WO2019234032A1

WO2019234032A1 - Microemulsion for dermal allergen immunotherapy

Info

Publication number: WO2019234032A1
Application number: PCT/EP2019/064506
Authority: WO
Inventors: Christopher KISELMANN; Christian MÖBS; Wolfgang PFÜTZNER; Frank Runkel; Thomas Schmidts; Christina ZINECKER
Original assignee: Technische Hochschule Mittelhessen; Philipps-Universität Marburg
Priority date: 2018-06-06
Filing date: 2019-06-04
Publication date: 2019-12-12
Also published as: DE102018113509A1

Abstract

The invention relates to the provision of a microemulsion containing at least one allergen for allergen immunotherapy of a human or animal patient in respect of allergens such as bee venom, wasp venom, pollen allergens, allergens of dustmites, animal allergens or food allergens, the microemulsion being suitable for dermal application of the allergens. The microemulsion comprises PEG-8 caprylic/capric glycerides, polyglyceryldioleate, and a water phase, an oil phase and at least one allergen dispersed therein. In vivo tests show that with this microemulsion the allergen penetrates the Stratum corneum and passes into the human or animal body. The induction of allergen tolerance by this innovative allergen immunotherapy technique, and thus the clinical efficacy, has already been demonstrated in vivo in the mouse model based on reduced drops in temperature.

Description

Patent application

Microemulsion for dermal hyposensitization with allergens

The invention relates to a microemulsion for introducing at least one allergen into the skin.

Allergens are substances that can cause hypersensitivity reactions of the immune system in the human or animal organism, so-called allergic reactions. Allergens are usually foreign molecules (usually proteins), which are recognized as harmless in a normal immune response and thus do not trigger an allergic reaction in a healthy organism. In contrast, the allergen is classified by the immune system of an allergic organism as dangerous and triggers an allergic reaction. The first contact with allergens causes sensitization, which is still largely symptomless. Repeated exposure to the same allergen results in a pathogenic allergic inflammatory reaction, which, depending on the nature of the allergen and the type of reaction, can cause typical allergic reactions such as rhinoconjunctivitis allergica ("hayfever"), bronchial asthma, or even anaphylactic shock.

The most well-known and clinically most relevant allergens are: Insect venom allergens, in particular bee and wasp venom, pollen allergens, especially birch and grass pollen, allergens of house dust mites as well as animal and food allergens. The term "allergen" is mostly used synonymously with the actual allergen source. For example, bee venom extract consists of a complex mixture of at least 13 known single allergens. These include various proteins such as phospholipase A2 (Api m 1), apamine, hyaluronidase (Api m 2), melittin (Api m 3), mast cell degranulating peptide, tertiapine, secamine and smaller molecules such as histamine, dopamine and norepinephrine. Wasp venom contains antigen 5 (Ves v 5) as the major allergen, as well as hyaluronidase, phospholipase A and wasp-specific phospholipase B. Among the IgE-mediated allergies, the insect venom allergy is one of the most dangerous allergies, as in allergy sufferers from a conspicuous stinging reaction with redness and swelling to very violent reactions with breathlessness, abdominal discomfort, dizziness, circulatory problems or even to unconsciousness or anaphylactic shock with possible lethal outcome.

Antihistamines and glucocorticoids are mainly used for the symptomatic treatment of an allergic reaction, and adrenaline is available for - especially severe allergic reactions. In contrast, allergen-specific immunotherapy, also known as hyposensitization, is the only recognized form of therapy that can treat allergy causally and thus prevent long-term overreactions of the immune system as part of an allergic reaction. The continuous administration of smaller amounts of allergen is trying to induce a tolerance of the immune system to the allergen in question, so that a renewed allergen contact causes only minor or at best no allergic symptoms. Colloquially, therefore, is sometimes spoken of an "allergy vaccine". In medical parlance, the terms "specific immunotherapy", "anergy immunotherapy" or "desensitization" are in addition to the above-mentioned terms. However, the term "desensitization" is not exactly exact and is considered obsolete because the sensitization to the allergen in question is not reversed by the therapy. It is still preserved, but the immune system reacts to the causative allergen after a successful hyposensitization by a Allergenignoranz and / or anergy either no longer allergic or only at very high allergen levels. Hyposensitization aims to gradually accustom the body to a specific allergen (allergen tolerance), so that allergic symptoms no longer occur when it comes into contact with this allergen. For this purpose, the patients receive over a longer period of time (usually 3-5 years) over and over again the respective allergen administered, whereby the dose is gradually increased during the therapy gradually more and more. If the individual maximum dose (maintenance dose) is reached, it will continue to be administered at regular intervals, depending on the treatment method, daily or every 4 to 6 weeks. By regularly repeated confrontation with the allergen, the immune system "learns" to classify the corresponding allergen again as harmless. Thus, the allergic symptoms decrease in the course of time with successful therapy noticeably and occur at best not at all. The duration of the application in the context of an allergen-specific immunotherapy may extend over several years and depends on how the allergy doses are administered. Currently, the following types of allergen-specific immunotherapy are known:

In subcutaneous immunotherapy (SCIT), the patient receives the allergen dose subcutaneously, ie injected under the skin where the allergen is either present in an aqueous solution or bound to a depot carrier. As soon as the individual maintenance dose is achieved by appropriate dose increases, the therapy is continued at regular intervals of about four to six weeks in order to achieve a tolerance of the immune system to the allergen.

Another method of hyposensitization is sublingual immunotherapy (SLIT). Here, the allergen dose is administered to the patient under the tongue and the allergen is absorbed through the oral mucosa. The administration can be done both in the form of drops and in the form of rapidly dissolving tablets. Tablet-based SLIT has been approved for the treatment of grass and house dust mite allergy. In contrast to the SCIT, the intake of allergens in SLIT must be done daily, but can also be continued by the patient after an initial instruction by the doctor. That's why fewer doctor's appointments are required compared to SCIT.

In SCIT and SLIT, a successful therapy and thus the development of an allergic tolerance is characterized in the early phase by the activation of interleukin-10-secreting regulatory T-lymphocytes, while in the late phase the reduction of the allergen-associated, allergen-specific T-helper (Th ) 2 cells is characterized. On a humorous level, it stands out Hyposensitization through the synthesis of allergen-specific IgG antibodies. These are called blocking antibodies because they prevent the binding of allergens to IgE and thus the IgE-dependent release of histamine from the allergy-relevant effector cells (mast cells and basophils). At the same time they also prevent the IgE-mediated allergen presentation to T cells and thus play an essential role in the maintenance of allergen tolerance. Since in allergies to bee venom or wasp venom a single sting can lead to a life-threatening anaphylactic shock for the allergy, a successful hyposensitization is a life-saving measure. However, redness, swelling or itching of the skin at the puncture site or at the sublingual therapy in the mouth as possible side effects occur. Normally, the side effects are generally low and resolve quickly. In severe cases, however, it can lead to severe swelling of the entire face and / or throat so that untreated laryngeal edema can lead to suffocation death. In addition, the administration of the allergy doses can also be a burden on the immune system, in which a feeling of fatigue in the patients is noticeable on the treatment days. As a side effect, albeit in rare cases, subcutaneous hyposensitization can lead to allergic shock, as described with potentially life-threatening disease.

There is therefore a need for a gentle alternative for hyposensitization, in particular in an allergen-specific immunotherapy with insect venom allergens.

The hyposensitization is also relevant for pets, because every 5th dog has an allergy including u.a. also insect poison allergy and also horses and cats suffer from allergies to insects, pollen, feed mites or mold spores. There is also a need here for a sparing alternative for the hyposensitization in veterinary medicine, in particular in an allergen-specific immunotherapy with insect venom allergens.

A gentle administration of active ingredients takes place dermal, over the skin. The person skilled in the art knows in the dermal application, for example, the bee venom ointment Apireven®, Forapin® or Apisarthron®. These contain highly concentrated bee venom, which is used against rheumatic complaints and joint pain. These ointments are applied to painful areas of the skin, they increase the skin temperature and promote blood circulation. Thus, the lack of oxygen in the painful zone is improved. At the same time there are local, superficial immune reactions, so that the immune cells responsible for the joint inflammation are "distracted" and the rheumatic complaints are temporarily alleviated. Some ointments are alternatively used to prevent wrinkles. In this application, apitoxin is supposed to support the production of the skin protein collagen VII, which promotes the supportive function of the skin and thus counteracts the formation of wrinkles. However, these ointments are expressly not suitable for allergy sufferers and can under no circumstances be used for hyposensitization, since on the one hand the exact composition of the components of the bees glycemic extract is not known, on the other hand the allergen concentration is very high and thus serious anaphylactic reactions can be triggered.

The skilled worker knows that an ointment, a cream but also a microemulsion for dermal application is suitable. A microemulsion is generally a composition (formulation) of water, oil and one or more surfactants that form small droplets such that visible light is not scattered on them. Microemulsions are as transparent as water, while normal emulsions look more like milk. By suitable choice of the components microemulsions can be developed so that they can solvate a variety of different substances. Solvation or solvation is known to those skilled in the art as a process in liquid solutions when molecules of the solvent are associated with molecules or ions of the solute. An interaction of the dissolved particles with the solvent leads to the stabilization of the dissolved particles in the solution. Depending on the amount and type of surfactants used, microemulsions can be penetration-promoting in comparison to conventional preparations (such as ointments, creams, macroemulsions, etc.). The prior art knows some microemulsions, many of which are also suitable for dermal application. However, these are mostly experimental microemulsions which contain components which, while underlining the penetration-promoting potential of microemulsions, are unsuitable for practical use on humans or animals, for example because of their dermato-toxic effects.

Thus, WO201313588A1 describes a microemulsion for dermal application, which has the poison of seafood as allergen. DE3844247A1 describes a microemulsion which is suitable as a patch for transdermal application. In the plaster, an allergen can be introduced. W02007070983A1 describes another microemulsion which is suitable for transdermal application and may generally have allergens. However, this is not suitable for hyposensitization and not for an allergen-specific immunotherapy. WO2014130761 A1 describes a drug delivery system comprising a microemulsion for transporting proteins. It consists of porous particles containing, for example, a toxin, which is then released. Although the toxin may also be an allergen, this system is not suitable for hyposensitization and not for allergen-specific immunotherapy. DE1020130151 12B4 also deals with the problem of desensitizing allergens. The proposal is for a allergen liposome delivery system based on PEG nanoparticles containing a polyethylene glycol-acetal-dimethacrylate (PEG-acetal-DMA) macromolecule. The system is suitable in principle for dermal application, but not for hyposensitization and not for allergen-specific immunotherapy. W020061 13227A1 describes another transdermal drug delivery system comprising a microemulsion for transporting proteins. EP2621499B1 discloses a bee venom composition for the prevention of an allergic reaction. The composition is not a microemulsion. This system is not suitable for hyposensitization and not for allergen-specific immunotherapy. EP2222337B1 discloses a composition for desensitizing against mellitin-free bee venom. The composition is not suitable for dermal application and there is no microemulsion. EP1322330B1 discloses a drug delivery system for transporting bee or wasp venom. The composition is not suitable for dermal application because it is injected directly into the lymph node. There is no microemulsion.

The prior art does not disclose a solution for dermal hyposensitization, in particular for allergen-specific immunotherapy, which is suitable for humans or animals. The known solutions are disadvantageous because:

• The surfactants are used in too high concentrations so that they are damaging to the skin

• Skin-friendly surfactants are used

• The allergen can not penetrate sufficiently deep into the skin, so that no allergy-protective immune response is induced

• Oil components are included, which are not suitable for dermal application to humans or animals

None of the microemulsions containing allergens described in the prior art is suitable for the hyposensitization of humans or animals.

task

The object of the invention is to provide an easily applicable, non-invasive possibility for hyposensitizing a patient (human or animal) against at least one specific allergen. In addition, the invention should be suitable for application via the skin and the particularly sensitive allergic skin and therefore contain components which are characterized by good skin compatibility.

solution

The object is achieved by a microemulsion containing at least one allergen for dermal application having the features of the main claim; advantageous developments of the invention are described in the subclaims. The microemulsion according to the invention comprises PEG-8 caprylic / capric glycerides, polyglyceryl dioleate and a water phase, an oil phase and at least one allergen dispersed therein, PEG-8 caprylic / capric glycerides and polyglyceryl dioleate being present in a ratio of 1: 8 (wt%) , The polyglyceryldioleate is selected from the group consisting of polyglyceryl-6-dioleate or polyglyceryl-3-dioleate. The water phase includes polyol, glycerol and PBS, with polyol selected from the group butylene glycol or propylene glycol. Alternatively, the water phase may additionally comprise a preservative, preferably phenoxyethanol, eg at a level of 1%. The oil phase includes cetearyl ethyl hexanoate and isopropyl palmitate. The at least one allergen is selected from the group of the recombinantly produced or purified individual allergens or from combinations of these single allergens, from purified total allergen extracts and from the group of the immunogenic peptides. The at least one allergen is the major allergen of bee venom (Api m 1) or the main allergen of wasp venom (Ves v 5). Alternatively, the at least one allergen is selected from the group of pollen allergens, house dust mite allergens, animal allergens or food allergens. Combinations of single allergens, eg the major allergen of bee venom with at least one other pollen allergen, animal allergen, food allergen or at least one dust mite allergen, are possible and may be solvated in the microemulsion and administered to a patient for dermal immunotherapy. The at least one allergen or the combination of allergens is used in a concentration of 0.05 mg / ml to 1 mg / ml. Alternatively, the microemulsion according to the invention comprises at least one lipophilic, hydrophilic or amphiphilic gelling agent. In one embodiment, the microemulsion according to the invention comprises at least one adjuvant from the group aluminum hydroxide or monophosphoryl lipid-A. Alternatively, the microemulsion according to the invention comprises at least one fragrance or flavoring agent, for example a bittering agent, when the microemulsion is to be used in animals or a pleasant-smelling fragrance when the microemulsion is used People should be used. Also advantageous is an application in the context of a deodorant.

It is shown a storage stability of at least three months. The microemulsion according to the invention is suitable for the hyposensitization of a human or animal patient, it penetrates the stratum corneum. The efficacy is shown in vitro and in vivo.

Surprisingly, a formulation for a microemulsion has been found in own scientific experiments, which has at least one allergen, preferably a bee venom or wasp venom, so that the allergen can be easily administered through the skin and the microemulsion can thus be used for hyposensitization.

The microemulsion is easy to use and provides a non-invasive way to hyposensitize a patient (human or animal) to the allergen. In addition, the microemulsion is suitable for the patient's extra-sensitive skin and has components with good skin tolerance, none Produces skin irritation. The advantage is that the administration of the at least one allergen can be non-invasive, and thus it can not be z.T. Painful injury to the skin barrier comes, so that the compliance is increased especially by anxious or sensitive patients. In addition, the potential for other risks, e.g. Infections, thrombosis, injury to adjacent tissue by cannulation and permanent damage such as paralysis of nerves, which are very rare but can not be completely ruled out. This makes the microemulsion of the invention particularly practical for patients and target groups, e.g. are particularly at risk due to immunodeficiency.

Another advantage is that the microemulsion, which has at least one allergen, is also suitable for animals, in particular pets, and thus hyposensitization against an allergen is possible at all for them. Skin application is simple, safe and reliable for a veterinarian or pet owner. It has been found that both the human and the animal sublingual application via the tongue and the Mucosa is possible because the microemulsion is very well tolerated there and there is a hyposensitization to at least one allergen.

In the following, the term "allergen" means at least one allergen selected from the group of the recombinantly produced or purified single allergens or combinations of these individual allergens, purified total allergen extracts and from the group of the immunogenic peptides. The at least one allergen is selected from the group of insect venom allergens, in particular bee and wasp venom or pollen allergens, eg birch and grass pollen, allergens of house dust mites and animal and food allergens. The term "allergen" is mostly used synonymously with the actual allergen source. Thus, for example, the bee venom extract consists of a complex mixture of at least 13 known single allergens. Including various immunogenic peptides and proteins such as phospholipase A2 (Api m 1), apamine, hyaluronidase (Api m 2), melittin (Api m 3), mast cell degranulating peptide, tertiapine, secamine and smaller molecules such as histamine, dopamine and norepinephrine. Wasp venom contains antigen 5 (Ves v 5) as the major allergen, as well as hyaluronidase, phospholipase A and wasp-specific phospholipase B. The allergen is often an immunogenic peptide or an immunogenic protein. The microemulsion according to the invention containing at least one allergen passes through the stratum corneum through the skin into the human or animal patient and triggers an immune reaction there. However, finding a suitable formulation for this microemulsion in which both relatively large allergens such as the major allergen of the bee (Api m 1) or the major allergen of the wasp venom (Ves v 5) can be stably formulated as well as the Penetration of sufficient for a clinically and immunologically protective effect sufficient amount of allergen. For this purpose, many different compositions, ingredients and amounts were tested. Surprisingly, it has been found that the microemulsion according to the invention is able to solvate a wide range of allergens of different origin, size and biochemical nature. In micro-emulsions which have been found to be particularly useful in further measurements, the following composition has been found to be particularly suitable:

In this microemulsion, the at least one allergen is introduced and depending on the concentration and amount thereby reduces the proportion of PBS buffer until the total is 100 wt%. The person skilled in the art knows PBS buffer (abbreviated from English phosphate buffered saline, phosphate-buffered saline solution), it is a saline solution containing sodium chloride, disodium hydrogen phosphate, potassium chloride and potassium dihydrogen phosphate:

Substance quantity [g

Sodium chloride 4.00

Potassium chloride 1, 00

Na ₂ HPC> 4 0.72

KH2PO4 0.12

Purified water 500.00

The microemulsion according to the invention is a liquid formulation which can be easily applied eg to a skin area of a human or animal patient by means of a pump spray applicator. In an alternative embodiment, the microemulsion according to the invention additionally comprises at least one lipophilic, hydrophilic or amphiphilic gelling agent, which causes thickening of the microemulsion, so that prolonged application to a skin area is made possible and flow away from the site of application is prevented. Also, an application as an ointment or cream is possible. In a further alternative embodiment, the microemulsion according to the invention additionally comprises an adjuvant, such as, for example, aluminum hydroxide or monophosphoryl lipid A, in order to provoke an increased unspecific immune response. These substances can eg be dispersed / solvated in the water or oil phase. This makes it very easy to adapt the microemulsion to the respective patient groups and to the at least one allergen. It is also possible to use gelling agent and adjuvant together. Depending on the concentration and amount, this reduces the proportion of the PBS buffer until the total sum of the microemulsion containing at least one allergen again yields 100 wt%. For comparative experiments and for control during the test phase, a microemulsion without allergen is also prepared.

The water phase includes polyol, glycerol and PBS, with polyol selected from the group butylene glycol or propylene glycol. Alternatively, the water phase may additionally comprise a preservative, preferably phenoxyethanol, e.g. in a concentration of 1%. In this case, the proportion of the PBS buffer is thereby reduced until the total sum of the microemulsion containing at least one allergen again yields 100 wt%.

The microemulsion according to the invention comprising at least one allergen was tested in the mouse model for its in vivo activity to trigger an immune reaction, and the antibodies IgE and IgG typical for the immune response were determined by means of ELISA. The quantification of the allergy-associated cytokines was carried out by means of ELISPOT and ELISA. Frequency analysis of T-cell subpopulations was performed by ELISPOT and flow cytometry. These methods are very well known to the person skilled in the art and were also carried out according to the standard protocol. From these experiments it can be shown that the dermal application of the allergen-containing microemulsion according to the invention in the mouse model leads to the induction of a clinical tolerance to the corresponding allergen. This result is substantiated by immunological experiments. This leads to the induction of protective, allergen-specific IgG antibodies and to a reduction of the total IgE. At the cellular level, the microemulsion does not cause any toxic effects. An increase in regulatory T cells can not be detected, so that the protective effect in the mouse model is due to the significant increase in IgG antibodies (see also "Exemplary embodiments" point 3).

Furthermore, the penetration behavior of the fluorescently labeled allergen on the pig's ear was demonstrated. It is clear that the microemulsion according to the invention containing at least one allergen penetrates into the stratum corneum, ie does not remain on the skin surface. The stratum corneum is the no longer living outermost skin layer, which is penetrated by the allergen, and then penetrate into the lower layers of skin to meet there on immune cells.

Exemplary embodiments 1. Preparation of the microemulsion

FIG. 1 shows a pseudo-ternary diagram. Pseudo-ternary diagrams are known to those skilled in the art as means for the preparation of various mixtures of oil phase, water phase and surfactant. Each point within the diagram describes a specific percentage mixing ratio of the three named phases. The solid lines represent turbid, anisotropic macroemulsions, whereas the dashed lines show translucent, isotropic microemulsion mixtures. The pseudo-ternary diagram is constructed using the PDMPD method, which is already known to the person skilled in the art from the publication "Development of an alternative, time and cost-saving method of creating pseudoternary diagrams using the example of a microemulsion" (Schmidt et al., 2009, Colloids and Surfaces A: Physicochemical and Engineering Aspects 340 (1 -

3), 187-192.). In addition, the optical density is measured at 600nm. This parameter is used in addition to the visual inspection to characterize the turbidity of a liquid or a mixture. FIG. 2 shows that the microemulsion according to the invention (emulsion A) is an optically clear liquid, while emulsions B and C appear milky-cloudy. The pseudo-ternary

Diagram was taken at about 22 ° C (+/- 2 ° C). The specification of the temperature condition is important, since the temperature can influence the interactions of the individual components of a mixture with each other partly strong; ie a pseudo-ternary diagram is only valid for a specific temperature range. The microemulsion according to the invention comprises PEG-8 caprylic / capric glycerides, polyglyceryl dioleate and a water phase, an oil phase and at least one allergen dispersed therein, PEG-8 caprylic / capric glycerides and polyglyceryl dioleate being present in a ratio of 1: 8 (wt%) , The polyglyceryldioleate is selected from the group consisting of polyglyceryl-6-dioleate or polyglyceryl-3-dioleate. The water phase includes polyol, glycerol, and PBS, with polyol selected from the group of butylene glycol or propylene glycol. The water phase may alternatively additionally

Preservatives preferably include phenoxyethanol.

The oil phase includes cetearyl ethyl hexanoate and isopropyl palmitate. The abbreviation S + CoS stands for surfactant + co-surfactant, in one embodiment for a mixture of PEG-8 caprylic / capric glyceride (Labrasol) as surfactant and polyglyceryl-6 dioleate (plural oleique) as co-surfactant.

2. Preparation of the microemulsion with allergen

The microemulsion according to the invention containing at least one allergen is prepared by a standard method known to the person skilled in the art. The at least one allergen may be lyophilized or dissolved in PBS.

When using at least one lyophilized allergen

a) In the first step, the PBS buffer is prepared

Weigh salts according to the above table into a suitable vessel. It is then filled with 500 ml of purified water. The mixture is stirred until all salts are dissolved without residue. In one embodiment, additionally at least one hydrophilic adjuvant is used. This is weighed into the PBS buffer.

b) In a further embodiment, at least one lipophilic adjuvant is used. In this case, a premix of the Components of the oil phase produced, in which then the adjuvant is incorporated.

c) In another embodiment, at least one hydrophilic or lipophilic preservative, e.g. Phenoxyethanol used. In this case, a premix is created and introduced.

d) Weighing of the individual components of the microemulsion according to the above table.

e) homogenization of the mixture e.g. using a magnetic stirrer (e.g., 1,000 rpm, 2-3 minutes).

f) Wait until any incorporated air bubbles have disappeared from the microemulsion.

g) dispersing the lyophilisate of the allergen (e.g., Api m 1) in the resulting microemulsion.

When using allergen in PBS buffer

a) In the first step, the PBS buffer is prepared: For this, the individual salts are weighed according to the above table in a suitable vessel. It is then filled with 500 ml of purified water. The mixture is stirred until all salts are dissolved without residue. In one embodiment, at least one additional hydrophilic adjuvant is used, which is weighed into the PBS buffer.

b) In a further embodiment, at least one lipophilic adjuvant is used. In this case, a premix of the components of the oil phase is prepared, in which then the adjuvant is incorporated.

c) Weighing of the individual constituents of the microemulsion according to the above table. The required amount of PBS buffer is reduced according to the desired concentration of allergen and supplemented with allergen in PBS buffer.

d) homogenization of the mixture, for example with a magnetic stirrer, until the mixture becomes clear. e) Wait until any incorporated air bubbles have disappeared from the microemulsion.

Depending on the allergen / allergen mixture used, a gentler form of homogenization may also be used instead of homogenization by means of a magnetic stirrer, or the intensity may be varied by adjusting the rotational speed.

Alternatively, at least one lipophilic, hydrophilic or amphiphilic gelling agent is additionally added. In a further embodiment, at least one flavor and / or fragrance is additionally added.

As an exemplary allergen, the native major allergen of the bee venom Api m 1 is used. In an alternative approach, the recombinant major allergen of the wasp venom Ves v 5 is used. Allergen concentrations between 0 to 1 mg / ml are introduced into the microemulsion. Own measurements already show an immunizing effect at an allergen concentration of 0.05 mg / ml, which increases at an allergen concentration of 0.25 mg / ml to 0.625 mg / ml. The immunizing effect is approximately equal from 0.625 mg / ml to 1 mg / ml, so that the preferred allergen concentrations are between 0.25 mg / ml to 0.625 mg / ml. The microemulsion according to the invention with an allergen concentration of 1 mg / ml is mainly used to represent the compatibility on the skin or the coat of the patient.

Possible further allergens are formulated in the microemulsion according to the invention:

All recombinantly produced or purified single allergens such as.

Insect venom allergens such as Api m 10, Ves v 1, pollen allergens such as Bet v 1, Phi p 1 or 5, allergens of house dust mites such as Der p 1, Der p 23, Der f 1, Animal allergens such as Fel d 1 or food allergens like Mal d 1, Cor a 1, Ara h 2, etc.

- Combinations of these single allergens

- purified total allergen extracts (eg bees' gum extract, wasp toxin extract, birch pollen extract, house dust mite extract, etc.) - Peptides, ie specific protein fragments on which the immune system has been shown to be a reaction and which are thus immunogenic, but have no allergenic effect.

As an additional control in the test phase, instead of the microemulsion according to the invention with and without at least one allergen, pure PBS buffer was mixed with the respective allergen concentration. For this purpose, the PBS buffer was prepared as shown above and then the required amount Allergenlyophilisat added in PBS.

In a further embodiment, at least one fragrance or flavoring agent is additionally added to the microemulsion according to the invention comprising at least one allergen. This causes the microemulsion to be better accepted and accepted by a patient. In the case of domestic use, the fragrance is e.g. from a bitter substance, so that the microemulsion is not licked again after the application on the skin or the coat.

3. Effect of the microemulsion on the skin

It can be shown by inventors that the microemulsion according to the invention not only allows a successful tolerance to the corresponding allergen, but also causes no skin irritation. Skin biopsies on the mouse model have shown that the microemulsion comprising at least one allergen, even at a high allergen concentration, does not cause any irritative effects and is therefore well tolerated. This is shown in FIG. 3 in an HE staining of a tissue section of the mouse. Hematoxylin-eosin staining (HE staining) is a dichromatic staining method known to those skilled in the art composed of a dye for the nucleus (hematoxylin) and a dye for the cytoplasm (eosin). Thus, the microemulsion according to the invention differs from known microemulsions in the cosmetic and medical field, all of which have a certain skin irritation-inducing potential. The developed microemulsion also differs from known microemulsions of research, which are merely intended to show the penetration-promoting principle of a microemulsion in general, but in some cases use substances which rule out a practical application to the skin or to a human or animal patient from the outset.

4. Dermal use of the microemulsion with allergen

a) mouse model

Balb / c wild-type mice are used for the mouse model. After application of the microemulsion containing the native major allergen of bee venom (Api m 1) or the recombinant major allergen of wasp venom (Ves v 5) on the skin of a shaved mouse, the immunological effects are tested. For this, the microemulsion comprising various allergen concentrations between 0 and 1 mg / ml allergen is applied to the previously shaved back surface (about 6 cm ² ). The control used in each case is PBS buffer with the corresponding allergen concentration. This should simulate the situation of prophylactic dermal allergen-specific immunotherapy, as it can be used in humans or animals. It is examined whether the application of the microemulsion prevents the induction of an insect venom allergy in the treated mice or the allergic symptoms can be reduced. The decisive clinical test parameter is the triggering of an anaphylactic reaction by the specific allergen provocation, which is measurable as a drop in rectal body temperature in the experimental mouse model used. By allergen challenge, the skilled person understands the administration of allergen. There is a phase of sensitization with administration of low allergen doses (here 5 pg) and a final provocation phase with administration of high doses of allergy (here 100 pg). The provocation serves to cause an allergic / anaphylactic reaction.

To check the effects induced by the dermal immunotherapy with microemulsion (or corresponding controls), the tested mice are prepared, ie there is a withdrawal of blood, spleen and lymph nodes. FIG. 5 shows the detailed treatment scheme using the example of the administration of a microemulsion with the allergen Api m 1. Over the entire experiment (days -28, -14, 0 and 14), blood is taken from the mice for immunological analysis (ie for the frequency analysis of regulatory T cells and antibody determination). During prophylactic dermal allergen-specific immunotherapy, the mice are dosed every 2-3 days, ie on each day -28, -26, -24, -21, -19, -17, -14, -12, -10, -7, - 5 and -3 with defined concentrations between 0 and 1 mg / ml allergen (here the example of Api m 1) treated, which is administered via the microemulsion to the mice. In parallel, PBS control groups are carried on mice. During the subsequent sensitization phase, the mice are treated 3 times every 7 days with 5 μg Api m 1 combined with 1 mg AI (OFI) 3 in PBS intraperitoneally (ip) (day 0, 7 and 14). On day 21, the allergen-specific provocation is performed with the administration of 100 pg Api m 1 per mouse and the body temperature measured (0, 15, 30, 60 and 90 min and 3 and 6 h after provocation). The preparation takes place on day 22.

By administering the efficacy of both the prophylactic dermal allergen-specific immunotherapy and the sensitization based on the temperature drop in vivo can be shown. The severity of the temperature drop as well as the recovery of the original body temperature (recovery) correlates with the severity of the anaphylactic reaction. The mice were given intraperitoneally 100 pg Api m 1 in PBS for provocation as described above, 7 days after the last sensitization (i.e., on day 21), followed by the body temperature drop of the mice over an observation period of 6 hours. Rectal measurement of body temperature was carried out using a thermometer suitable for temperature measurement in mice (Therm 2241-1 from Ahlborn).

In addition, further analyzes are carried out with the samples obtained during the dermal immunotherapy, in the course of the sensitization phase as well as after the provocation (serum, blood, secondary lymphatic organs) in order to draw conclusions about dermal allergen - specific immunotherapy Microemulsion induced to be able to draw immunological effects. These are the determination of IgE and IgG antibodies (by ELISA), the quantification of allergy-associated cytokines (using ELISPOT and ELISA) as well as the frequency analysis of T-cell subpopulations (using ELISPOT and flow cytometry). The highest immunological effects are found for the microemulsion with 0.625 mg / ml Api m 1 in the mouse model, ie at this concentration the greatest tolerance to the allergen is induced. Therefore, the following results are shown using the example of this allergen concentration compared to the negative control (PBS buffer without allergen) and the corresponding PBS control (PBS with 0.625 mg / ml Api m 1).

FIG. 6 shows the results of the in vivo test of the microemulsion according to the invention with the preferred allergen concentration for Api m 1 in a mouse model. The temperature profiles for mice receiving dermal immunotherapy with microemulsion without allergen or with different allergen concentrations in FIG PBS buffer were treated. There is a significant reduction in the allergen-induced anaphylactic temperature drop. This is concentration-dependent, since at lower concentrations of allergen tolerance and the protective immunological mechanisms are less pronounced. However, higher concentration experiments show no improvement in effects induced by dermal immunotherapy. In addition, it should be noted that even further increases in the allergen concentration can even lead to negative effects. Thus, it is known to the person skilled in the art that too high concentrations, especially at the beginning of the therapy, can trigger anaphylactic reactions.

While in the control group with microemulsion without allergen treated mice a maximum temperature drop of 4.5 ° C can be observed, this is significantly reduced in the treated with 0.625 mg / ml allergen mice by about half. In addition, it can be shown that with increasing allergen concentration in the microemulsion faster recovery of the mice comes. It seems the allergen tolerance on one by the dermal allergen-specific immunotherapy induced synthesis of allergen-specific IgG antibodies to be due. On the one hand, this depends on the allergen concentration used, on the other hand on the microemulsion used, since application of 0.625 mg / ml of allergen or higher concentrations in PBS does not lead to any synthesis of allergen-specific antibodies (FIG. 7). On the one hand, the increase in allergen-specific IgG levels in the context of dermal immunotherapy (day -28 to day 0, Figure 7 above) with microemulsion as a function of allergen concentration, on the other hand, the disproportionate increase in allergen-specific IgG level during the sensitization phase (day 0 to 22 Figure 7 below).

This class of antibodies is known to be capable of inhibiting the binding of allergens to IgE, thus preventing the IgE-dependent release of histamine from mast cells and basophils as well as the IgE-mediated allergen presentation to T cells. The increase in allergen-specific IgG antibodies thus leads to a blockade of these immunological mechanisms and prevents the allergic or anaphylactic reaction.

In the case of the mouse model used, this blocking function explains the reduction in temperature drop and the better recovery of the mice with increasing allergen concentration in the microemulsion. Other cell-level immune mechanisms described in the literature (eg, the induction of regulatory T cells (see FIG. 8) or the decline of allergy-associated Th2 cells) discussed in the induction of allergen tolerance by allergen-specific immunotherapy appear to be of concern dermal immunotherapy with the microemulsion used here to play no or a minor role. b) Penetration experiment on the pig's ear

Since the human skin is very similar to the pigskin, in vivo experiments are carried out on the pig's ear for the microemulsion according to the invention. The native major allergen of the venom of the European honeybee (Api m 1) is labeled with a fluorescent dye eg Alexa Fluor-647 (Thermo Fisher Scientific). Subsequently, the penetration behavior of the labeled allergen investigated by means of a Franz diffusion cell experiment. The person skilled in the art knows this standard method. For this purpose, both PBS buffer and microemulsion with fluorescence-labeled allergen is added, the target concentration is 0.08pg / pl. Fresh pig ears are sourced from a local butcher, gently cleaned and dried under lukewarm water. Subsequently, skin samples are prepared, clamped in the Franz cells and loaded with microemulsion with allergen or as control PBS with allergen. After a penetration time of 24 hours, the fluff preparations are embedded in cryogenic medium after rinsing off excess substances and shock-frozen in isopentane. Cryo sections are made and then evaluated by confocal laser scanning microscopy (CLSM). This standard method is known to the person skilled in the art.

FIG. 9 shows the results and images of the prepared pig ear prepared penetration studies by confocal laser scanning microscopy of pig ear cross-sections after 24-hour penetration studies using Franz cells. The scale bar corresponds to 100 pm. The fluorescence signal (left), a transmitted light image (center) as well as the overlay of fluorescence and transmitted light image (right) can be seen. The upper row of the pictures shows fluorescently labeled Api m 1 in microemulsion. The bottom row of the pictures shows as a control fluorescence-labeled Api m 1 in PBS buffer. It can be seen that the microemulsion according to the invention (top row) allows penetration of Api m 1 into the stratum corneum, while the PBS buffer (lower row) does not allow allergen to penetrate the flaut.

5. Storage stability of the microemulsion

In a 3-month stability study, it was shown that the microemulsion remained stable under storage at 25 ° C, 60% relative humidity and 40 ° C, 75% relative humidity. The person skilled in the art knows as features of a stable microemulsion the optical inspection for clarity and / or phase separation. In addition, the optical density is measured at 600nm (ODeoo). Figure 4 shows that over the period of three months of storage, the optical density of the microemulsions has remained nearly constant. A Clouding of the microemulsion could have been detected by increasing OD6O values. Thus, longer-term storage beyond the observed period seems realistic.

pictures

Fig. 1 shows a pseudo-ternary diagram taken at about 22 ° C. S + CoS: mixture consisting of capoylocaproylmacrogol-8-glycerides EP and polygylceryl-6-dioleate in the ratio 1: 1 (mass percent); Oil phase: cetearyl ethylhexanoate, isopropyl palmitate; Water phase: butylene glycol, glycerol, PBS.

FIG. 2 shows a photograph of three selected mixtures (= emulsions) A, B and C, marked in FIG. 1. Mixture B and C clearly show macroemulsions while Mixture A shows the microemulsion according to the invention.

3 shows the HE staining of the biopsy of a Balb / c wild-type mouse (male) in the 8th week after the start of treatment with the maximum dose of Api m 1 (1 mg / ml) used in the microemulsion. The mouse has no skin lesions.

Fig. 4 shows the results of the ODeoo when stored under different temperatures and humidities (rH) of up to three months.

FIG. 5 shows a treatment scheme in the mouse model for a prophylactic dermal allergen-specific immunotherapy.

Fig. 6 shows an allergen tolerance induced by dermal allergen-specific immunomodulation with microemulsion. At an allergen concentration of 0.625 mg / ml in microemulsion, a significantly reduced temperature drop can be seen after provocation with Api m 1 in the bee venom-specific mouse model. Also, the recovery of the mice is significantly increased compared to controls (PBS-treated mice at 0 and 0.625 mg / ml and microemulsion-treated mice without allergen, respectively). Figure 7 shows the increase in allergen-specific IgG levels in dermal immunotherapy (day -28 to day 0, above) as a function of microemulsion, and the disproportionate increase in allergen-specific IgG levels during the sensitization phase (days 0 to 22, bottom).

Figure 8 shows that no induction of regulatory T cells could be detected during dermal immunotherapy (day -28 to day 0) as well as during the sensitization phase (day 0 to day 22). Shown are the results CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells by flow cytometry.

9 shows the result of in vivo penetration studies on the pig's ear with the microemulsion according to the invention and fluorescence-labeled allergen Api m 1 (upper row) and PBS (lower row).

Claims

claims

1. Microemulsion for the dermal application of at least one allergen

Hyposensitization to the at least one allergen comprising PEG-8 caprylic / capric glycerides, polyglyceryl dioleate and a water phase, an oil phase and at least one dispersed allergen therein.

2. microemulsion according to claim 1, characterized in that PEG-8

caprylic / capric glycerides and polyglyceryl dioleate in a ratio of 1: 8 (wt%).

3. microemulsion according to claim 1 or 2, characterized in that

Polyglyceryldioleate is polyglyceryl-6-dioleate or polyglyceryl-3-dioleate.

4. Microemulsion according to one of the preceding claims characterized

characterized in that the oil phase cetearylethylhexanoate and

Isopropyl palmitate includes.

5. Microemulsion according to one of the preceding claims characterized

characterized in that the water phase comprises polyol, glycerol and PBS.

6. microemulsion according to claim 5, characterized in that the

Water phase comprises the preservative phenoxyethanol.

7. microemulsion according to claim 5 and 6, characterized in that the polyol comprises butylene glycol or propylene glycol.

8. Microemulsion according to the preceding claims characterized

in that the at least one allergen is selected from the group of recombinantly produced or purified single allergens, from combinations of these single allergens, from purified whole

Allergen extract and from the group of immunogenic peptides.

9. Microemulsion according to one of the preceding claims, characterized in that the at least one allergen is the main allergen of the bee venom (Api m 1) or the main allergen of the wasp venom (Ves v 5).

10. Microemulsion according to one of the preceding claims characterized

in that the at least one allergen is selected from the group of pollen allergens, allergens of dust mites, animal allergens or food allergens.

11. Microemulsion according to one of the preceding claims characterized

in that the at least one allergen is used in a concentration of 0.05 mg / ml to 1 mg / ml, in particular 0.25 mg / ml to 0.65 mg / ml.

12. Microemulsion according to one of the preceding claims characterized

in that it has at least one lipophilic, hydrophilic or amphiphilic gelling agent.

13. Microemulsion according to one of the preceding claims characterized

characterized in that these are at least one adjuvant from the group

Aluminum hydroxide or monophosphoryl lipid-A.

14. Microemulsion according to one of the preceding claims characterized

characterized in that it comprises at least one fragrance or flavor.

15. Use of a microemulsion according to one of the preceding

Claims for hyposensitization of a human or animal patient.