WO2000071154A2 - Stability, biocompatibility optimized adjuvant (sba) for enhancing humoral and cellular immune response - Google Patents

Abstract

The invention relates to a stability and biocompatibility-optimized adjuvant (SBA) for enhancing the humoral and cellular immune response by jointly injecting said adjuvant with one or more antigens. The adjuvant consists of particles based on solid lipids or solid lipid mixtures and can be used in the production of more efficient and compatible vaccines, the inoculation of human beings and animals and for obtaining antibodies. The strength of the immune response can be modulated in a targeted manner and additionally adapted to the specific species by selecting the size, charge and surface characteristics of the particles. Other adjuvants, e.g. molecular adjuvants such as GMDP, can be added to the SBA, whereby the cellular immune response is additionally enhanced. The SBA is more effective and cost-efficient and easier to use than existing products and is well tolerated in vivo.

Description

 Stability-, biocompatibility-optimized adjuvant (SBA) to increase the humoral and cellular immune response

1. Background of the Invention

Antigens are applied to animals and humans to produce antibodies. The goals are, for example, the immunization of humans or animals to protect against diseases or the production of antibodies, which are subsequently isolated and processed into products. The most common application route for antigens is parenteral administration, alternative routes are e.g. oral, nasal and topical application.

A common problem is that often the strength of the immune response to the administered antigen is insufficient for the intended purpose. Sometimes the problem can be solved by greatly increasing the administered dose of the antigen. A serious problem, however, is that antigens are often very expensive and the dose increase leads to a corresponding increase in the price of the vaccine. These costs lead to a heavy burden on the health system, for many sections of the population - especially in the Third World - the vaccine becomes too expensive and desirable mass vaccinations cannot be carried out.

An alternative solution is the administration of antigens together with an adjuvant, which increases the antigenic effect and thus leads to a higher antibody titer. The adjuvant principle was first lig 1948 described by Jules Freund [Freund, JJ Immunology 1948, 60, 383-398] and realized by two emulsions based on mineral oil. Freund's Incomplete Adjuvant (Freund's Incomplete Adjuvant - FIA) is a blend of mineral oil with mannide monooleate (Montanide, Arlacel). To use it, mix it with the antigen solution and inject the emulsion formed. The resulting strengthening of the immune response is so great that FIA is still used as the "gold standard" when new adjuvants are developed and tested. Their efficiency and quality are assessed by comparison with FIA, although many newly developed adjuvants remain well below the efficiency of FIA (e.g. at 0.2, FIA = 1.0).

The addition of killed mycobacteria to FIA (e.g. M. tuberculosis) further increased the immune response. This mixture is called Freund's Complete Adjuvant (Freund's Complete Adjuvant - FCA). Freund's oil emulsion, however, produces inflammatory and ulcerating tumors (granulomas) at the injection site, in some cases they break open and spread into spreading abscesses. According to today's standard, the use of Freund's oil emulsion on humans is therefore out of the question. It is sometimes used in animals. The general well-being of the animals is often so severely affected that there are uneconomical failures. The mycobacteria M. tuberculosis or M. butyricum proposed by Freund to complete the adjuvant are also poorly tolerated. They also cause granulomas and fever as well as abscesses at the injection site [Brown, E.A. Rev Allergy 1969 23 (5): 389-400].

The task therefore is to find a more tolerable adjuvant, in particular from the point of view of inexpensive vaccines. make desirable worldwide vaccinations against certain diseases that can only be financed with an inexpensive vaccine and the increasing importance of immunological production techniques and products. Even with increasing awareness of the need for protection of animals and corresponding pressure from legislation, a more compatible, (ie biocompatible) equally efficient, but at the same time inexpensive adjuvant must be found. In addition, it must be physically stable so that admixing before the injection can be omitted and fluctuations in efficiency - for example due to different dispersions - are eliminated.

The most commonly used adjuvant approved for human use today is a fine suspension of aluminum hydroxide, the roots of which are even more archaic than those of Freund's emulsion [Glenny, A.T. et al. J. Pathol. 1926, 29 31-40]. It is based on the assumption that antigens are better recognized by the immune system if they are adsorbed on the surface of the aluminum hydroxide particles. However, it is disadvantageous that aluminum hydroxide is less efficient than Freund's adjuvant and it can also cause granulomas.

Starting from Freund's emulsion, emulsions have recently been described which consist of more compatible materials such as, for. B. Squalan and Sqalen, [Sanchez-Pestador, L. et al. J. Immunol. 1988 141, 1720-1727, Masihi KN, Lunge, W., Bremer W., Ribi, E. Int. J. Immunopharmacol. 1986 8 (3), 339-45, Hunter RL, Bennett, B. Scand J Immunol. 1986 23 (3), 287-300, Allison AC, et al. Semin Immunol. 1990 2 (5), 369-74]. One of these systems is MF59 (European Patent Application 0399843A2). Based on the suspensions according to Glenny, particles from different polymers up to various inorganic and organic particles (e.g. carbon) were used [O'Hagan, DT, Jeffrey, H., Roberts MJ, McGee, JP, Davies, SS Vaccine . 1991 9 (10), 68-71, Glenny, AT et al. J. Pathol. 1926, 29 31-40]. Disadvantages of these systems are the cytotoxicity of some polymers (e.g. formaldehyde formation in polyalkylcyanoacrylates), missing or too slow degradation rate in the organism (e.g. polystyrene or polymethacrylate derivatives), insufficient biocompatibility (e.g. capsule formation in PLA particles, pH shift up to pH2) and inadequate approval by the registration authorities (e.g. soot particles).

An alternative way to use particulate adjuvants such as oil drops or solid particles is to use macromolecular adjuvants. It has been shown that the mycobacteria described by Freund can be replaced by building blocks from their capsule substance, which have greater physical tolerance. MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) [Adam, A., Lederer, E. Med. Res. Rev. 1984 4, 111-152] Thr-MDP (threonyl analogue of the MDP ) [Byars NE, et al. Vaccine. 1987 5 (3), 223-8] and the GMDP (N-acetylglucosaminyl-N-acetylmuramyl dipeptide) derived from the yoghurt bacillus [Grubhofer, N. Immunology Letters 1995 44, 19-24]. GMDP is a homologue of the MDP, an increased immunostimulatory effect has now been demonstrated for GMDP (patent specification DE 19611235 Cl).

Other approaches in the development of an adjuvant were the use of molecular adjuvants in high concentrations so that the solubility was exceeded and a suspension was obtained (e.g. DDA (dimethyldioctadecylammonium bromide) [Grubhofer, N. Immunology Letters 1995 44, 19-24] and to increase the humoral response, the mixture of macromolecular adjuvants such as glycopeptides with solid particles (e.g. GMDP with colloidal particles) [Grubhofer, N. Im- Munology Letters 1995 44, 19-24].

So far, however, no solution has been found that has the same effectiveness as Freund's adjuvant and at the same time meets the other requirements: the new emulsions are by no means always tolerated without reaction, the synthetic macromolecules such as glycopeptides have so far not been shown to be superior to the mycobacteria in addition, they are far too expensive. Solid particles have approval problems. Many adjuvants show too high toxicity and thus too little biocompatibility. There are also problems with physical stability (avoidance of aggregation or coalescence). The FIA emulsion must be freshly prepared and injected, long-term storage is not possible. Sufficient physical storage stability is, however, the essential prerequisite for a marketable product - especially for pharmaceuticals.

The goals are thus the production of a new adjuvant with:

1. sufficient physical stability

2. low cytotoxicity and sufficient biocompatibility

3. Efficiency comparable to FIA 4. Cost-effective production. 1. Description of the invention

Up to now, GMDP had to be used in a mixture with colloidally disperse solid lipid particles in a particle size <200 nm to increase the immunostimulating effect (US Patent Gerbu, processing number of the US Patent Office is 08 / 816,787). The structure of the particles was systematically modified in studies for this invention (e.g. surfactants, stabilizers) to find lipid particles that could further increase the immunostimulating effect of GMDP. More surprising

It was found that lipid particles alone are just as efficient as the combination of lipid particles and GMDP (Example 9). Thus, the expensive GMDP can be dispensed with in the invention and a comparably high humoral immune stimulation can be achieved by an inexpensive lipid particle alone.

The strength of the immunostimulating effect depends on the surface properties of the particles (surfactants used, particle charge) and the particle size. If the lipid particle is negatively charged, the efficacy is approx. 1/3 of FIA; if a positively charged lipid particle is generated by adding EQ 1, the efficacy is comparable to FIA (no significant difference, t-test) (Example 9). The desired potency can thus be set in a targeted manner via the composition of the lipid particles used. This avoids over-reactions of the organism.

From previous research into adjuvants on inorganic and polymer suspensions it is known that there is a particle size with maximum efficiency for each antigen. knows. For example, the most suitable particle size of aluminum hydroxide suspensions for a vaccine is determined empirically; polymer particles as adjuvants for immunization had a different effect as a function of their size [Kreuter J, et al. Vaccine. 1986, 4, 125-9]. The same was surprisingly found for the lipid particles despite the very different matrix material, so that the potency can be modulated by changing the particle size in an antigen-specific and species-specific manner.

The lipid particle dispersions of the stable biocompatible adjuvant

(SBA) consist of in water or aqueous liquids or non-aqueous, e.g. oily liquids dispersed lipid particles, which can, but need not, be stabilized by surfactants or polymers. With a sufficiently high viscosity of the outer phase or fineness of the particles, a physically stable dispersion is obtained. The same applies to low-viscosity outer phases, if the particles carry a sufficiently high charge in the same direction and the sediments formed can be easily redispersed.

The SBA is produced by dispersion or precipitation, generally known methods described in textbooks in pharmacy and process engineering being used. During dispersing, coarsely dispersed lipids are broken up by mechanical methods. The lipids can be in the solid state (e.g. mortar mill) or in the liquid state (eg emulsification of molten lipids by stirrers). To produce the SBA dispersion, the lipids can first be comminuted and then dispersed in the outer (eg aqueous) phase or alternatively can be comminuted directly in the outer phase. For the production of SBA dispersions by dispersing tion can z. B. used include: piston-gap homogenizers (e.g. APV homogenizers, French Press), jet stream homogenistors (e.g. microfluidizers), rotor-stator stirrers (e.g. Ultraturax, Silverson homogenizers), static mixers on a microscale and macroscale (e.g. mixers from Sulzer), gas jet mill, rotor-stator colloid mill and mortar mill (example 15).

SBA dispersions show sufficient long-term physical stability. Determination of the particle size with photon correlation spectroscopy (PCS) and laser diffractometry (LD) showed no or negligible particle growth over periods of 1 to 3 years (example 3). The stability of SBA dispersions is far superior to that of Freund's incomplete adjuvant (FIA) (example 1); higher stability is also shown compared to newer adjuvant emulsions (example 2).

Stability: SBA dispersions represent a simple system. In contrast to glycopeptides, the additives used are chemically simple and robust. The use of heat in sterilization processes does not lead to chemical decomposition, the dispersion remains physically stable and particle aggregation does not occur. Example 4 shows an example of the sterilization of SBA dispersions by autoclaving (121 ° C., 15 minutes, 2 bar). FIA shows phase separation under the same conditions.

Many vaccines are stored at a low temperature (4 - 6 ° C). With optimized SBA dispersions, storage at room temperature and even higher temperatures is possible without physical destabilization occurring (Example 11). This simplifies the handling of products based on SBA, especially for hotter climates (eg use as an adjuvant for mass vaccinations in Third World countries).

Biocompatibility: Low toxicity and good biocompatibility are essential for a widely used adjuvant. In a sheep study, no abnormalities were observed at the application site after application of SBA dispersion (example 6). The good tolerance is explained by the extremely low toxicity of lipids observed in vitro in cell cultures. Compared to polymers approved by the German regulatory authorities and the FDA for parenteral administration, they show a viability at least approximately 20 times higher at high particle concentrations (example 5). The good biocompatibility is attributed to the fact that body proteins generally only adsorb to a small extent on the particle surface - in contrast to other particles (Example 7). In addition, there are no proteins on the surface that promote intolerance reactions.

For widespread use of an adjuvant, it is advisable for cost reasons to produce an adjuvant preparation which is admixed before the antigen solution is used. Ideally, the properties of the adjuvant should be such that it can be mixed with a number of different antigens. To reduce the pain of injection, the mixture should be applied in physiological saline or other isotonized solution. Due to the reduction of the zeta potential, physiological saline leads to destabilization in dispersions and subsequent aggregation [Lucks, JS et al., Int. J. Pharm 1990 58, 229-235]. The SBA adjuvant should be physically stable for a sufficiently long time after being added to the antigen in isotonic solution. Example 8 shows that the lipid particles according to Zumi to physiological saline are stable for over 6 hours, measurable aggregation does not occur.

In addition to the particle size, surface properties such as loading can also be set in a targeted manner and the SBA adjuvant can thus be produced in a species-specific manner. Positive and negative charges can be generated by adding appropriately charged surfactants or stabilizers. The strength of the load can be adjusted via the concentration of the additive. The ideal addition is charged substances, which, like cetylpyridinium chloride, are approved as preservatives for parenteral application (example 12).

A variety of different lipids can be used to produce SBA dispersions. These are both chemically uniform lipids and their mixtures. The lipids are characterized in that they are present in the end product SBA dispersion in the crystalline state (e.g. β-, ßi-modification) or in the liquid-crystalline state (α-modification) or in a mixture thereof. When lipid mixtures are used, liquid lipids (e.g. oils, lipophilic hydrocarbons, lipophilic organic liquids such as oleyl alcohol) can also be mixed with the solid lipids (e.g. glycerides, lipophilic hydrocarbons such as hard paraffin) (so-called "lipid blends").

Find z. B. the following lipids as a dispersed phase and can be used as an individual component or as a mixture: natural or synthetic triglycerides or mixtures thereof, monoglycerides and diglycerides, alone or mixtures thereof or with, for example, triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols, including their esters and ethers as well as in the form of lipid peptides, or any mixtures thereof. Synthetic monoglycerides, diglycerides and triglycerides are particularly suitable as individual substances or as a mixture (eg hard fat), Imwitor 900, triglycerides (eg glycerol trilaurate, glycerol myristate, glycerol palmitate, glycerol stearate and glycerol behenate) and waxes such as cetyl palmitate and white. Also hydrocarbons, such as hard paraffin.

The proportion of the inner or lipid phase based on the total formulation is 0.1% to 80% (m / m) and is preferably in the range from 1% to 40% (m / m). Should the addition of dispersion stabilizing additives be necessary or desirable, e.g. Emulsifiers, in order to be able to produce stable dispersion, can be incorporated in the form of pure substances or in the form of mixtures in order to stabilize the particles.

The amount of such additives, which can be added in relation to the total weight of the aqueous dispersion, is in the range from 0.01% to 30% and preferably in the range from 0.5% to 20%. The surfactants, stabilizers and polymers which are generally known from the production of dispersions can be used to stabilize the SBA dispersions or to specifically modify their surfaces. Examples include:

sterically stabilizing substances such as poloxamers and poloxamines (polyoxyethylene-polyoxypropylene block copolymers), ethoxylated sorbitan fatty acid esters, especially polysorbates (eg Polysorbate 80 or Tween 80®), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and esters and ethers of sugars or of Sugar alcohols with fatty acids or fatty alcohols (eg sucrose monostearate, sucrose distearate, sucrose cocoat, sucrose stearate, sucrose dipalmitate, sucrose palmitate, sucrose laurate, sucrose octanoate, sucrose oleate).

2. charged ionic stabilizers such as diacetyl phosphates, phosphatidylglycerol, lecithins of various origins (eg egg lecithin or soy lecithin), chemically modified lecithins (eg hydrogenated lecithins), as well as phospholipids and sphingolipids, mixture of lecithins with phospholipids, sterols (eg cholesterol Cholesterol derivatives, as well as stigmasterol) and also saturated and unsaturated fatty acids, sodium cholate, sodium glycocholate, sodium taurocholate, sodium deoxycholate or their mixtures, amino acids or anti-flocculants, such as Sodium citrate, sodium pyrophosphate, sodium sorbate [Lucks, J.S. et al. Int. J. Pharm., 1990, 58, 229-235]. Zwitterionic surfactants such as (3 - [(3-cholamidopropyl) dimethylammonio] -2-hydroxy-l-propanesulfonate) [CHAPSO], (3 - [(3-cholamidopropyl) dimethylarnmonio] - 1-propanesulfonate) [CHAPS] and N-dodecyl- N, N-dimethyl-3-ammonio-propane sulfonate. Cationic surfactants, especially compounds used as preservatives, e.g. Benzyldimethylhexadecylammonium chloride, methylbenzethonium chloride, benzalkonium chloride, cetpyridinium chloride.

3.Viscosity-increasing substances such as cellulose ethers and cellulose esters (e.g. methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose), polyvinyl derivatives and polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate, alginates, polyacrylates (e.g. carbopol), xanthans and xanthans and xanthans . If necessary or desired, the charged stabilizers are preferably present in the SBA dispersion in an amount of 0.01% to 20% (m / m) and in particular in an amount of 0.05% to 10%. If necessary or desired, viscosity-increasing substances are incorporated in the formulation in a similar ratio, preferably in an amount of 0.01-20% and in particular in an amount of 0.1% to 10% (m / m) and preferably in the range between 0.5% and 5%.

The outer phase (dispersion medium, continuous phase) can be water, aqueous solutions or liquids miscible with water, as well as glycerin or polyethylene glycol and oily liquids such as miglyols (medium chain triglycerides - MCT) and other oils (castor, peanut, soybean, cotton seeds -, rapeseed, linseed, olive, sunflower, and safflower oil can be used.

Surfactant-free SBAs are produced by dispersing the lipid phase in an aqueous solution which contains one or more viscosity-increasing substances, either alone or in combination with other substances, and also sugars, sugar alcohols, especially glucose, mannose, trehalose, mannitol, sorbitol and others. Furthermore, it is possible to use a combination of the viscosity-increasing substances or the combination of these with sugars or sugar alcohols, or in a further combination with charge stabilizers or anti-flocculants.

SBA dispersions can be used as an adjuvant to many different antigens for vaccination against various diseases. Examples include: Glycoproteins such as Gonococcal Protein I, Brucella abortus antigen, Tetanus toxoid, Diphteria toxoid, Listeria monocytogenes, virus antigens such as Semliki Forest Virus, Enceohalomyocarditis virus, Porcine rarovirus, Pseudorabies virus, Newcastle desease virus, Bovine virus, HIV, Influenza diarrheal virus , Herpes simplex, hepatitis C, measles, parasites such as malaria, Eimeria spp.,

In summary, it can be said that the SBA dispersions provide an adjuvant that:

1. has sufficient physical stability to be manufactured as a product and especially as a pharmaceutical,

2. has low toxicity and good biocompatibility, especially if biodegradable lipids such as glycerides are used,

3. has a comparable effect as Freund's incomplete adjuvant (FIA) and

4. can be produced inexpensively from low-cost auxiliaries using inexpensive manufacturing processes.

SBA dispersions can be widely used to reduce the antigen dose and thus the costs with toxicologically acceptable excipients, since the addition of SBA at a low antigen dose achieves the same immunostimulating effect.

Antigens with previously insufficient antigenicity for a vaccine can be converted into an efficient vaccine by adding immune response-stimulating SBA. Due to the inexpensive manufacture of existing comparable

Efficiency to FIA, SBA dispersions are suitable as an adjuvant for vaccinations in the veterinary field, where for profitability reasons only very low-priced

Vaccines can be used.

Adjuvants used to date have focused on increasing the humoral immune response. In view of the efficiency of action of SBA dispersions, it is no longer necessary to add another adjuvant to the SBA lipid particles, or to add adjuvants such as GMDP bring no further increase in the humoral immune response. Obviously the immune system is at its maximum capacity of response, additional adjuvant can no longer bring an additional effect. Thus, additives to SBA have no advantage for the humoral response, additives as described in the Gerbu patent (patent specification DE 1961 1235 C1) are superfluous due to the surprisingly found potency of the SBA described in the invention.

Surprisingly, however, it was found that after application of GMDP in combination with SBA dispersions, a renewed later vaccination resulted in a significantly increased cellular immune response than if only SBA dispersion alone had previously been used as an adjuvant. It was thus newly found that a combination of SBA dispersion is specifically suitable for increasing the cellular immune response when vaccinated again. There is an increased immune response in the case of a subsequent second vaccination if an additional adjuvant such as GMDP was used beforehand in a mixture with SBA dispersion (example 13).

To produce an adjuvant with the aim of increasing the cellular immune response, it is therefore advantageous to use the SBA dispersion with a to combine another adjuvant. Possible adjuvants for a combination are: N-acetylglucosaminyl- (ßl-4) -N-acetylmuramyl-L-alanyl-D-isoglutamine [GMDP], dimethyldioctadecylammonium bromide [DDA], N-acetylmuramyl-L-alanyl-D-isoglutamine [ ], N, N di- (ß-stearoylethyl) - N, N-dimethylammonium chloride [EQ 1], glycopeptides, components of the

Cell wall of mycobacteria, saponins, quaternary amines, such as, for example, cetylpyridinium chloride and benzalkonium chloride, zwitterionic amines, such as CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] - l-propanesulfonate), dextran sulfate, dextran, 3-odesophyl-4'-monosacyl-4 ^' lipid A [MPL®], N-acetyl-L-alanyl-disoglutaminyl-L-alanine-82) - 1, 2 dipalmitoyl - sn glycero - 3- (hydroxy-phosphoryloxy)) ethylamide, monosodium salt [MTP-PE], Gra - Nulocyte-macrophage colonies stimulating factor [GM-CSF], block copolymers, for example P1205, Poloxamer 401 (Pluronic L121), dimyristoyl-phosphatidylcholine [DMPC], dehydroepiandrosterone-3ß-01- 17-one [DHEA], dimyristoylcerolphosphat DMPG], deoxycholic acid sodium salt, cytokines, imiquimod, DTP-GDP, saponins, 7-allyl-8-oxoguanosine, Montanide ISA 51, Montanide ISA 720, MPL, murametide, murapalmitin, D-murapalmitin, 1-monopalmitoloyl-rac-glycerol , Dicetyl phosphate, methyl methacrylate [PMMA], PEG sorbitan fatty acid esters such as Polysorbate 80 (TWEEN® 80), Quil A Saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN®85, Arlacel85), DTP-DPP, stearyl-tyrosine, N, N dioctadecyl-N ', N'- bis (2hydroxyethyl) propanediamine, calcitriol.

In addition to increasing the humoral immune response through SBA dispersions, the invention opens up the possibility of increasing the cellular immune response in the case of a second vaccination by combining SBA dispersions with other adjuvants. Instead of admixing adjuvants to increase the cellular immune response, the adjuvants can also be incorporated into the lipid particles. Incorporation is possible by incorporation in the solid particle matrix, enrichment in the interface in the case of amphiphilic adjuvants or by simple adsorption on the particle surface. Adjuvants can be incorporated during particle production or subsequently (for example in the case of incorporation, example 16). For incorporation during manufacture, adjuvants are dissolved, solubilized or dispersed in the molten lipid phase and the lipid phase containing adjuvants is then further processed. In the case of amphiphilic adjuvants, these can also be dissolved in the outer phase of the SBA dispersion and then accumulate in the particle interface or by adsorption on the surface. Incorporation of adjuvants leads to prolonged release by diffusion or in the course of particle degradation by enzymes. Delayed release over a longer period increases the immune response.

Another attractive area of application is antibody production in animals. The antibody yield can be significantly increased by adding the adjuvant.

Examples

Example 1: Determination of the physical stability of SBA versus Freund's Incomplete Adjuvant (FIA): An aqueous SBA dispersion was prepared by high pressure homogenization at 95 ° C. from 20% beeswax, 2% Tween 80 (PCS diameter 289 nm polydispersity index 0.101). FIA was produced according to the method described by Freund [Freund, JJ Immunology 1948, 60, 383-398]. SBA and FIA were stored at the temperatures of the climatic zones that are used in the stability testing of drugs [EMEA guideline CPMP / QWP / 159/96, January 1998)]. The storage temperatures were: 5 ° C, 25 ° C, 40 ° C. The physical stability was determined by measuring the particle size with laser diffractometry, characterization parameters were the diameter 50% and the diameter 95% (50% and 95% of the particles are below the specified size, sensitive parameters for particle aggregation). FIA showed significant particle growth after just a few minutes of storage - even at room temperature - so FIA is not a storage-stable adjuvant. In contrast, the particle sizes of SBA remain unchanged over a period of 1 year under the 3 storage conditions (Table 1).

Table 1: Investigation of the stability of SBA (20% beeswax, 2% Tween 80) in comparison with Freund's incomplete adjuvant (FIA)

Example 2: Determination of the physical stability of SBA versus squalene adjuvant: SBA was prepared as described under 1, it contained 10% cetyl palmitate and 1.2% miranol (PCS diameter 210 nm, PCS polydispersity index 0.189). Squalene adjuvant was prepared as described in European patent application 0 399 843 with filing date May 25, 1990 (adjuvant MF59). Particle size measurement was carried out using laser diffractometry, storage was carried out as in Example 1 at 3 temperatures (Table 2). In addition, an accelerated stability test was carried out, SBA and MF59 dispersions were shaken at 40 ° with a frequency of 50 Hz (Table 3). SBA shows increased stability both in normal storage and in the stress test. Table 2: Investigation of the stability of SBA (10% cetyl palmitate, 1, 2% miranol) in comparison with MF59

Table 3: Stability of MF 59 against SBA at 40 ° C and a shaking frequency of 50 Hz

Example 3. Long-term stability of SBA: SBA dispersion consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C for one year. The PCS data and laser diffractometer diameter showed little or no change (Table 4).

Table 4: Long-term stability of SBA: SBA dispersion consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C for one year.

Example 4. Heat stability of SBA on autoclaving (heat, pressure) versus FIA and MF59: The SBA dispersion was composed of 18% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water. Sterilization of 20 mL was carried out in injection vials according to the standard conditions of the European Pharmacopoeia (121 ° C, 2 bar, 15 minutes). Particle size was determined using PCS and laser diffractometry (LD 95%) (PI: polydispersity index, measure for the width of the particle distribution, MW: mean from 3 measurements, staff: standard deviation, PI polydispersity index) (Table 5). The FIA emulsion showed after autoclaving Phase separation, MF59 a clear particle growth. SBA is physically stable and can be sterilized by accretion.

Table 5: Heat stability of SBA upon autoclaving (heat, pressure) versus FIA and MF59: The SBA dispersion was composed of 18% hard paraffin, 4% Tween 89 / Span 85, 7/3 and water. Sterilization of 20 mL each in injection vials at 121 ° C, 2 bar, 15 minutes. Particle size was determined using PCS and laser diffractometry.

Example 5. Physiological tolerance: To assess the tolerance, the cytotoxicity of SBA in cell cultures was determined (human granulocytes, HL60 cells). To quantify the toxicity, the viability of the cells was assessed using the MTT test [Mosmann, T., J. Immunol. Meth. 1993, 65, 55-63]. The SBA dispersion was composed of 10% cetyl palmitate, 0.5% poloxamer 188 and water. The number of cells per well was 200,000 for human granulocytes and 200,000 for HL60 cells. Incubation was for 12 hours. In SBA the viability was 80% for the granulocytes and 85% for the HL60 cells. The viability of nanoparticles made of PLA was only 5%, for nanoparticles made of PLA / GA it dropped to 0%. The tolerance of SBA in cell cultures is at least a factor of about 20 better than that of the FDA-approved polymers for parenteral administration.

Example 6: Compatibility after parenteral application: Aqueous SBA dispersion with the composition 5% hard paraffin, 5% Tween 80 / Span 85 (7/3) and water was used. Parenteral injection was carried out in sheep (n = 30), the injection site was the lateral chest wall, the injection volume was 5 mL distributed over 4 injection sites. The sheep showed no abnormalities, neither at the injection site nor in their behavior.

Example 7: Biocompatibility - Interaction with Body Proteins: The SBA dispersion was composed of 10 compritol, 2.5% Poloxamer 407 and water. Production was carried out with high pressure homogenization. The particles were incubated for 5 minutes with human plasma, then separated from the plasma and the body proteins adsorbed on the particle surface using two-dimensional polyacrylamide gel electrophoresis [Blunk, T et al. Electrophoresis 14, 1382-1387 (1993)]. In case of Similar particle surfaces adsorbed a very small amount of protein on SBA with 96.41 cpm (counts per minute) compared to emulsions (comparison values: 472 cpm on emulsion, 390 cpm on polystyrene particles [Harnisch, S. et al. Elektrophoresis 1998, 19, 349-354 , Blunk, T., Electrophoresis 1993, 14, 1382-1387]. Complement factors that promote intolerance were not detected on the SBA surface.

Example 8: Stability in phosphate-buffered physiological saline (PBS): SBA composed of 20% hard paraffin, 5% Tween 80 / Span85 (7/3) and water were mixed with PBS (2 mL SBA + 2 mL saline). The physical stability in the physiological saline solution was determined as a function of time using laser diffractometry. There was no increase in particle size over 6 hours

(Diameters 90% and 95%, Table 6) (staff: standard deviation).

Table 6: Stability in phosphate buffered saline (PBS) SBA (20% hard paraffin. 5% Tween 80 / Span85 (7/3)) was mixed with PBS (2 mL SBA + 2 mL saline). Determination of physical stability in physiological saline using laser diffractometry as a function of time.

Example 9: Adjuvant effect in comparison to molecular adjuvant (GMDP - N-acet lglucosaminyl-N-acetylmuramyl dipeptide) and FIA: sheep were vaccinated with the strain Mycoplasma Bovis PG 45 R9. The vaccine antigen was cultivated in stand culture over 72 hours under microaerophilic conditions in Hayflick medium. Inactivation was carried out by adding 0.1% β-propiolactone. The cells were separated, washed with phosphate buffer pH 7.4 and adjusted to a content of 1 x 10 ¹⁰ CFU / mL. The sterility of the preparation was checked in accordance with the German Pharmacopoeia Edition 10. The dry matter determination showed a content of 1 mg / mL Mycoplasma Bovis antigen. The adjuvant SBA, GMDP and FIA were mixed equally with the antigen in buffer. Injection volume was 5mL, spread over 4 injection sites.

The composition of SBA was 4% hard paraffin, 1% EQ 1 (N, N di- (β-stearoylethyl) - N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3). The composition of the GMDP adjuvant was 5% lipid and 0.5% surfactant. FIA was made as in Example 1.

Blood was drawn on day 0 before vaccination, on day 35 and on day 63. The antibodies were determined using ELISA. A commercially available marked anti-IgG sheep from Sigma was used for the ELISA test.

SBA showed a comparable intensity of action as the combination of lipid particles with GMDP. Furthermore, SBA was comparable in effectiveness to FIA (Figure 1). There was no significant difference in the intensity of action between the three adjuvants.

Figure 1: Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA. Composition of SBA was 4% hard paraffin, 1% EQ 1 (N, N di- (β-stearoylethyl) - N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3). The composition of the GMDP adjuvant was 5% EQ 1 and 0.5% Montanide 888. FIA according to Example 1.

Example 10: Effect of SBA composition on immune titer: SBA dispersions were prepared with identical lipid but different surfactants on the surface, ie. H. they differ in their surface properties. The formulation SBA-1 consists of 4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water, the formulation SBA-2 contains 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3) and water. The efficiency for increasing the immune titer was tested analogously to Example 9, FIA served as a comparison. Depending on the surface properties, there are different levels of immune responses

CORRECTED SHEET (RULE 91) ISA / EP for the two SBA dispersions (Figure 2). The strength of the desired immune response can thus be adjusted by varying the surface properties (surfactants, stabilizers, charge, etc.).

Figure 2: Effect of SBA composition on immunotiter: Formulation SBA-1 consists of 4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water, formulation SBA-2 contains 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3) and water.

Example 11: Storage stability of SBA-2: The SBA dispersion SBA-2 from Example 11 was stored at different temperatures and the physical stability was determined by measuring the particle size with PCS. Particle growth did not occur (Figure 3).

Figure 3: Storage stability of SBA-2: The SBA dispersion SBA-2 from Example 11 was stored at different temperatures and the physical stability was determined by measuring the particle size with PCS.

Example 12: Surface modification of SBA dispersions: To modify the surface charge, SBA dispersions were prepared with surface-active positively charged stabilizers (EQ 1 - distearoylethyldiamonium chloride, cetylpyridinium chloride) and negatively charged stabilizers (sodium lauryl sulfate, (SDS)). The composition of the SBA dispersions was: SBA-EQ 1 (20% cetyl palmitate, 4% Tween 80 / Span 85 (7/3), 1% EQ1), SBA-CPC (18% lipid, 10% surfactant, 0.1) % Cetylpyridinium chloride) and SBA-SDS (20% cetyl palmitate, 1% SDS 80). The zeta potential was measured in millivolts (mV) as a measure of the charge

CORRECTED SHEET (RULE 91) (Electrophoresis measurement), Zetasizer4 (Malvern Instruments, UK). The Helmholtz-Smoluchowski equation was used to convert the electrophoretic mobility into the zeta potential. The zeta potentials were +40 mV, +28 mV and -35 mV for the 3 SBA dispersions.

Example 13: Increased cellular immune response in booster chickens treated with GMDP-containing adjuvant (SBA) at the time of primary immunization. SBA (5% EQ 1, 0.5% Montanide 888 was mixed 1: 1 with the antigen (IgG from rabbit) and injected subcutaneously. SBA contained 5 µg GMDP and the immunization schedule was as follows: first immunization and two boosters on day 14 and day 28. The antibody determination took place on day 42 and the IgY titer was measured in the egg yolk. To check for a strengthened cellular immune response, antigen contact was repeated on day 100 (renewed vaccination) and the antibody determination on day 120. The results show that in the case of the first immunization (day 14 and 28) with SBA containing GMDP, with renewed contact with the antigen, significantly increased antibody production takes place.

Figure 4: Antibody production in chickens after basic immunization and renewed antigen contact on day 100. The last antibody determination of the basic immunization took place on day 42, that of the booster vaccination (day 100) on day 120. The composition of the vaccines of Examples 1-5 as follows:

CORRECTED SHEET (RULE 91) ISA / EP 1. Day 42: antigen in PBS, day 100: antigen in PBS (n / a: no adjuvant, antigen in PBS)

Day 42: Antigen in SBA with GMDP, Day 100: Antigen in SBA

3. Day 42: Antigen in SBA with GMDP, Day 100: Antigen in FCA (FCA = Freund's complete adjuvant)

4. Day 42: Antigen in SBA with GMDP, Day 100: Antigen in PBS

5. Day 42: antigen in FCA, day 100: antigen in PBS

Example 14: The adsorption of GMDP on SBA particles was investigated using PCS. GMDP was mixed with SBA (4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and left for 30 minutes at room temperature to allow adsorption of the GMDP to the particles. The final concentration was 1.435 mg / ml The size increase is 3.9 nm. (PCS diameter without GMDP: 99.4 nm, standard deviation: 0.764, diameter with GMDP: 103.3 nm, standard deviation: 0.755).

Example 15: Modification of the particle size: The SBA dispersion with EQ 1 from Example 12 (SBA-EQ 1) was produced using various production processes in order to vary the particle size. The particle size was measured using laser diffractometry (laser diffractometer LS 230, Coulter Electronics-Germany, measuring range: 40 nm - 2000 μm). The diameter 50% of the particles is given as the characterization parameter. The following manufacturing methods were used:

a) High pressure homogenization: The lipid was melted, added to the aqueous surfactant solution, dispersed with a stirrer and the crude emulsion obtained is homogenized at 80 ° C. using a high-pressure homogenizer (Micron LAB 40, APV Gaulin Homogeniser GmbH, Germany). Homogenization parameters were 500 bar pressure, 3 homogenization cycles. The particle diameter 50% was 0.15 μm.

b) The raw emulsion was prepared as described under a) and homogenized with a micro fluidizer (device type 1 10N, Microfluidix lnc, USA). Homogenization parameters were 700 bar, 10 minutes circulation time. The average particle diameter was 0.452 μm.

c) Rotor-stator dispersion: The raw emulsion was prepared as described under a) and then with an Ultraturrax (type T25, Jahnke and Kunkel, Staufen, Germany) at one

Speed of rotation of 10,000 revolutions per minute for 1 minute and 10 minutes dispersed, dispersion temperature 80 ° C. The particle diameters were 7.5 and 1.2 μm

d) Static mixer: Lipid and aqueous surfactant solution from a) were heated to 80 ° C. and mixed in a static mixer (Sulzer, Germany). The particle size was 15.8 μm.

a) Gas jet mill: behenic acid triglyceride was air jet milled (Jetmill, Mosokawa Alpine AG) and then dispersed with stirring in the aqueous surfactant solution at room temperature. The particle diameter 50% was 37.03 μm. e) Mortar mill: The coarsely powdered lipid was ground in a mortar mill with the addition of liquid nitrogen for 3 minutes and 15 minutes (Retsch Mörsermühle, Reetsch, Germany). The lipid was dispersed in water as in e). The average particle size was 40 μm.

Example 16: Molecular adjuvant to increase the cellular immune response incorporated in SBA: GMDP was dissolved in Span 85 (W / O) emulsifier and cetyl palmitate was added. The mixture was melted at 70 ° C. and, after re-cooling, was ground in a mortar mill with the addition of liquid nitrogen. The ground lipid-GMDP mixture was dispersed in a 2.5 percent Tween 80 solution and predispersed with the Ultraturrax for 1 minute at 8000 rpm. This dispersion was homogenized at 4 ° C. by means of high pressure homogenization in 3 cycles at 1000 bar. The PCS diameter is 260 nm with a polydispersity index of 0.430.

Example 17: Molecular adjuvant for increasing the cellular immune response incorporated in the interface: Saponins are generally known for increasing the cellular immune response. The particles were produced using a rotor stator analogous to Example 15. The composition of the SBA dispersion is 5% cetyl palmitate, 0.5% saponin (Quil A saponin) and water. The saponin was dissolved in the aqueous phase, this was heated to 80 ° and the melted lipid was added. Production was carried out with an Ultraturrax, stirring at 10,000 RPM for 5 minutes. The diameter 50% determined with the laser diffractometer was 2.28 μm. Example 18: Production of SBA in the presence of an amphiphilic adjuvant. The amphiphilic surfactant CHAPS has been described in the literature as an agent for increasing the immune response. The particles consist of 5% cetyl palmitate and 0.5% CHAPS. The particles were produced analogously to example 20. The diameter 50%, determined by means of laser diffractometry, is 1,897 μm.

Example 19: Comparison of SBA versus pure molecular adjuvant: SBA dispersion No. 2 from example 10 (SBA-2) was tested in sheep (conditions as in example 9) against molecular adjuvant, ie. H. pure GMDP (N-acetylglucosaminyl-N-acetylmuramyl dipeptide). The concentration of GMDP (0.1 mg / ml) was analogous to Example 9. The in vivo testing was carried out as described in Example 9. SBA 2 shows a higher active intensity than pure GMDP (Fig. 5). Composition of SBA-2: 4% hard paraffin, 1% EQ 1 (N, N Di- (ß-

Stearoylethyl) - N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3).

Example 20: Effect of charge (surface property) on the immune response (sheep study analogous to Example 9): There is no difference in the potency between the positively charged particles SBA 4 and SBA 2. EQ 1 from SBA 2 can be replaced without loss of effectiveness by the toxicologically examined and approved as a pharmaceutical preservative cetylpyridinium chloride (SBA 4). One observes in relation to the negatively charged particles of the formulation SBA 5 a stronger effect of the positively charged particle formulations (Fig. 6).

The SBA formulations have the following composition: SBA 4: 4% hard paraffin, 4% Tween 80 / Span 85 (7/3), 0.5% cetylpyridinium chloride. SBA 5: 4% hard paraffin, sodium deoxycholate 0.2%, sodium cholate 0.2%, sodium oleate 1%, lipoid E80 2%. SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3). The surface charge (zeta potential) was determined in conductivity water with a conductivity of 50μS / cm: SBA 4: +41, 2 mV, SBA 2: +40.5 mV, SBA 5: -36.4 mV. The sizes (PCS diameter and polydispersity index (P.I.)) were: SBA 4: 103 nm (P.I. 0, 1 10), SBA 5: 107 nm (P.I. 0, 115), SBA 2: 10 nm (P.I. 0, 101).

Example 21: Species independence of the effect (chickens): The antigen from Example 9 was mixed with SBA 1 and SBA 2 in a ratio of 1: 1 and 0.5 ml was injected per chicken. The antibody titers were determined from the hen's eggs. An ELISA test was used for quantification. In contrast to the description in Example 9, a labeled anti-IgG chicken was used in the ELISA test. The first immunization took place on day 0. A booster with the same preparations took place on day 31. Analogous to the results in example 10, the formulation SBA 2 had a stronger effect (FIG. 7). Composition SBA 1: 4% hard paraffin and 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (PI 0, 112) and SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: lOnnm (PI 0.101). Example 22: Influence of the lipid matrix on the adjuvant effect: In the formulation SBA 2, the non-biodegradable hard paraffin was exchanged for the biodegradable glycerol tribehenate (SBA 3). The intensity of the effect does not differ; Hard paraffin can be replaced by glycerol tribehenate (Fig. 8)

Composition SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: 10 lnm (PI 0, 101) SBA 3: 4% glycerol tribehenate, 1% EQ 1 and 4 % Tween 80 / Span 85 (7/3), PCS diameter: 105nm (PI 0, 1 12).

Example 23: Formulations SBA 1 and SBA 2 were tested in comparison to aluminum hydroxide (procedure analogous to Example 9). The effect of SBA 1 and SBA 2 is the same as that of aluminum hydroxide (control: antigen in PBS). Analogous to Example 9, SBA 2 is more effective than SBA 1 (Fig. 9).

Composition SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: 10 lnm (PI 0, 101) SBA 1: 4% hard paraffin, 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (PI 0, 112).

List of figures:

Figure 1: Adjuvant effect compared to molecular adjuvant (GMDP N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA.

Figure 2: Effect of SBA composition on immune titer.

Figure 3: Storage stability of SBA-2.

Figure 4: Antibody production in chickens after basic immunization and renewed antigen contact on day 100, the last antibody determination of the basic immunization took place on day 42, that of the booster vaccination (day 100) on day 120.

Figure 5: Adjuvant effect of SBA compared to molecular adjuvant GMDP (Example 19)

Figure 6: Effect of different particle charge (Example 20)

Figure 7: Adjuvant effect in chickens (Example 21)

Figure 8: Influence of the lipid matrix on the adjuvant effect (Example 22)

Figure 9: Comparison of the effect of SBA 1 and SBA 2 with aluminum hydroxide (Example 23)

CORRECTED SHEET (RULE 91)

Claims

claims

1. Means to increase the immune response in vaccinations of humans and animals and to increase the yield of antibodies in immunology and antibody production, characterized in that it is solid at room temperature (= 20 ° C) lipid particles which are in a for the respective subject to be immunized, the optimal dose, as well as the optimum particle size, particle charge and surface properties

(stabilizing surfactant layer) are applied, whereby they are simply mixed into the solution of the antigen.

2. Composition according to claim 1, wherein it is particles in the range of 10- 1000 nm.

3. Composition according to claim 1, wherein it is particles in the range of 1- 10 microns.

4. Composition according to claim 1, wherein it is particles in the range of 10-200 microns, in particular particles in the range of 10-100 microns.

5. Composition according to claim 1, wherein it is particles in the range of 200-1000 microns, in particular particles in the range of 200-500 microns.

6. Composition according to claims 1-5, characterized in that the lipids used to produce the lipid particles are solid at room temperature, for example ethyl stearate, octadecane, DDA, natural before or synthetic triglycerides or mixtures thereof, monoglycerides and diglycerides, alone or mixtures thereof or with, for example, triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols, including their esters and ethers, and in the form of lipid peptides, or any

Mixtures of the same. Synthetic monoglycerides, diglycerides and triglycerides are particularly suitable as individual substances or as a mixture (e.g. hard fat), Imwitor 900, triglycerides (e.g. glycerol trilaurate, glycerol myristate, glycerol palmitate, glycerol stearate and glycerol behenate) and waxes such as e.g. Cetyl palmitate and white

Wax (DAB), which can be used individually or in mixtures, and also hydrocarbons, such as Hard paraffin.

7. Composition according to claim 6, characterized in that liquid lipids such as liquid gycerides (miglyols), peanut oil, castor oil, soybean oil, cottonseed oil, rapeseed oil, linseed oil, olive oil, sunflower oil, safflower oil are added to the solid lipids used to produce the lipid particles the particles made from it are solid at room temperature (20 ° C).

8. Composition according to claims 1-7, characterized in that they are positively charged.

9. Composition according to claim 8, characterized in that to produce the positive charge in the production of the lipid particles, the substances benzyldimethylhexadecylammonium chloride, methylbenzethonium chloride, benzalkonium chloride, cetylpyridinium chloride, N, N di- (ß-stearoylethyl) -N, N- dimethylammonium chloride, dimethyldiocta- decylammonium bromide can be added individually or in a mixture with one another.

10. Composition according to claims 1-7, characterized in that they are negatively charged

1 1. Agent according to claim 10, characterized in that to generate the negative charge in the production of the lipid particles, the following compounds: diacetyl phosphates, phosphatidylglycerol, lecithins of different origins (eg egg lecithin or soy lecithin), chemically modified lecithins (eg hydrogenated lecithins), just like phospholipids and sphingolipids, mixture of lecithins with phospholipids, sterols (eg cholesterol and cholesterol derivatives, just like stigmasterol) and also saturated and unsaturated fatty acids, sodium cholate, sodium glycocholate, sodium taurocholate, sodium deoxycholate, individually or in a mixture are added together.

12. Composition according to claims 1-7, characterized in that they are uncharged or only slightly charged

13. Composition according to claim 12, characterized in that for their production in the production of the lipid particles uncharged substances such as polyethylene glycol sorbitan fatty acid esters (in particular the Tween series such as Tween 80), polyoxyethylene polyoxypropylene copolymers

(in particular the Poloxamer series and the Poloxamine series), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and esters and ethers of sugars or of sugar alcohols with fatty acids or fatty alcohols (e.g. saccharas rose monostearate) may be added individually or as a mixture with one another.

14. Composition according to claims 1- 13, characterized in that one or more adjuvants are added to them.

15. Composition according to claim 14, characterized in that there are molecular adjuvants such as N-acetylglucosaminyl- (ßl-4) -N- acetylmuramyl-L-alanyl-D-isoglutamine [GMDP], dimethyldioctadecylammonium bromide [DDA], N- acetylmuramyl-L-alanyl-D-isoglutamine

[MDP], N, N di- (ß-stearoylethyl) - N, N-dimethylammonium chloride [EQ 1], glycopeptides, components of the cell wall of mycobacteria, saponins, quaternary amines such as cetylpyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] - 1-propanesulfonate), dextran sulfate, dextran, 3-odesacyl-4 ^' -monophosphoryl lipid A [MPL®], N-acetyl-L-alanyl-disoglutaminyl-L-alanine 82) - 1,2 dipalmitoyl - sn glycero - 3- (hydroxy-phosphoryloxy)) ethylamide, monosodium salt [MTP-PE], granulocyte-macrophage colony stimulating factor [GM-CSF], block copolymers, for example P1205, poloxamer 401 (Pluronic

L121), Dimyristoylphosphatidylcholine [DMPC], Dehydroepiandrosterone-3ß-01- 17-one [DHEA], Dimyristoylphosphatidylglycerol [DMPG], Deoxycholic Acid Sodium Salt, Cytokines, Imiquimod, DTP-GDP, Sapuanosine, 7-Aloglyosine, 7-Aloglyosine, 7-Aloglyosine Montanide ISA 51, Montanide ISA 720, MPL, Murametid, Murapalmitin, D-Murapalmitin, 1-

Monopalmitoyl-rac-glycerol, dicetyl phosphate, polymethyl methacrylate [PMMA], PEG sorbitan fatty acid esters such as Polysorbate 80 (TWEEN® 80), Quil A saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN®85, Arlacel85), DTP-DPP, stearyl-tyrosine, N, N dioctadecyl-N ', N'-bis (2hydroxyethyl) propanediamine, calcitriol.

16. Composition according to claim 14, characterized in that it contains particulate adjuvants such as e.g. Aluminum hydroxide, polymer particles,

Liposomes.

17. Composition according to claim 14, characterized in that they increase the effectiveness of adjuvants substances from the group of divalent transition metal ions, quaternary amines, dextran, dextran sulfate, vitamin E derivatives such as vitamin E phosphate and vitamin E Hemisuccinate, and isoprinosine were added.

18. Agent according to claims 1-17 prepared in that the lipid particles were produced by comminution of lipids in the solid state, e.g. Mortar mill, gas jet mill, electro sputtering, high pressure homogenization, microfluidization.

19. Composition according to claims 1- 17, characterized in that the lipid particles by comminution of lipids in the molten state with dispersion in an external phase (eg high-speed stirrers, static mixers on a microscale and macroscale, rotor-stator mills, colloid mills, High pressure homogenization, microfluidization, spray processes such as spray drying and electro-spray processes) can be produced, the outer phase being liquid

(Water, organic liquids, oils or their mixtures) or gaseous (air, nitrogen, noble gas).

20. Composition according to claims 1- 19, characterized in that the lipid particles and optionally the additional adjuvant are dispersed in an outer phase, e.g. aqueous liquids such as water, isotonic sugar solutions and isotonic sodium chloride solution, non-aqueous liquids such as PEG 400 or 600, organic liquids such as oils (miglyols, peanut oil, castor oil, soybean oil, cottonseed oil, rapeseed oil, linseed oil, olive oil, sunflower oil, safflower oil and other oils or their oils Mixtures.

21. Composition according to claim 20, characterized in that viscosity-increasing substances have been added to the outer phase, e.g.

22. Composition according to claims 1- 19, characterized in that the lipid particles and optionally the additional adjuvant are in a dry form, e.g. as a lyophilisate, spray-dried product, solid

Dispersion, pellet or tablet.