria in the different Divided into leukocyte subpopulations.

d) Measurement of OVA-specific antibodies of the isotype IgE, IgG1 and IgG2a

The Awareness of a. Allergen can be determined by the determination of Allergen-specific antibodies of classes IgG1, IgG2a and IgE, for their quantification, a laboratory internal standard serum is used. In the used murine allergy model correlates However, the increase in the specific immunoglobulin titer also with the increase in total immunoglobulin concentration, as already has been shown in the literature. Therefore, for the description of the Sensitization degree the total immunoglobulin titer by means of commercially available "sandwich" ELISA Test systems quantified. For the measurement of IgE was used an optEIA test system from BD Biosciences. For the determination of IgG1 and IgG2a were test systems of the company Bethyl-Laborstories used. The test procedure was based on the Manufacturer's instructions.

To determine the antibodies in the serum of the mice, blood was taken from the tail vein of the mice two days after the last application of OVA aerosol. After clotting the blood, the serum was collected by centrifugation. To determine the antibodies in the BAL fluid of the mice this was won as described above.

to Quantitation was a dilution series on each ELISA plate a standard serum with known IgG1, IgG2a or IgE concentrations carried. By comparing the measured values for the unknown samples with those of the standard, the concentration can in the serum and BAL samples.

e) Measurement of in vitro IL-5 production of spleen cells after restimulation with OVA

Respiratory eosinophilia, as a result of allergic sensitization, is dependent on the secretion of T _H 2 -type cytokines, specifically IL-5, by the lymphocytes. Thus, the secretion of IL-5 correlates with the intensity of eosinophilic airway inflammation. In the respiratory tract of healthy animals no IL-5 is produced.

To determine the ability of the spleen cells to produce the cytokine IL-5, the mice were sacrificed three days after the last OVA aerosol exposure and spleens removed. By mechanical comminution, a cell suspension was produced therefrom and the erythrocytes were dissolved. The spleen cells were then incubated in cell culture medium (RPMI-1640 with 10% fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin / all biochrome) (cell density of 5 x 10 ⁶ cells per mL of medium).

to Stimulation of specific cytokine production was up to OVA a final concentration of 50 ug / mL was added. After 48 h incubation, the supernatants were removed and the IL-5 concentration with an optEIA kit (BD Biosciences) accordingly the product description determined.

f) Treatment of mice with Lipopeptide solutions via the respiratory tract

The Lipopeptides in DMSO were added to a 5 mg / mL stock solution and then with PBS buffer on the required concentrations (0.1 mg / mL, 0.4 mg / mL, 1 mg / mL) diluted.

Around a dose-dependent effect of the immunostimulatory To capture the effect, groups were formed with different Doses of the lipopeptide (5 μg, 20 μg, 50 μg) were treated. 5 animals were treated per concentration level. A control group (5 animals) was treated with a lipopeptide-free DMSO solution (4% in PBS buffer), another (5 animals) treated with pure PBS buffer instead of the lipopeptide.

The Lipopeptides according to the invention were given to the mice applied intranasally in dissolved form. For this purpose, the Animals anesthetized with a mixture of ketamine and Rompun. Subsequently 50 μL solution per animal were administered intranasally.

The treatments were carried out in parallel with the sensitization with OVA, a total of 14 times to the treatment regimen ( ) specified times.

g) Statistical evaluation

The Results of the experiments were made using the methods of robust Statistics evaluated. The significance of the effects achieved was tested with the Mann-Whitney U-Test. In the diagrams the individual values as well as the median were shown:

Example D - Detection of Allergy Protective Potency of the Lipopeptides of the Invention in the Standardized Animal Model - Results

In order to demonstrate the effectiveness of the lipopeptides according to the invention in the treatment of allergic diseases, they were tested in the standardized mouse model for allergic asthma with the methods described in Example C. Exemplary of the lipopeptides according to the invention, the effects of a treatment with RR-Pam ₃ Cys-AQEKEAKSELDYD (LPM, SEQ ID NO 3) are shown here.

a) Asthma symptoms

The most extensive attempt to demonstrate the biological activity of the lipopeptides according to the invention is the investigation of the asthma symptoms triggered by the OVA aerosol, in particular the bronchial hyperreactivity.

shows that the allergic sensitivity of the respiratory tract in the asthmatic mice could be greatly reduced by the parallel to the OVA sensitization treatment with the lipopeptide of the invention. The greatest effect was achieved with a lipopeptide dose of 5 μg.

This Result indicates that the lipopeptide is already in lower Concentration extremely effective for prevention and treatment of allergic diseases. Too high dosages limit the success by additional Irritation of the respiratory tract slightly. However, too at 50 ug lipopeptide still a significant reduction of Asthma symptoms visible to the control group.

b) inflammation in the bronchi and leukocytes

at Asthmatic mice reflect eosinophilic granulocytes in the bronchi, in addition to bronchial hyperreactivity, the condition of an airway inflammation. In sensitized mice Increases their numbers greatly, while in healthy mice are not detectable in the respiratory tract.

demonstrates that by treating the mice with the lipopeptide according to the invention, the number of eosinophilic granulocytes infiltrating into the lung fluid can be greatly reduced. For all application doses, these are only slightly to be found in the BAL.

As a result of sensitization, some lymphocytes, which are also undetectable in healthy, non-sensitized mice, were infiltrated into the respiratory tract. The treatment with the lipopeptide solutions showed a slight decrease in the secreted cells of this leukocyte type ( ).

By contrast, the lipopeptide treatment increasingly detects neutrophilic granulocytes as part of the innate rapid immune defense in the respiratory tract. The neutrophils of the sensitized mice did not respond to treatment with PBS or the DMSO control solution ( ).

Macrophages, which on the one hand eliminate foreign pathogens and on the other hand initiate the adaptive immune response through antigen presentation, are the only ever detectable leukocyte subtype in healthy animals. Their number is only marginally increased as a result of sensitization. No significant difference was detectable between the animals treated with the lipopeptide solutions and the control groups ( ).

c) allergic sensitization

The Asthma symptoms in the mice were due to the sensitization triggered with an allergen, resulting in an allergen-specific formation IgE antibody leads.

and show that the intensity of sensitization as a precursor of the acute allergic disease can be influenced by the lipopeptides according to the invention. The concentration of IgE antibodies in the BAL could be greatly reduced by the lipopeptide treatment. The greatest effect was again observed for the lowest lipopeptide dose. On the other hand, the treatment has only a minor effect on the serum IgE level.

Obviously The applied lipopeptide solutions preferably act local. This sets the inhalation of the invention Lipopeptides close as the most effective form of administration.

A significant influence of the lipopeptide treatment on the release of the IgG1 antibodies as part of the T _H 2-stimulated immune response could not be detected both in the serum and in the BAL ( . ).

The influence on the IgG2a antibodies as part of the T _H 1-stimulated immune response was comparable in the BAL and serum ( . ). The slightly increased release caused by the lipopeptides could be expected because it simulates bacterial components.

d) IL-5 production by the spleen cells (in vitro)

The secretion of eosinophilia into the lumen of the respiratory tract as a result of allergic sensitization is dependent on the secretion of T _H 2 -type cytokines, especially IL-5, by the lymphocytes. The release of IL-5 correlates with the intensity of the eosinophilic inflammatory response. The immune cells of non-sensitized animals do not release IL-5.

As with the secretion of eosinophilia into the lung fluid, the IL-5 production of spleen cells could almost be stopped by treatment with the lipopeptides according to the invention ( ). The most effective effect was achieved with the application amounts 5 μg and 20 μg.

All in all could by treatment with the inventive Lipopeptides significantly reduced the allergic reaction to the OVA become. The biochemical effects correlate excellently with the phenomenological observations.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (29)

Lipopeptide with the following general structure

wherein R 1 and R 2, which may be the same or different, represent a saturated or unsaturated acyl radical having from 7 to 25 carbon atoms, R 3 is a saturated or unsaturated acyl radical having from 7 to 25 carbon atoms or H and R 4 consisting of 1 to 25 amino acid residues, physiologically acceptable and in the treated species not per se immunogenic peptide or a pharmaceutically acceptable salt, a stereoisomer, a stereoisomer mixture or a prodrug compound thereof, characterized in that it has an immunostimulatory effect having. Lipopeptide according to claim 1, characterized in that that the peptide R4 from the germination protein GerD of Bacillus cereus spores is derived. Lipopeptide according to Claim 2, characterized that the peptide R4 a) the amino acid sequence AQEKEAKSELDYDQT (SEQ ID NO 1), b) the amino acid sequence AQGKETKSELDYDQT (SEQ ID NO 2), c) the amino acid sequence AQEKEAKSELDYD (SEQ ID NO 3), d) the amino acid sequence AEEKEAEEK (SEQ ID NO 4), e) the amino acid sequence GEEKEEK (SEQ ID NO 5), f) one by replacement of 1-5 amino acids obtained analog amino acid sequence, g) one of them analogs obtained by deleting 1-5 amino acids Amino acid sequence or h) one by extension 1-5 amino acids analog amino acid sequence having. Lipopeptide according to claim 3, characterized in that that the resulting analog peptides R4 with respect to the starting sequences a degree of homology of greater than 55%, preferably about 80%, in particular about 90%. Lipopeptide according to one of the preceding claims, characterized in that the amino acid at position 1 of the peptide R4 a small, sterically undemanding amino acid, preferably alanine or glycine. Lipopeptide according to one of the preceding claims, characterized in that R1 and R2, the same or different can be for a saturated acyl radical with 7 to 25 carbon atoms and R3 for a saturated one Acyl residue with 7 to 25 carbon atoms or H stand. Lipopeptide according to claim 6, characterized in that R1, R2 and R3 represent a saturated acyl radical with 16 C atoms. Lipopeptide according to claim 6, characterized in that that R1 and R2 represent a saturated acyl radical with 16 C atoms and R3 stand for H. Lipopeptide according to one of the preceding claims, characterized in that the dihydroxypropylcysteine as RR stereoisomer is present. Lipopeptide according to any one of claims 1-8, characterized in that it is a mixture of any possible Stereoisomers is present. Lipopeptide according to one of the preceding claims, characterized in that it is synthetically produced or made a Bacillus cereus clone has been isolated. Lipopeptide according to one of the preceding claims, characterized in that the immunostimulatory effect on the Toll-like receptors TLR 1, TLR 2 and / or TLR 6 mediates becomes. Lipopeptide according to claim 12, characterized in that that the immunostimulatory effect on the toll-like receptors TLR 1, TLR 2 and / or TLR 6 the therapy and prophylaxis of allergic Diseases possible. Process for the preparation of a lipopeptide according to one of the preceding claims, characterized that the unusual amino acid is Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [R] -cysteine (Fmoc-RS-Dhc-OH) in solution or on solid phase with a also on solid phase, side-chain protected Peptide is linked and then acylated and that while the target compound obtained as a stereoisomer mixture becomes. Process for the preparation of a lipopeptide according to one of the preceding claims, characterized that the synthetic building block Nα-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2R) -propyl] - [R] -cysteine (Fmoc-RR-Dhc-OH) in solution or on solid phase with a Side-chain protected peptide constructed at this stage linked and then acylated and that thereby the target compound as a stereochemically defined RR derivative is obtained. Use of a lipopeptide according to any one of the claims 1-13, characterized in that it is an immune stimulant in the mammal, preferably in humans. Use of a lipopeptide according to any one of the claims 1-13, characterized in that it is for prophylactic or therapeutic treatment of allergies, preferably allergic Asthma, in the mammal, preferably in humans, suitable is. Pharmaceutical preparation containing at least a lipopeptide according to any one of claims 1-13, characterized characterized in that it optionally contains other ingredients such as pharmaceutical active ingredients, adjuvants and customary, pharmaceutically acceptable carriers and / or adjuvants contains. Pharmaceutical preparation according to claim 18, characterized in that for its preparation, the lipopeptides mixed with at least one or no other ingredient and / or brings in solution. Pharmaceutical preparation according to claim 18, characterized in that the lipopeptides with a physical or biological carriers are associated or associated. Pharmaceutical preparation according to claim 18, characterized in that the lipopeptides are incorporated in liposomes are. Pharmaceutical preparation according to one of Claims 18-21, characterized in that it in one for the oral or parenteral application suitable formulation is present. Pharmaceutical preparation according to claim 22, characterized in that it is in the form of an ointment, a lotion, a suspension, an emulsion, a solution or a Plaster. Pharmaceutical preparation according to one of Claims 22-23, characterized in that it by inhalation, mucosal, intranasal, by applying to the Skin, by subcutaneous, intramuscular or intravenous Injection or directly into the lymph nodes can be applied. Use of a pharmaceutical preparation according to any one of claims 18-24, characterized that it is suitable for simulating a natural microbial environment for the purpose of maturing the immune system. Use of a pharmaceutical preparation according to one of claims 18-24, characterized that it is used to prevent or treat allergic diseases suitable is. Use according to any of claims 25-26, characterized in that the pharmaceutical preparation for the prevention or treatment of hay fever, atopic dermatitis or allergic asthma is suitable. Use according to any one of claims 25-27, characterized in that the pharmaceutical preparation for the prevention of allergic diseases in patients due to a positive family history, a significantly increased Have a risk of developing an allergic disease is. Use according to any one of claims 25-27, characterized in that the pharmaceutical preparation for treating allergic diseases in patients, the first have manifested allergy symptoms is appropriate.

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