WO2002049668A1

WO2002049668A1 - Use of compatible solutes as adjuvants for vaccines

Info

Publication number: WO2002049668A1
Application number: PCT/DE2001/004836
Authority: WO
Inventors: Georg Melmer; Thomas Schwarz
Original assignee: Bitop Gesellschaft Für Biotechnische Optimierung Mbh
Priority date: 2000-12-21
Filing date: 2001-12-21
Publication date: 2002-06-27
Also published as: DE10195473D2; AU2002229491A1

Abstract

The invention relates to a pharmaceutical formulation containing proteins, peptides, polypeptides and/or nucleic acids, which contains one or more compounds from the group of the compatible solutes.

Description

USE OF COMPATIBLE SOLUTES AS ADJUVANTS FOR VACCINE

The invention relates to pharmaceutical formulations containing proteins, peptides, polypeptides and / or nucleic acids. It also affects vaccines, adjuvants and stabilizers for pharmaceutical formulations. Furthermore, the invention relates to the use of compatible solutes as stabilizers for pharmaceutical formulations and as adjuvants.

Vaccines traditionally consist of living, weakened pathogens, completely inactivated organisms or inactivated proteins. Although these forms have proven successful in the past, there have been significant disadvantages for serious illnesses:

Certain weakened live viruses in immunosuppressed individuals can nevertheless lead to the corresponding clinical pictures

Completely inactivated vaccines can have pathogenic components that can lead to undesirable side effects.

Some pathogens (such as hepatitis B, C, papillomavirus) are very difficult or impossible to keep in culture.

When using vaccines, unsatisfactory efficacy is often observed in practice, which manifests itself, for example, in an insufficiently high level of antibody training and clinical manifestation despite vaccination. This can be caused by low immunity of the antigens. Possibilities to increase this would be e.g. B. a higher antigen concentration per dose. However, this often fails because the virus multiplication in the finish line can often only achieve a limited virus concentration. One way out would be to multiply in primary cell cultures, where higher virus concentrations can often be achieved. However, this option is not applicable in many cases due to legal regulations.

Therefore, adjuvants are generally added to the vaccines, which are intended to increase the immune response in other ways. Such adjuvants are known.

Factors that influence the choice of adjuvants for vaccines are the cost and the complicability of the preparation, the effects and / or side effects in use and the acceptance as a component in medicinal products.

A group of particulate adjuvants are mineral compounds. From this group, only aluminum compounds, for example aluminum hydroxide, are approved and have been used for a long time and very widely. The advantages of aluminum hydroxide are its few side effects and the inexpensive, simple availability. A depot function with delayed release of the antigen, a possible stabilizing effect on the antigen conformation and the simulation of the humoral immune response were identified as mechanisms of action for aluminum hydroxide.

Disadvantages of aluminum hydroxide as a relatively weak adjuvant are that it is not equally effective for all antigens and the cellular immune response is not affected because Langerhans cells are not reached. Furthermore, aluminum hydroxide can only be used in conjunction with relatively strong antigens. Aluminum hydroxide is also suspected to be involved in the formation of allergies and also neurological diseases such as Alzheimer's disease. Another group of particulate adjuvants are hydrophobic and surface-active substances. These include mineral oils which are used to produce emulsions of various types (e.g. water-in-oil, oil-in-water, water-in-oil-in-water). These types of emulsion have different immunomodulatory effects, stabilizing effects on the antigen presentation on effector cells and depot effects when the antigen is released in the body. Such adjuvants cannot be used in humans, but only in animals.

Various new approaches to vaccine development have been undertaken in the past 10 years and are expected to have significant benefits. These new approaches use recombinant proteins or protein subunits, synthetic peptides or plasmid DNA. Although these methods have significant advantages such. B. have reduced toxicity, they are often not very immunogenic. This is especially true for vaccines based on recombinant proteins or peptides. There is therefore a great need for new immunological adjuvants that can be combined effectively and compatible with new vaccines.

Immunological adjuvants were originally defined as substances that, when combined with certain specific antigens, produce more immunity than the antigen itself. However, despite intensive research, only one adjuvant is approved by the American Food & Drug Administration (FDA). These are aluminum-based mineral salts (generic alum). Although alum was previously considered to be very safe, there is a strong need to remove alum as an adjuvant. Aluminum is suspected to be involved in Alzheimer's disease and possibly also other diseases. But above all, alum is only a very weak adjuvant. There is also great concern because alum can trigger an (IgE) response that is associated with some allergenic reactions in humans.

Safety is of great importance for the development of adjuvants. Although a number of adjuvants were in advanced clinical trials and were highly effective, they were found to be too toxic for one Routine use. In addition to safety, biostability, administration, costs, biodistribution and universality are also important when using a new adjuvant.

The use of adjuvants is often empirical in nature, so there is no clarity about the mode of action. It is still difficult to make predictions about suitable adjuvants.

Vaccines / vaccines within the scope of the invention mean both vaccines / vaccines for active as well as for passive immunization. However, the invention preferably relates to vaccines / vaccines for active immunization.

Compatible solute

Extremophilic microorganisms synthesize certain small molecules to protect against extreme living conditions. These connections are called Compatible Solutes. These compatible solutes from extremophilic microorganisms include di-sugar alcohols from C3 to C6 sugars (especially di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG) and 1,1-di-glycerol phosphate (DGP)), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A), ectoin, hydroxyectoin and / or di-mannosyl-di-inositol phosphate (DMIP) and / or derivatives, e.g. B. acids, salts or esters of these compounds.

Traditional vaccines are mostly killed or weakened pathogens. These vaccines are generally relatively stable and can be used without any problems. In contrast to traditional vaccines, most of the modern vaccines contain synthetic, recombinant or highly purified antigens in the form of proteins or peptides. These vaccines are considered to be safer in their targeted vaccination, but generally have the disadvantage of lower stability and immunogenicity. In order to compensate for this disadvantage, additives are added to the vaccines in order to strengthen and prolong the stability and optionally also the specific immune response to antigens.

Vaccines that serve to protect the resistance situation must have a high stability almost more than other pharmaceuticals. Transport and distribution of the goods to the end user are particularly problematic. The consumer also expects that the purchased product does not spoil immediately after purchase, but can be kept in stock for a few days or weeks, depending on the product. Conventionally, labile vaccines or pharmaceuticals are therefore preserved with stabilizing agents or by lyophylization, which are mostly used in the form of chemical monosubstances or their combinations. The aspect of bioavailability and the optimal biological half-life is often taken into account in addition to galenics by certain formulations.

Traditional vaccines are mostly killed or weakened pathogens. These vaccines are generally relatively stable and can be used without any problems. In contrast to traditional vaccines, most of the modern vaccines target the use of synthetic, recombinant and highly purified antigens in the form of proteins or peptides. These vaccines are considered to be safer in their targeted vaccination, but generally have the disadvantage of lower stability and immunogenicity.

The basic requirement for using a stabilizer is safety. Other important factors which influence the selection of stabilizers for vaccines are the cost and the complicability of the preparation, the effects and / or side effects in use and the acceptance as a component in medicinal products.

A large number of additives for stabilizing pharmaceutical products are known from the prior art. These include e.g. B. Additives based on flavorings, alcohols, organic acids, aldehydes, phenolic substances and essential oils. Some of the known stabilizers significantly improve durability. However, they are rejected by many consumers because their effects on the health of the consumer are not known and harmful effects, especially if they are repeated over a long period of time, cannot be excluded. However, most of the stabilizers used so far have side effects, and these substances also do not meet the requirements placed on the safety of stabilizers, such as stability with regard to the stabilizing effect, minimal toxicity without interaction with the antigen, further degradability in the organism and the lack of one own immunogenic effect.

If unstable protein-based vaccines are stored, the proteins can also become cloudy or adsorbed on the walls of the vessels, which considerably deteriorate the quality of these products. In order to ensure the durability of labile components such as antigens and vaccines, they can be lyophilized and / or stored at a low temperature, preferably (4 ° C; -2 to - 8 ° C; -20 ° C; -80 ° C). However, the above instabilities can increase during the lyophilization, freezing, solubilization and thawing process.

Safety is of great importance for stabilizer development. Although a number of adjuvants have been in advanced clinical trials and have shown high efficacy, they have been found to be too toxic for routine use. In addition to safety, biostability, administration, costs, biodistribution and universality are also important when using a new stabilizer.

The aforementioned problems also relate to pharmaceutical formulations based on proteins, peptides, polypeptides and / or nucleic acids.

There is therefore a need for novel pharmaceutical formulations of the type mentioned at the outset. This object is achieved by pharmaceutical formulations according to claim 1. Preferred embodiments of the invention are the subject of the dependent claims.

The present invention relates to an adjuvant or an adjuvant combination for vaccines comprising at least one substance from the group of compatible solutes. The group of compatible solutes named in the invention comprises various low-molecular substances from extremophilic microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate ( DGP), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A) and / or di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, salt or ester of these compounds. The invention further comprises crude extracts of compatible solutes from microorganisms and mixtures of compatible solutes. The invention further relates to the production of combination preparations from compatible solutes with adjuvant effect and vaccines or vaccines, including the associated intermediate stages, or constructs, production processes and uses.

The present invention relates to an adjuvant or an adjuvant combination for vaccines comprising at least one substance from the group of

Compatible Solute. The group of the compatible named in the invention

Solute includes various low molecular weight substances from extremophiles

Microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate

(DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A) and / or

Di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, salt or ester of these compounds. The invention further relates to the

Production of combination preparations from compatible solutes with adjuvant effect and vaccines or vaccines, including the associated intermediate stages or constructs,

Manufacturing process and uses. It has surprisingly been found that the above-mentioned compounds from the group of compatible solutes as individual substances and / or in combination with one another and / or in combination with conventional adjuvants (eg aluminum hydroxide, mineral oil) increase the immune response in a synergistic manner. By using compatible solutes, a high level of effectiveness could be achieved even when using a comparatively small amount of antigen.

This has the advantage that less amount of antigen can be used for one vaccination, as a result of which the costs per vaccine dose can be considerably reduced.

It is advantageous if the adjuvant combination according to the invention is in the form of an emulsion. Emulsions suitable according to the invention are, for example, water-in-oil, oil-in-water or water-in-oil-in-water emulsions. An adjuvant for vaccines in the form of an oil-in-water emulsion is preferred according to the invention.

Another object of the present invention is a method for producing an adjuvant for vaccines, comprising a combination of vaccine and Compatible Solut.

The preparation takes place according to the invention in such a way that the antigens are prepared, purified and stored in a stable manner in the presence of the compatible solute (s). This method offers the advantage that production and storage advantages can be combined with the advantage of the adjuvant effect of compatible solutes.

Another object of the present invention are vaccines containing an adjuvant according to the invention. The vaccines can optionally contain other pharmaceutically acceptable excipients, carriers and / or solvents. The vaccines according to the invention are produced in a manner known per se. The vaccines according to the invention are notable for the fact that the immune response is substantially greater with the same amount of antigen. This makes it possible, with the same immune response, to reduce the amount of antigen without impairing the vaccination protection achieved. This leads to a much cheaper vaccination.

According to a preferred embodiment, the invention relates to combination preparations from compatible solutes and vaccines. Combination preparations with compatible solutes, conventional adjuvants (aluminum hydroxide, recombinant proteins, protein subunits, synthetic proteins, plasmid DNA and hydrophobic and surface-active substances such as mineral oil) and vaccines are particularly preferred. Raw extracts of compatible solutes from microorganisms are particularly suitable as adjuvants. According to a further preferred embodiment, adsorbate vaccines are used as vaccines.

The present invention relates to the field of vaccines / vaccines and the use of compatible solutes from extremophilic microorganisms for the production and / or the stable storage and transportation of vaccines / vaccines.

The present invention relates to a pharmaceutical composition or a vaccine which comprises the mixture of a vaccine and one or more compounds from the group of compatible solutes from extremophilic microorganisms. The group of compatible solutes named in the invention comprises various low-molecular substances from extremophilic microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate ( DGP), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A) and / or di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, salt or ester of these compounds. The invention further relates to the production of combination preparations from compatible solutes and vaccines or vaccines and / or other chemical or biological substances or structures for immunization, including the associated intermediate stages, or constructs, production processes and uses.

These compounds are added to vaccines to increase the stability during manufacture, transport, storage and / or in use.

The present invention relates to the field of vaccines and the use of compatible solutes from extremophilic microorganisms for the production and / or stable storage and / or transport of vaccines and vaccines. The present invention thus relates to a method of using compatible solutes to improve the shelf life and / or stabilize vaccines and vaccines.

The present invention relates to a pharmaceutical composition or a vaccine which comprises the mixture of a vaccine and one or more compounds from the group of compatible solutes from extremophilic microorganisms. The group of compatible solutes named in the invention comprises various low-molecular substances from extremophilic microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate ( DGP), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A) and / or di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, salt or ester of these compounds.

The invention further relates to the production of combination preparations from compatible solutes and vaccines or vaccines, or other chemical or biological substances or structures for immunization, including the associated intermediates, or constructs, production processes and uses. These compounds are added to vaccines to increase the stability during manufacture, transport, storage and / or in use.

The present invention had for its object to provide a vaccine formulation that the stability of vaccines z. B. on the basis of antigens in the form of peptides or proteins. With regard to the peptide or protein antigens, the vaccine according to the invention is not subject to any restrictions. The antigens can be naturally occurring immunogenic proteins, e.g. B. act from viral or bacterial pathogens originating proteins or fragments or cellular degradation products in the form of peptides; or tumor antigens or fragments thereof. In a preferred embodiment, the antigen is a tumor antigen or a natural or synthetic peptide derived therefrom, in this case the vaccines are present as tumor vaccines.

Surprisingly, the addition of compatible solutes to vaccine preparations has shown stabilization of the vaccine against a variety of stress conditions such as lyophylization, freezing, thawing, resuspending, elevated temperature, etc., without the substances themselves being unstable, toxic, interacting with the antigen, non- have been shown to be degradable in the organism or immunogenic. Surprisingly, no side effects were found when using the Compatible Solute. Even after lyophilization with compatible solutes mixed vaccine solutions, a clear product is obtained after dissolution.

The present invention relates to the use of compatible solutes from extremophilic microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP), ß -Mannosylglycerate (Firoin), ß-Mannosylglyceramid (Firoin-A) and / or Di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, a salt or ester of these compounds for the long-term stabilization of biological liquids, especially those obtained by lyophilization and subsequent reconstitution. The present invention relates to a pharmaceutical composition or a vaccine which comprises the mixture of a vaccine and one or more compounds from the group of compatible solutes. The group of compatible solutes named in the invention comprises various low-molecular substances from extremophilic microorganisms, namely ectoin, hydroxyectoin, di-myo-inositol phosphate (DIP), cyclic 2,3-iphosphoglycerate (cDPG), 1,1-di-glycerol phosphate ( DGP), ß-mannosylglycerate (Firoin), ß-mannosylglyceramide (Firoin-A) and / or di-mannosyl-di-inositol phosphate (DMIP) and / or a derivative, e.g. B. an acid, salt or ester of these compounds. The invention further relates to the production of combination preparations from compatible solutes and vaccines or vaccines, including the associated intermediate stages, or constructs, production processes and uses.

In the sense of the invention, such biological liquids can also be synthetically composed solutions from an artificial or natural liquid matrix (e.g. serum or phosphate-buffered NaCl solution) known to the person skilled in the art and added biological substances, which in turn can be produced by genetic engineering.

The amount of active antigen in the vaccine according to the invention can vary over a wide range. The amount of peptide may vary. a. depending on the type of processing or the respective formulation. The amount of peptide to be administered can be approximately 0.1 μg to approximately 5000 μg per vaccination dose, generally 1.0 μg to approximately 1000 μg, in particular approximately 10 μg to approximately 500 μg.

The addition of compatible solutes to pharmaceutical formulations, in particular vaccines, vaccines and / or other chemical or biological substances, liquids or structures also serves to stabilize them against stress conditions. For example, vaccines / vaccines and / or combination preparations in isolated components of organisms are stabilized against stress conditions. The vaccines / vaccines are preferably protein and / or peptide-based Are vaccine. The vaccines can also contain synthetic, recombinant or highly purified antigens. The stress conditions are in particular elevated temperature, lyophilization, freezing, solubilization and / or thawing processes. By adding the Compatible Solute, the specific immune response to antigens can be strengthened and / or extended. Stabilization against stress conditions is preferably carried out during manufacture, transport, storage and / or in use. In particular, the stabilization against stress conditions is a long-term stabilization. The use of the compatible solutes for a tumor vaccine, in particular based on one or more tumor-associated or tumor-specific antigens, is particularly preferred.

The invention further relates to vaccines, adjuvants and stabilizers for pharmaceutical formulations. Furthermore, the invention relates to the use of compatible solutes as stabilizers for pharmaceutical formulations and as adjuvants.

The invention will be explained below using an exemplary embodiment as an example.

example 1

The adjuvant according to the invention is e.g. B. suitable for vaccines for pigs. The adjuvant according to the invention is preferred for a vaccine against parvovirus infection in pigs. The optimal concentration of the compatible solute in the vaccine preparation is between 0.05 M and 0.5 M. A concentration of 0.1 M is particularly preferred.

Example 2

The stabilizer according to the invention is e.g. B. suitable for vaccines in humans. The stabilizer according to the invention is preferred for a vaccine against polio (infantile paralysis) in humans, which is caused by picomaviruses of the genus enteroviruses. The Compatible Solute is used alone or together with the known stabilizers such as magnesium chloride or sorbitol. The optimal concentration of the compatible solute in the vaccine preparation is between 0.01 M and 1 M or better between 0.05 M and 0.5 M. A concentration of 0.1 M is particularly preferred.

Example 3

A protein vaccine (recombinant protein A; internal nomenclature) cannot be stored stably at T = 0 ° C. If no stabilizers are found, it must therefore be stored at T = -70 ° C. However, this is not possible due to the logistical framework. A way must therefore be found to store the protein vaccine stably at refrigerator temperature (T = 5 ° C) over long periods of time. Here certain stabilizers have to be added. The following formulation A or formulation B is used:

Formulation A:

Standard stabilization solution 1 (polysaccharides and other components) 10% (w / v) hydroxyectoin

Formulation B:

Standard stabilizing solution 2 10% (w / v) hydroxyectoin

Denatured recombinant protein A and protein stored at -70 ° C. without additives are used as controls. The activity of the vaccine is determined using antigen binding tests (relative unit; scale 0-10). Different structures of the recombinant protein A (type AA - AB - AC), which vary slightly in the amino acid sequence, are tested. Result:

It is observed that hydroxyectoin and formulations containing hydroxyectoin have a positive impact on vaccine titers of protein vaccines after storage at refrigerator temperature. The use of hydroxyectoin alone has advantages over formulation B since the addition of multicomponent stabilizing solution is no longer necessary.

Ectoin and Firoin are registered trademarks.

- Expectations

Claims

claims

1. Pharmaceutical formulation containing proteins, peptides, polypeptides and / or nucleic acids, characterized in that it contains one or more compounds from the group of compatible solutes.

2. Formulation according to claim 1, characterized in that it contains one or more of the following compounds as compatible solutes: ectoin, hydroxyectoin, di-sugar alcohol phosphates from C3 to C6 sugar alcohols,? -Mannosylglycerate (Firoin), iff-mannosylglyceramide (Firoin-A) , Di-mannosyl-di-inositol phosphate (DMIP) and / or physiologically compatible derivatives thereof.

3. Formulation according to claim 2, characterized in that the di-sugar alcohols are di-myo-inositol phosphate (DIP), cyclic 2,3-diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP).

4. Formulation according to claim 3, characterized in that the compatible solute or solids therein are present in a concentration of 0.05 to 0.5 M and preferably 0.1 M.

5. Vaccine for active or passive immunization according to one of the preceding claims.

6. Vaccine according to claim 5, characterized in that the antigen (s) contained therein are synthetic, recombinant and / or highly purified antigens.

7. Vaccine according to claim 5 or 6, characterized in that the antigen or one or different proteins, peptides or nucleic acids or a mixture thereof.

8. Vaccine according to one of claims 5 to 7, characterized in that a vaccination dose has an antigen content of 0.1 μg to 5 mg, preferably 1.0 μg to 1.0 mg and particularly preferably 10 μg to 0.5 mg.

9. Vaccine according to one of the preceding claims 5 to 8, characterized in that it further contains one or more of the following adjuvants: aluminum hydroxide, recombinant proteins, protein subunits, synthetic proteins, nucleic acids, in particular plasmid DANN, and hydrophobic and / or surface-active substances such as B. mineral oil.

10. Tumor vaccine according to one of the preceding claims 5 to 9, preferably containing one or more tumor-associated and / or tumor-specific antigens.

11. Adsorbate vaccine according to one of the preceding claims 5 to 10.

12. adjuvant, characterized in that it contains one or more of the compatible solutes according to any one of claims 1 to 4.

13. Adjuvant according to claim 12, characterized in that at least one of the compatible solutes is present as a crude extract from microorganisms.

14. adjuvant according to one of claims 12 or 13, characterized in that it is present as a water-in-oil, water-in-oil-in-water and preferably as an oil-in-water emulsion.

15. Use of one or more of the compatible solutes according to one of claims 1 to 4 as a stabilizer for pharmaceutical formulations, preferably for vaccines.

16. Use according to claim 15 in the manufacture, transport, storage and / or use of the formulations.

17. Use according to one of claims 15 or 16, characterized in that the formulation is a vaccine according to one of claims 5 to 11.

18. Use according to any one of claims 15 to 17, characterized in that the vaccine is prepared, purified and / or stored in the presence of the compatible Solute / s.

19. Use of one or more of the compatible solutes according to one of claims 1 to 4 as an adjuvant for combined administration with a vaccine, in particular for administration as a vaccine-adjuvant combination preparation or for isolated administration.

20. Stabilizer for pharmaceutical formulations containing one or more of the compatible solutes according to one of claims 1 to 4.

21. Stabilizer according to claim 20, characterized in that the compatible solutes are present as water-in-oil, water-in-oil-in-water and preferably as an oil-in-water emulsion.