KR20020012221A - Stability, biocompatibility optimized adjuvant(sba) for enhancing humoral and cellular immune response - Google Patents

Abstract

The present invention relates to an adjuvant (SBA) with optimized stability and biocompatibility for enhancing humoral and cellular immune responses when injected with one or more antigens. Such adjuvants consist of particles based on solid lipids or solid lipid mixtures. Possible applications include the manufacture of more efficient and tolerated vaccines, vaccination of humans and animals, and antibody production. Through selection of particle size, particle charge and surface properties, the intensity of the immune response can be specifically controlled and additionally species-specific. Additional adjuvants, for example molecular adjuvant such as GMDP, can be added to the SBA to further increase cellular immune response. The SBA is more efficient than conventional products, is inexpensive (effective), simpler to manipulate and well tolerated in vivo.

Description

STABILITY, BIOCOMPATIBILITY OPTIMIZED ADJUVANT (SBA) FOR ENHANCING HUMORAL AND CELLULAR IMMUNE RESPONSE} to Enhance Humoral and Cellular Immune Responses

Antigens are administered to animals and humans to produce antibodies. The purpose of doing so is, for example, to protect humans or animals from disease, to immunize them or to produce antibodies, which subsequently separate them and then process them into products. The most frequently used mode of antigen administration is the parenteral mode of administration, as another method, for example, oral, intranasal and topical administration.

A frequently occurring problem is that the effect of an immune response on the administered antigen is often not as intended. Often, this problem can be solved by significantly increasing the dose of antigen administered. However, since antigens are often very expensive, increasing their dose raises the serious problem of correspondingly increasing the cost of the vaccine. Since these costs place a significant burden on the health system, for many social classes, especially in the third world, vaccines are too expensive to achieve the desired mass vaccination.

Another approach is to administer an adjuvant with the antigen that enhances the antigenic effect and thus provides higher antibody titers. This adjuvant principle was first reported in 1948 by Jules Freund (Freund, J.J. Immunology 1948, 60, 383-398], which was achieved with two mineral oil-based emulsions. Freund's incomplete adjuvant (FIA) is a mixture of mineral oil and mannide monooleate (Montanide, Arlacel). To apply it, it is mixed with the antigen solution and then injected with the emulsion formed. Since the resulting increase in immune response was significant, FIA is still referred to as a "reliable standard" when developing and testing new adjuvants.

Their efficacy and properties are assessed by comparing FIA with a number of newly developed adjuvants, but their efficacy is much lower than that of FIA (for example, they are 0.2 compared to FIA of 1.0).

Inactivated mycobacteria such as M. a. The addition of M. tuberculosis to the FIA further enhanced the immune response. This mixture is termed Freund's Complete Adjuvant (FCA). However, Freund's oil emulsions cause inflammation and ulcerative swelling (granulomas) at the injection site, some of which rupture into extensive abscesses. According to current standards, the use of Freund's oil emulsions in humans is not a problem at all. It is often used in animals, but sometimes the general health of the animal is severely damaged, resulting in uneconomic losses. M. Freund suggested for the completion of the adjuvant. Tuberculosis or M. Butyricum mycobacteria are also poorly tolerated. They not only cause granulomas and fever, but also produce abscesses at the injection site. Brown, E.A. Rev Allergy 1969 23 (5): 389-400.

Therefore, it is possible to produce vaccines that are particularly cost-effective, to enable the desired global immunization for certain diseases, which can only be achieved economically with low-cost vaccines, and to increase the importance of immunologically generated technologies and products. In this regard, a widely tolerated adjuvant should be developed. In addition, due to increased awareness of the need for animal care and the corresponding increase in legislative pressure, it is necessary to develop an adjuvant that is widely tolerated (ie biocompatible) and equally efficient but at the same time low cost. In addition, it must be physically stable so that it does not need to be mixed before injection and there is no variation in efficacy caused by, for example, different fine dispersants.

Currently, the most frequently used adjuvant, approved for human use, is a fine suspension of aluminum hydroxide, the source of which dates back much more than the source of Freund's emulsion. Glenny A.T. et al., J. Pathol. 1926, 29 31-40. This is based on the hypothesis that when antigens are adsorbed on the surface of aluminum hydroxide particles, they are better recognized by the immune system. However, aluminum hydroxide has the disadvantage that it is less efficient than Freund's adjuvant and may cause granulomas.

Starting from Freund's emulsion, recently more commonly used substances have been reported, such as emulsions consisting of squalane and squalene. Sanchez-Pestador, L. et al., J. Immunol. 1988 141, 1720-1727, Masihi KN, Lunge W., Bremer W., Ribi E. Int. J. Immuno-Pharmacol. 1986 8 (3), 339-45, Hunter R. L., 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 (see European Patent Application No. 0399843A2).

On the basis of the suspending agent according to Glenny, particles ranging from different polymers to various organic and inorganic particles (eg carbon) have been used (see O'Hagan, DT, Jeffrey, H., Roberts MJ, McGee, JP, Davies, SS Vaccine. 1991 9 (10). 68-71, Glenny, A. T. et al., J. Pathol. 1926, 29 31-40. Disadvantages of these systems are that some polymers are cytotoxic (e.g., formaldehyde is formed in the case of polyalkylcyanoacrylates) and are not degraded or degraded too slowly in the organism (e.g., Polystyrene or polymethacrylate derivatives), inadequate biocompatibility (e.g., in the case of PLA particles, capsules are formed, and the pH is shifted to pH 2), and registered from a registration authority (e.g., Boot) Particles] cannot be obtained.

Another way to use specific adjuvants such as oil droplets or solid particles is to use macromolecular adjuvant. It has been found that the mycobacteria reported by Freund can be replaced by units from their capsule material, which is more tolerant to the body. Essentially described and synthesized are the following: MDP (N-acetylmural-L-alanyl-D-isoglutamine) [Adam, A., Lederer, E., Med. Res. Rev. 1984 4, 111-152], GMDP derived from Thr-MDP (threonyl homologue of MDP) (Byars NE, et al. Vaccine, 1987 5 (3), 223-8) and yoghurt bacillus (N-acetylglucosaminyl-N-acetylmurayl-dipeptide) (Grubhofer, N. Immunology Letters 1995 44. 19-24). GMDP is a homologue of MDP; Increased immune system stimulating effects are currently demonstrated for GMDP (see patent specification DE 19611235 C1).

Additional steps in adjuvant development included the use of molecular adjuvant at high concentrations, resulting in better solubility and suspending agents [eg, DDA (dimethyldioctadecyl ammonium bromide)] [Grubhofer, N. Immunology Letters 1995 44, 19-24], in order to enhance the humoral immune response, involves the use of a mixture of macromolecular adjuvant and solid particles, such as glycopeptides (eg, colloidal particles). GMDP with Grubhofer, N. Immunology Letters 1995 44, 19-24.

To date, however, no solution has been found that is equal in efficacy to Freund's adjuvant and meets other requirements; New emulsions are not always taken without side effects, synthetic macromolecules such as glycopeptides have never been found to be superior to mycobacteria, and they are much more expensive. In the case of solid particles, there is a registration problem. Many adjuvants show a fairly high level of toxicity, so the level of biocompatibility is extremely low. There are also problems in terms of physical stability (eg avoiding aggregation and / or merging). Therefore, long-term storage is not possible because FIA emulsions must be freshly produced and injected. However, sufficient physical stability during storage is an essential prerequisite for marketable products, especially drugs.

It is therefore an object of the present invention to produce a novel adjuvant having the following:

1. Sufficient physical stability

2. Low cytotoxicity and sufficient biocompatibility

3. Efficacy comparable to the efficacy of FIA

4. Low production cost.

1 is a diagram showing the adjuvant effect of comparing FIA with molecular adjuvant (GMDP-N-acetyl glucosaminyl-N-acetyl muramil-dipeptide).

2 shows the effect of SBA composition on antibody titer.

3 shows the storage stability of SBA-2.

Figure 4 shows antibody production in hens after primary immunization and resumed antigen contact on day 100. Final antibody determination of basal immunization was performed on day 42 and its booster injection (day 100) on day 120.

5 shows the adjuvant effect of SBA (Example 19) compared to molecular adjuvant GMDP.

6 shows the effect of different particle charges (Example 20).

7 shows the adjuvant effect (Example 21) in hens.

8 shows the effect of the lipid matrix on the adjuvant effect (Example 22).

9 is a view showing an effect comparison between SBA 1 and SBA 2 and aluminum hydroxide (Example 23).

To date, in order to increase the immune system stimulating effect, GMDP has been used in a mixture with colloid-dispersible solid lipid particles having a particle size of <200 nm [USA Patent Gerbu; US patent application Ser. No. 08 / 816,787]. The structure of these particles was systematically modified in the study of the present invention to identify lipid particles that could further enhance the immune system stimulating effects of GMDP (eg, surfactants, stabilizers were used). Surprisingly, at the same time, it was found that the lipid particles alone were nearly as efficient as the combination of lipid particles with GMDP (Example 9). Thus, in the present invention, there is no need to use expensive GMDP, and even low-cost lipid particles alone can achieve comparable levels of humoral immune system stimulation.

The intensity of the immune system stimulating effect herein depends on the surface activity (surfactant used, particle charge) and particle size of the particles. For negatively charged lipid particles, the effective amount is about one third of the FIA; When positively charged lipid particles are produced by adding EQ1, its efficacy is comparable to that of FIA (no significant difference, t-test) (Example 9). Thus, the desired efficacy can be adjusted through the composition of the lipid particles used. This eliminates overreaction of the organism.

From adjuvant studies conducted to date on organic and polymeric suspending agents, it is known that for each antigen there is a particle size that exhibits maximum potency, and species dependency is likewise known. Therefore, the particle size of the aluminum hydroxide suspending agent best suited for the vaccine is determined experimentally; Polymer particles as immunization adjuvants show different effects depending on their size (Kreuter J, et al., Vaccine, 1986, 4, 125-9). Although found to be surprisingly identical for lipid particles, despite being extremely different matrix materials, efficacy can be controlled by changing their particle size antigen-specifically and species-specifically.

The lipid particle dispersant of a stable and biocompatible adjuvant (SBA) consists of lipid particles dispersed in water or in an aqueous liquid or a non-aqueous (eg oily) liquid, which particles are not necessary but possibly interfacial. Stabilized by an active agent or polymer. A sufficiently high viscosity of the outer phase or the fineness of the particles are used to obtain a physically stable dispersant. If the particles have the same sufficiently high charge so that the precipitate formed can be easily redispersed, it can be applied as above even in the case of low viscosity external phases.

The production of SBA is accomplished through dispersion techniques or precipitation methods using generally known methods described in pharmaceutical or process engineering textbooks. During dispersion, the coarsely dispersed lipids disintegrate by mechanical processes. Such lipids may be in solid aggregate state (eg mortar grinder) or in liquid aggregate state (eg emulsification of lipid melt by stirrer). To prepare an SBA dispersant, the lipid is first milled and then dispersed in an external (eg aqueous) phase, or alternatively directly in an external phase. To prepare SBA dispersants by dispersing, for example, the following may be used: piston-gap homogenizers (eg APV homogenizers, French presses), jet stream homogenizers (eg microfluidizers), rotors- Stator shaker (e.g. Ultraturrax, Silverson Homogenizer), small and large scale static mixers (e.g. mixer from Sulzer company), gas-jet grinder, rotor-stator colloid grinder and mortar grinder (Example 15 ).

SBA dispersants exhibit sufficient long-term physical stability. Particle size determination using photon correlation spectroscopy and laser diffraction (LD) showed no particle growth or negligible particle growth over one to three years (Example 3). The stability of the SBA dispersant is much better than Freund's incomplete adjuvant (FIA) (Example 1), and also shows greater stability compared to more recent adjuvant emulsions (Example 2).

Stability : SBA dispersants represent a simple system. Unlike glycopeptides, the adjuvant materials used are chemically simple and robust in structure. No chemical decomposition takes place when heat is applied in the sterilization process, and the dispersant is still physically stable and no particle aggregation occurs. One example of sterilizing the SBA dispersant by autoclaving (122 ° C., 15 minutes, 2 bar) is shown in Example 4. FIA shows phase separation under the same conditions.

Many existing vaccines are stored at low temperatures (4-6 ° C.). In the case of optimized SBA dispersants, it can be stored at room temperature as well as even at higher temperatures without inducing a physical destabilization process (Example 11). This simplifies the manipulation of SBA-based products, especially in tropical climate regions (eg used as adjuvants in high volume vaccinations in the Third World).

Biocompatibility : Low toxicity and good biocompatibility are essential for widespread application of adjuvant. In the studies using sheep, no strange symptoms were observed at the site of application after application of the SBA dispersant (Example 6). The fact that this is widely tolerated is evident by the fact that the toxicity of lipids has been observed to be extremely low in in vitro cell culture. Compared to polymers approved for parenteral administration by the German registrar and the FDA, they exhibit approximately 20 factors higher viability for high particle concentrations (Example 5). This good biocompatibility is due to the fact that, in general, body proteins are adsorbed only to a slight extent on the surface of the particles as opposed to other particles (Example 7). In addition, no protein was detected on the surface that stimulates intolerant (hypersensitive) responses.

For widespread application of the adjuvant, its own solution in terms of cost is to prepare an adjuvant formulation that is mixed into the antigen solution prior to application (administration). Ideally, it is capable of mixing with many different antigens. Adjuvant with properties should be prepared. To reduce injection pain, the mixture should be administered in physiological sodium chloride solution or other isotonic solution. Due to the decrease in zeta potential, physiological sodium chloride solution causes destabilization in the dispersant and causes agglomeration continuously. Lucks, J.S. et al., Int. J. Pharm. 1990 58, 229-235. The SBA adjuvant must be physically stable for a sufficiently long time after mixing with the antigen in an isotonic solution. Example 8 showed that the lipid particles were stable even over 6 hours after mixing with physiological sodium chloride solution and no measurable levels of aggregation occurred.

In addition to particle size, surface properties such as charge can be specifically adjusted to allow species-specific SBA adjuvants to be produced at effective intensities. Positive and negative charges can be generated by mixing the correspondingly charged surfactants or stabilizers. The charge intensity can be adjusted through the concentration of the additive, an ideal additive being an ionic material that is acceptable as a preservative for parenteral administration, such as cetyl pyridinium chloride (Example 12).

Many different lipids can be used in the preparation of SBA dispersants. These are chemically homogeneous lipids and mixtures thereof. The lipids are characterized in that they exist in a crystalline state (eg β-, βi-modified) or liquid-crystalline state (α-modified), or a mixture thereof in the SBA dispersant final product. When lipid mixtures are used, liquid lipids such as oils, lipophilic hydrocarbons, and lipophilic organic liquids such as oleyl alcohol, may be used as solid lipids such as glycerides, lipophilic hydrocarbons such as hard Paraffin) ("lipid blend").

The following lipids are used, for example, as the disperse phase and can be applied as individual components or as a mixture: natural or synthetic triglycerides and / or mixtures thereof, monoglycerides and diglycerides alone or mixtures thereof, Or, for example, mixtures with triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols (including their esters and ethers, and forms of lipid peptides), or mixtures thereof. Particularly suitable are synthetic monoglycerides, diglycerides and triglycerides as individual substances or mixtures (e.g. hard fats), Iwitor 900, triglycerides (e.g. glycerol trilaurate, glycerol myristate, glycerol palmi) Tate, glycerol stearate and glycerol behenate), and waxes such as cetyl palmitate and white wax (DAB-German Pharmacopeia). Also suitable are hydrocarbons such as hard paraffin.

The proportion of the internal or lipid phase based on the total formulation is in the range of 0.1 to 80% (m / m), preferably 1 to 40% (m / m). In order to be able to produce stable dispersants, addition of additives which stabilize the dispersant, for example emulsifiers, is necessary or desirable, so that it can be incorporated in the form of a pure substance or in the form of a mixture to stabilize the particles. .

Based on the total weight of the aqueous dispersant, the amount of the additive that can be added is in the range of 0.01 to 30%, preferably 0.5 to 20%. For stabilizing SBA dispersants or for their specific surface modifications, surfactants, stabilizers and polymers generally known from dispersant preparation can be used. Examples of these are:

1. Stereoscopically stabilized materials such as poloxamers and poloxamines (polyoxyethylene-polyoxypropylene-block-copolymers), ethoxylated sorbitan fatty acid esters, in particular polysorbates such as polysols Bait 80 / Twin 80 ), Ethoxylated mono and diglycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and esters and ethers of sugars or sugar alcohols with fatty acids or fatty alcohols such as sucrose monostearate, sucrose distearate , Sucrose cocoate, sucrose stearate, sucrose dipalmitate, sucrose palmitate, sucrose laurate, sucrose octanoate, sucrose oleate).

2, charged ionic stabilizers such as diacetylphosphate, phosphatidyl glycerin, lecithin of various origins (eg egg lecithin or soy lecithin), chemically modified lecithin (eg hydrogenated lecithin) as well as phospholipids and Sphingolipids, mixtures of lecithin and phospholipids, sterols (such as cholesterol and cholesterol derivatives, and stigmasterol), and saturated and unsaturated fatty acids, sodium cholate, sodium glycolate, sodium taurocholate, sodium deoxycholate or their Mixtures, amino acids or anti-agglomerates such as sodium citrate, sodium pyrophosphate, sodium sorbate [Lucks, JS et al., Int. J. Pharm. 1990 58, 229-235. Zwitterionic Surfactants, for example, (3-[(3-colamidopropyl) -dimethylammonio] -2-hydroxy-1-propane sulfonate) [CHAPSO], (3-[(3- Colamidopropyl) -dimethylammonio] -1-propane sulfonate) [CHAPS], and N-dodecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate. Cationic surfactants, especially compounds used as preservatives, for example benzyldimethyl-hexadecylammonium chloride, methylbenzetonium chloride, benzalkonium chloride, cetyl pyridinium chloride.

3. Materials that increase viscosity, such as cellulose ethers and cellulose esters (eg methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose), polyvinyl derivatives as well as polyvinyl alcohol, polyvinyl Pyrrolidone, polyvinyl acetate, alginates, polyacrylates such as carbopol, xanthan and pectin.

The charged stabilizers, if necessary or desired, are preferably contained in SBA dispersants in amounts ranging from 0.01 to 20% (m / m), in particular from 0.05 to 10% (m / m). Materials which increase the viscosity, if necessary or desired, are in a similar proportion in the formulation, preferably in amounts ranging from 0.01 to 20%, in particular from 0.1 to 10% (m / m), more preferably from 0.5 to 5%. It is incorporated.

As external phase (dispersion medium, continuous phase), water, aqueous solution, or liquids miscible with water, such as glycerin or polyethylene glycol and oily liquids, such as glycols (medium chain triglycerides-MCT) And other oils (castor oil, peanut oil, soybean oil, cottonseed oil, seed oil, linseed oil, olive oil, sunflower oil and safflower oil) can be used.

Surfactant-free SBAs contain one or more viscosity increasing substances alone or in combination with other substances such as sugars, sugar alcohols, in particular glucose, mannose, trehalose, mannitol, sorbitol and the like. Prepared by dispersing the lipid phase in an aqueous solution. Moreover, combinations of viscosity increasing substances, combinations of these with sugars or sugar alcohols, or further combinations with charged stabilizers or anti-agglomerates may be used.

SBA dispersants can be used as an adjuvant to many different antigens for immunization against various diseases. An example of this is:

Glycoproteins such as gonococal protein I, brucella abortus antigen, tetanus toxoid, diphtheria toxoid, listeria monocytogenes,

Viral antigens such as Semliki Forest virus, cerebral myocarditis virus, swine rarovirus, pseudo-rabies virus, Newcastle disease virus, bovine viral diarrhea, HIV, influenza, cytomegalovirus, herpes simplex, hepatitis C, margins,

Parasites such as malaria, Emerasian species, and the like.

In summary, using an SBA dispersant,

1. have sufficient physical stability to be prepared as a specific product, in particular a drug,

2. It has good biocompatibility and low toxicity, especially when biologically degradable lipids such as glycerides are used,

3. show comparable effect to Freund's incomplete adjuvant (FIA),

4. Using a low cost manufacturing method, an adjuvant can be obtained which can be produced cost effectively from an excipient having a low cost.

SBA dispersants can be used extensively to reduce antigen dose, thereby reducing costs and by using toxicologically acceptable excipients and adding SBA, the same immune system stimulating effect is achieved at lower antigen doses.

To date, antigens that have insufficient antigenicity against the vaccine can be made effective by adding an immune response stimulating SBA thereto.

SBA dispersants are suitable as veterinary vaccination adjuvant when only a very low cost vaccine can be used in terms of profitability because it can be produced cost effectively with comparable efficacy to FIA.

Adjuvant, which has been used until now, has focused on enhancing the humoral immune response. In terms of efficacy of the SBA dispersant, it is no longer necessary to add additional adjuvant to the SBA lipid particles and / or the addition of an adjuvant such as GMDP does not further increase the humoral immune response. Clearly, using the immune system as its maximum response capacity, no additional adjuvant can bring additional effects. Thus, due to the surprising efficacy found for the SBA described herein, addition to the SBA does not provide an advantage for the humoral response, but rather an additive as described in Gerbu's patent (patent specification DE 19611235 C1). Is unnecessary.

Surprisingly, however, for subsequent reinoculations (boosters), after administering the SBA dispersant in combination with GMDP, it was found that a markedly enhanced cellular immune response was achieved than if the SBA dispersant was previously used alone as an adjuvant. . Thus, a combination of SBA dispersants has been found to be particularly suitable for increasing cellular immune responses in case of reinoculation. When an additional adjuvant such as GMDP was previously used in a mixture with the SBA dispersant, the immune response was increased at subsequent second inoculations (Example 13).

In order to produce an adjuvant for the purpose of enhancing a cellular immune response, it is advantageous to combine the SBA dispersant with additional adjuvant. Additional adjuvants for the combination are as follows: N-acetyl-glycosaminyl- (β1-4) -N-acetylmuramyl-L-alanyl-D-isoglutamine [GMDP], dimethyldiooctadecylammonium Bromide [DDA], N-acetyl muramil-L-alanyl-D-isoglutamine [MDP], N, N-di- (β-stearoyl ethyl) -N, N-dimethyl ammonium chloride [EQ1], Glycopeptides, cell wall components of mycobacteria, saponins, quaternary amines such as cetyl pyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3-[(3-colamido Propyl) -dimethylammonio] -1-propane sulfonate), dextran sulfate, dextran, 3-odesyl-4'-monophosphoryl lipid A [MPL ], N-acetyl-L-alanyl-disoglutaminyl-L-alanine-82) -1,2-dipalmitoyl-glycero-3- (hydroxy-phosphoryloxy) ethyl amide, monosodium salt [MTP-PE], granulocyte-macrophage colony stimulating factor [GM-CSF], block copolymers such as P1205, poloxamer 401 (Pluronic L121), dimyristoyl-phosphatidyl choline [DMPC] , Dehydroepiandrosterone-3β-01-17-one [DHEA], dimyristoyl-phosphatidyl glycerol [DMPG], deoxycholic acid sodium salt, cytokines, imiquimod, DTP-GDP, saponin, 7-allyl -8-Oxoguasonine, Montanide ISA 51, Montanide ISA 720, MPL, Murametid, Murapalmitin, D-murapalmitin, 1-Monopalmitotoyl-rac-glycerol, Dicetyl Phosphate, Polymethyl Methacrylate [PMMA], PEG-sorbitan fatty acid esters such as polysorbate 80 (twin 80), quill A saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN 85, Arlacel 85), DTP-DPP, stearyl-tyrosine, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine, calcitriol.

In addition to the humoral immune response induced by the SBA dispersant, the present invention opens up the possibility of enhancing the cellular immune response in the case of secondary inoculation by combining the SBA dispersant with other adjuvants.

Instead of a mixture of adjuvant to enhance the cellular immune response, the adjuvant may be incorporated into lipid particles. Incorporation is possible by enclosing in a solid particle matrix, in the case of an amphiphilic adjuvant, at the interface or by simply adsorbing onto the particle surface. Incorporation of the adjuvant may occur during or after particle generation (eg, Example 16 for incorporation). For incorporation during production, the adjuvant is dissolved, solubilized or dispersed in the lipid melt phase, and then the adjuvant containing lipid phase is further processed. In the case of amphiphilic adjuvant, they are dissolved by dissolving on the outer phase of the SBA dispersant and then by adsorbing onto or adsorbing onto the particle surface. Due to the incorporation of the adjuvant, prolonged release occurs either by diffusion or by enzymes during the particle degradation process. Prolonged delayed release promotes an immune response.

An additional field of interest is antibody production in animals. Antibody yield can be obviously increased by the addition of adjuvant.

Example 1 Determination of Physical Stability of SBA to Freund Incomplete Adjuvant (FIA)

Aqueous SBA dispersants are prepared by high pressure homogenization from 20% beeswax, 2% Tween 80 (PCS diameter 289 nm, polydispersity index 0.101) at 95 ° C. FIAs are prepared according to the methods described in Freund, J. J. Immunology 1948, 60, 383-398. SBA and FIA are stored at the temperature of the climatic zone used for drug stability testing [EMEA Directive CPMP / QWP / 159/96, January 1998]. Storage temperature is 5 degreeC, 25 degreeC, and 40 degreeC. Physical stability is determined by measuring particle size using laser diffractometer (LD), with the stellar identification parameters being 50% diameter and 95% diameter (50% / 95% of the particles, which are sensitive to particle aggregation, are indicated size Is under). Even after several minutes of storage at room temperature, FIA exhibits apparent particle growth, so FIA is not a stable adjuvant for storage. On the other hand, the particle size of SBA remained unchanged under three storage conditions over a year (Table 1).

Stability testing of SBA (20% beeswax, 2% Tween 80) compared to Freund's incomplete adjuvant (FIA) LD 50% (㎛) LD 95% (㎛) SBA 5 ℃ 0 days 1 day 30 days 365 days 0.320.320.330.33 0.750.770.780.79 SBA 25 ℃ 0 days 1 day 30 days 365 days 0.310.320.320.33 0.730.770.790.83 SBA 40 ℃ 0 days 1 day 10 days 30 days 0.320.320.320.34 0.730.770.780.78 FIA 25 ℃ 5 minutes 30 minutes 60 minutes 120 minutes 29.5430.2938.3438.85 63.0567.3975.8975.91

Example 2: Determination of Physical Stability of SBA to Squalene Adjuvant:

SBA was prepared as described in Example 1, which contained 10% cetyl palmitate and 1.2% miranol (PCS diameter 210 nm, PCS polydispersity index 0.189). Squalene adjuvant is prepared as described in European Patent Application No. 0 399 843, filed May 25, 1990 (adjuvant MF59). Particle size is measured by laser diffraction and storage is performed at three temperatures as in Example 1 (Table 2). In addition, accelerated stability tests are performed and the SBA and MF59 dispersants are shaken at 40 ° C. at a frequency of 50 Hz (Table 3). SBA shows increased stability in both normal storage and stress tests.

Stability test of SBA (10% cetyl palmitate, 1.2% miranol) compared to MF59 LD 50% (㎛) LD 95% (㎛) SBA 5 ℃ 0 days 1 day 30 days 365 days 0.320.320.330.33 0.750.770.780.79 SBA 25 ℃ 0 days 1 day 30 days 365 days 0.310.320.320.33 0.730.770.790.83 SBA 40 ℃ 0 days 1 day 30 days 365 days 0.320.320.320.34 0.730.770.780.78 MF59 5 ℃ 0 days 1 days 2 days 5 days 0.1190.1130.1110.115 0.2230.2280.2310.226 MF59 25 ℃ 0 days 1 days 2 days 5 days 0.1190.1280.1300.158 0.2230.3290.4800.859 MF59 40 ℃ 0 days 1 days 2 days 5 days 0.1190.3470.4630.741 0.2232.8093.5635.638

Stability of MF59 compared to SBA at 40 ° C and vibration frequency 50 Hz LD 50% (㎛) LD 95% (㎛) SBA 0 days 1 days 2 days 5 days 0.320.330.30.32 0.750.760.810.83 MF59 0 days 1 days 2 days 5 days 0.1190.5970.3020.451 0.2233.8487.7218.784

Example 3: Long-term Stability of SBA:

SBA dispersant consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C. for 1 year. PCS data and laser diffractometer diameters showed little or no change (Table 4).

Long term stability of SBA: SBA dispersant consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C. for 1 year. 0 days 10 days 30 days 60 days 90 days 365 days LD 50% [㎛] 0.35 0.36 0.35 0.35 0.35 0.39 LD 95% [㎛] 0.85 0.93 0.92 0.92 0.91 1.12 Average PCS Diameter [μm] 0.316 0.327 0.337 0.336 0.346 0.365 P.I. 0.138 0.192 0.172 0.16 0.159 0.25

Example 4: Thermal Stability of SBA to FIA and MF59 During Autoclaving (Heat, Pressure):

SBA dispersant was made of 18% hard paraffin, 4% Tween 80 / span 85 (7/3) and water. 20 ml each is sterilized in vials for injection according to the standard conditions of the European Pharmacopoeia (121 ° C., 2 bar, 15 minutes). Particle size was determined using PCS and laser diffraction (LD 95%) (PI: polydispersity index, measurement for particle size distribution width; AV: average from three measurements; St.dev: standard deviation; PI : Polydispersity index) (Table 5). Phase separation appeared after autoclaving the FIA emulsion, and MF59 showed apparent particle size growth. SBA is physically stable and can be sterilized by autoclaving.

Thermal Stability of SBA to FIA and MF59 during Autoclaving (Heat, Pressure): The SBA dispersant was made of 18% hard paraffin, 4% Tween 80 / span 85 (7/3) and water. Sterilize 20 ml each at 121 ° C., 2 bars, 15 minutes. Particle size was determined using PCS and laser diffraction. PCS diameter before sterilization [nm] P.I. before sterilization LD 95% [㎛] before sterilization PCS diameter after sterilization [nm] P.I. after sterilization LD 95% [μm] after sterilization SBA 101100104 0.1010.110.112 0.150.150.152 103101101 0.1090.1110.12 0.1540.1550.154 AV 102 0.108 0.151 102 0.113 0.154 St.dev. 2.042 0.006 0.001 1.436 0.006 0.001 MF59 246234254 0.1210.1030.144 0.2230.2230.222 859912899 0.3210.3050.389 5.0525.0565.055 AV 244.67 0.11 0.22 890.00 0.34 5.05 St.dev. 10.07 0.01 0.00 27.62 0.04 0.00

Example 5: Physiological Tolerance

To assess tolerability, cytotoxicity of SBA is determined in cell culture (human granulocytes, HL60 cells). To quantify this toxicity, MTT assay was used to determine the viability of the cells. See Mosmann, T., J. Immunol. Meth. 1993, 65, 55-63. SBA dispersant consisted of 10% cetyl palmitate, 0.5% poloxamer 168 and water. The number of cells per well was measured at 200,000 for human granulocytes and 200,000 for HL60 cells. Incubate for 12 hours. SBA growth was measured at 80% for granulocytes and 85% for HL60 cells. For nanoparticles of PLA, growth was only 5%, whereas for nanoparticles of PLA / GA, it dropped to 0%. SBA tolerance is at least 20 factors superior to the tolerability of polymers approved by the FDA for parenteral administration in cell culture.

Example 6: Tolerability After Parenteral Administration:

An aqueous SBA dispersant was used, the composition of which was as follows: 5% hard paraffin, 5% Tween 80 / span 85 (7/3) and water. Parenteral injection to sheep (n = 30), this injection site is the chest side wall, the injection volume is 5ml, divided into four fractions and injected. The amounts did not show any abnormalities in the injection site or in their behavior.

Example 7: Biocompatibility-Interaction with Body Proteins:

SBA dispersant consisted of 10% compritol, 2.5% poloxamer 407 and water. Prepared by high pressure homogenization. The particles are incubated with human plasma for 5 minutes, then separated from the plasma and determined by two-dimensional polyacrylamide gel electrophoresis of body proteins adsorbed onto the particle surface. Blunk, T. et al. , Electrophoresis 14, 1382-1387 (1993). For comparable particle surface areas, an extremely small amount of protein is adsorbed onto the SBA, which is 96.41 cpm (count per minute) compared to emulsion [comparative value: 472 cpm on emulsion and 390 cpm on polystyrene particles]. Harnisch, S. et al., Electrophoresis 1998, 19, 349-354, Blunk, T., Electrophoresis 1994, 14, 1382-1387]. Complement factors that increased intolerance were not detected on the SBA surface.

Example 8: Stability in Phosphate Buffered Physiological Sodium Chloride Solution (PBS):

SBA made with 20% hard paraffin, 5% Tween 80 / span 85 (7/3) and water is mixed with PBS (2 ml SBA + 2 ml salt solution). Using laser diffraction measurements, the physical stability in physiological sodium chloride solution is determined as a function of time. Over 6 hours particle size did not increase at all (90% and 95% diameter, Table 6) (St.dev .: standard deviation).

Stability in phosphate buffered physiological sodium chloride solution (PBS): SBA (20% hard paraffin, 5% Tween 80 / span 85 (7/3)) is mixed with PBS (2 ml SBA + 2 ml salt solution). Using laser diffraction measurements, the physical stability in physiological sodium chloride solution is determined as a function of time. LD 90% [㎛] St. dev. LD 95% [㎛] St.dev. SBA 0.233 0.01 0.304 0.012 SBA / PBS1 + 110 minutes 0.189 0.011 0.224 0.01 SBA / PBS1 + 16 hours 0.202 0.009 0.24 0.007

Example 9 Adjuvant Effect Comparing Molecular Adjuvant (GMDP-N-Acetyl Glucosamineyl-N-Acetyl Muramil-Dipeptide) with FIA

Sheep are inoculated with strain Mycoplasma Vorbis PG 45 R9. This inoculated antigen is incubated in culture for 72 hours under aerobic conditions in Hayflick medium. Deactivation is achieved by adding 0.1% β-propiolactone. The cells are isolated and washed with phosphate buffer (pH 7.4) and then adjusted to the content of 1 × 10 ¹⁰ CFU / ml. The sterility of the formulations is tested according to German Pharmacopoeia Edition 10. Anhydrous mass measurement showed the content of 1 mg / ml Mycoplasma Vorbis antigen. Adjuvant SBA, GMDP and FIA are mixed in equal portions with antigen in buffer. The injection volume is 5 ml, which is divided into 4 doses.

The SBA composition is as follows: 4% hard paraffin, 1% EQ1 (N, N-di- (β-stearoyl ethyl) -N, N-dimethyl ammonium chloride) and 4% Tween 80 / span 85 (7 / 3). The composition of GMDP adjuvant is 5% lipid and 0.5% surfactant. FIA is prepared as in Example 1.

Blood samples are taken on days 0, 35 and 63 before inoculation. Antibodies are determined using ELISA. For the ELISA test, a commercial anti-IgG-amount (source: Sigma) was used.

SBA showed potency intensity compared to the efficacy of the combination of lipid particles with GMDP. Moreover, the efficacy of SBA is compared with that of FIA (FIG. 1). There was no significant difference in efficacy intensity between the three adjuvants.

1: Adjuvant effect comparing molecular adjuvant (GMDP-N-acetyl glucosaminon-N-acetyl muramil-dipeptide) with FIA. The SBA composition is as follows: 4% hard paraffin, 1% EQ1 (N, N-di- (β-stearoyl ethyl) -N, N-dimethyl ammonium chloride) and 4% Tween 80 / span 85 (7 / 3) and water. The composition of the GMDP adjuvant is 5% EQ1 and 0.5% Montanide 888. FIA is prepared as in Example 1.

Example 10 Effect of SBA Composition on Antibody Titer:

SBA dispersants are prepared from the same lipids but different surfactants on the surface (ie, they have different surface properties). 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% EQ1, 4% Tween 80 / Span 85 (7/3) and water. Using FIA as a comparative, antibody titer efficiency was tested similarly to Example 9. Depending on the surface properties, different levels of immune response appeared for the two SBA dispersants (FIG. 2). Thus, the intensity of the desired immune response can be adjusted by varying the surface properties (surfactants, stabilizers, charges, etc.).

Figure 2: Effect of SBA composition on antibody titer: 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% EQ1, 4% Tween 80 / Span 85 (7/3) and water.

Example 11: Storage Stability of SBA-2:

The SBA dispersant SBA-2 from Example 10 was stored at different temperatures and physical stability was determined by measuring particle size using PCS. No particle growth occurred (FIG. 3).

Figure 3: Storage stability of SBA-2: SBA dispersant SBA-2 from Example 10 was stored at different temperatures and physical stability was determined by measuring particle size using PCS.

Example 12 Surface Modification of SBA Dispersants

To modify the surface charge, using a positively charged stabilizer of surfactant (EQ1-distearoyl ethyl diammonium chloride, cetyl pyridinium chloride) and a negatively charged stabilizer (sodium lauryl sulfate (SDS)) Prepare SBA dispersant. The composition of this SBA dispersant is as follows: SBA-EQ1 (20% Cetyl Palmitate, 4% Tween 80 / Span 85 (7/3), 1% EQ1), SBA-CPC (18% Lipid, 10% Surfactant) , 0.1% cetyl pyridinium chloride) and SBA-SDS (20% cetyl palmitate, 1% SDS 80). As a measure of charge, zeta potential was measured in millivolts (mV) (electrophoresis measurement), Zetasizer 4 (Malvern Instruments). Using the Helmholtz-Smoluchowski equation, the electrophoretic mobility is converted to the zeta potential. Zeta potentials for the three SBA dispersants were measured at +40 mV, +28 mV and -35 mV.

Example 13; In hens treated with GMDP-containing adjuvant (SBA) upon initial immunization, increased cellular immune response with booster injection:

SBA (5% EQ1, 0.5% Montanide 888) is mixed 1: 1 with antigen (IgG from rabbit) and injected subcutaneously. SBA contains 5 μg GMDP and the immunization schedule is as follows: Two booster injections are taken on days 14 and 28 following initial immunization. Antibodies were determined on day 42 and IgY titers were measured in egg yolk. To investigate the enhanced cellular immune response, new antigen contact (additional booster injection) was performed on day 100 and antibody determination was performed on day 120. As a result, when antigen contact was resumed and initially immunized with GMDP containing SBA (day 14 and 28), antibody production was found to increase significantly (columns 2, 3 and 4 of FIG. 4).

Figure 4: Antibody production in hens after primary immunization and resumed antigen contact on day 100. Final antibody determination of basal immunization was performed on day 42 and its booster injection (day 100) on day 120. The composition of the vaccines of Examples 1 to 5 is as follows:

1. 42 days, antigen in PBS, 100 days: antigen in PBS (n.a .: no adjuvant, antigen in PBS).

2. 42 days, antigen in SBA with GMDP, 100 days: antigen in SBA.

3. 42 days, antigen in SBA with GMDP, 100 days: antigen in PCA (Freundant Complete Adjuvant).

4. 42 days, antigen in SBA with GMDP, 100 days: antigen in PBS.

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

Example 14: The adsorption of GMDP onto SBA particles was examined using PCS. GMDP was mixed with SBA (4% hard paraffin, 4% Tween 80 / span 85 (7/3)) and left to stand at room temperature for 30 minutes to allow GMDP to adsorb onto the particles. Final concentration was determined to be 1.435 mg / ml. Size growth was measured at 3.9 nm (PCS diameter without GMDP: 99.4 nm, standard deviation: 0.764, diameter using GDP: 103.3 nm, standard deviation: 0.755).

Example 15 Modification of Particle Size

Using a variety of manufacturing processes that varied particle size, SBA dispersants (SBA-EQ1) with EQ1 from Example 12 were prepared. Particle size was measured using a laser diffractometer (Laser diffractometer LS 230, Coulter Electronics Germany, measuring range: 40 nm-2000 μm). A diameter of 50% of the particles is provided as a parameter for determining the appearance. The following manufacturing method was used:

a) High pressure homogenization method: The lipid is melted, poured according to the aqueous surfactant solution, dispersed with a stirrer and the obtained crude emulsion is homogenized at 80 ° C. with a high pressure homogenizer (Micron LAB40, APV Gaulin Homogeniser GmbH, Germany). . Homogenization parameters are 500 bar pressure, 3 homogenization cycles. It was determined that the particle 50% diameter was 0.15 mu m.

b) A crude emulsion is produced as described in a) and homogenized with a microfluidizer (Device type 110-Y, Microfluidix Inc., USA). Homogenization parameters are 700 bar, 10 minute cycle time. The average particle diameter was measured to 0.452 탆.

c) Rotor-Stator Dispersion: A crude emulsion is produced as described in a) and then ultraturrax (Type T25, Jahnke und Kunkel, at a dispersion temperature of 80 ° C. for 1 minute and 10 minutes). Staufen, Germany). Particle diameters were measured at 7.5 and 1.2 μm.

d) Static mixer: The aqueous surfactant solution and lipid from a) are heated to 80 ° C. and mixed in a static mixer (Sulzer, Germany). Particle size was measured at 15.8 μm.

e) Gas-Spray Mill: The behenic acid triglycerides are air-sprayed (Jetmill, Mosokawa Alpine AG) and then dispersed by stirring in an aqueous surfactant solution at room temperature. 50% particle diameter was measured to be 37.03 mu m.

f) Mortar grinder: The coarsely ground lipids are ground in a mortar grinder with the addition of liquid nitrogen for 3 and 15 minutes (Retsch mortar grinder, Retsch, Germany). The lipid is dispersed in water as in e). The average particle size is 40 μm.

Example 16: Molecular Adjuvant Incorporated into SBA to Enhance Cellular Immune Response: GMDP is dissolved in Span 85 (W / O) emulsifier and cetyl palmitate is added. The mixture is melted at 70 ° C., cooled again and then ground in a mortar mill with the addition of liquid nitrogen. This polished lipid-GMDP mixture is dispersed in a 2.5% Tween 80 solution and predispersed at 8000 rpm for 1 minute with Ultraturax. This dispersion is homogenized in 3 cycles at 4 ° C. and 1000 bar using a high pressure homogenization method. PCS diameter is 260 nm and polydispersity index is 0.430.

Example 17 Molecular Adjuvant Incorporated at Interfacial Surface to Increase Cellular Immune Response: Saponins are generally known to increase cellular immune responses. Similar to Example 15, the rotor-stator is used to generate particles. The composition of the SBA dispersant is 5% cetyl palmitate, 0.5% saponin (Quill A saponin) and water. The saponin is dissolved in the aqueous phase, it is heated to 80 ° C. and the molten lipid is added. The production process was carried out using UltraTrex while stirring at 10,000 rpm for 5 minutes. The 50% diameter determined by the laser diffractometer is 2.28 mu m.

Example 18: Preparation of SBA in the presence of amphiphilic adjuvant. Amphiphilic surfactant CHAPS is described in this document as a means of enhancing an immune response. The particles consist of 5% cetyl palmitate and 0.5% CHAPS. Similar to Example 20, the preparation of the particles is carried out. The 50% diameter, determined by laser diffraction, is 1.897 μm.

Example 19 Comparison of SBA to Pure Molecular Adjuvant: SBA dispersant No. 2 (SBA-2) from Example 10 was prepared with a molecular adjuvant, ie, pure GMDP (N-acetyl glucosaminyl-N-acetyl mura). Sheep in comparison to wheat-dipeptide) (same conditions as in Example 9). The concentration of GMDP (0.1 mg / ml) is similar to Example 9. In vivo testing is performed as described in Example 9. SBA 2 shows higher potency intensity than pure GMDP (FIG. 5).

The composition of SBA-2 is as follows: 4% hard paraffin, 1% EQ1 (N, N-di- (β-stearoyl ethyl) -N, N-dimethyl ammonium chloride) and 4% Tween 80 / span 85 (7/3).

Example 20 Effect of Charge (Surface Characteristics) on the Immune Response (Amount Study Similar to Example 9): No difference in potency intensity could be detected between the positively charged particles SBA 4 and SBA 2. EQ1 from SBA 2 can be replaced with cetyl pyridinium chloride approved toxicologically and as a pharmaceutical preservative without any loss in potency (SBA 4). In contrast to the negatively charged particles of the formulation SBA 5, the positively charged particle formulations were observed to have a stronger effect (FIG. 6).

The SBA formulation has the following composition: SBA 4: 4% hard paraffin, 4% tween 80 / span 85 (7/3), 0.5% cetyl pyridinium chloride. SBA 5: 4% hard paraffin, 0.2% sodium deoxycholate, 0.2% sodium cholate, 1% sodium oleate, 2% lipoid E80. SBA 2: 4% hard paraffin, 1% EQ1 and 4% twin 80 / span 85 (7/3). Surface charge (zeta potential) was determined in conductive water with a conductivity of 50 μS / cm: SBA 4: +41.2 mV, SBA 2: +40.5 mV, SBA 5: -36.4 mV. The size (PCS diameter and polydispersity index (P.I.)) is as follows: SBA 4: 103 nm (P.I. 0.110); SBA 5: 107 nm (P.I. 0.115); SBA 2: 101 nm (P.I. 0.101).

Example 21 Species-Independence of the Effect (Hen): The antigen from Example 9 is mixed with SBA 1 and SBA in a ratio of 1: 1 and 0.5 ml per hen is injected. Antibody titers are determined from the eggs of these hens. ELISA test was used to quantify. Contrary to that described in Example 9, commercially available anti-IgG-chicken was used for the ELISA test. Initial immunization was performed on day 0. Boosters using the same formulations are given on day 31. Similar to the results in Example 10, Formulation SBA 2 proved to have a stronger effect (FIG. 7).

Composition of SBA 1: 4% hard paraffin, and 4% tween 80 / span 85 (7/3), PCS diameter: 107 nm (P.I. 0.112); And composition of SBA 2: 4% hard paraffin, 1% EQ1 and 4% Tween 80 / span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101).

Example 22 Influence of Lipid Matrix on Adjuvant Effect: For Formulation SBA 2, non-biodegradable hard paraffins were exchanged for biodegradable glycerol tribehenate (SBA 3). The intensity of the effect is not different; Hard paraffin may be replaced with glycerol tribehenate (FIG. 8).

Composition of SBA 2: 4% hard paraffin, 1% EQ1 and 4% tween 80 / span 85 (7/3), PCS diameter: 101 nm (P.I. 0.102); And composition of SBA 3: 4% glycerol tribehenate, 1% EQ1 and 4% Tween 80 / span 85 (7/3), PCS diameter: 105 nm (P.I. 0.112).

Example 23 Formulations SBA 1 and SBA 2 are tested in comparison to aluminum hydroxide (similar to Example 9). The effect of SBA 1 and SBA 2 is the same as that of aluminum hydroxide (control: antigen in PBS). As in Example 9, SBA 2 has a stronger effect than SBA 1 (FIG. 9).

Composition of SBA 2: 4% hard paraffin, 1% EQ1 and 4% tween 80 / span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101); And composition of SBA 1: 4% hard paraffin, 4% tween 80 / span 85 (7/3), PCS diameter: 107 nm (P.I. 0.112).

SBA is more efficient, cheaper (effective) than conventional products, simpler to manipulate, and well tolerated in vivo.

Claims (22)

Solid at room temperature (= 20 ° C.); Applied at the optimal dose characteristic for the subject to be immunized, and at the optimal particle size, particle charge and surface properties (stabilizing the surfactant layer); A formulation for enhancing the immune response and increasing antibody yield and antibody production in immunology in the case of vaccination of humans and animals, characterized in that it comprises a lipid that is simply mixed with the antigen solution. The formulation of claim 1, wherein the particles are in the range of 10 to 1000 nm. The formulation of claim 1, wherein the particles are in the range of 1-10 μm. The formulation according to claim 1, wherein the particles are in the range of 10 to 200 μm, especially in the range of 10 to 100 μm. The formulation according to claim 1, wherein the particles are in the range of 200 to 1000 μm, in particular in the range of 200 to 500 μm. The lipid according to any one of claims 1 to 5, wherein the lipid used to prepare the lipid particles is solid at room temperature, for example ethyl stearate, octadecane, DDA, natural or synthetic triglycerides and / or mixtures thereof. , Monoglycerides and diglycerides alone or mixtures thereof, or for example mixtures with triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols (esters and ethers thereof, and lipid peptides) Or mixtures thereof, and particularly suitable are synthetic monoglycerides, diglycerides and triglycerides as individual substances or mixtures (e.g. hard fats), Imwitor 900, triglycerides (e.g. glycerol tri Laurate, glycerol myristate, glycerol palmitate, glycerol stearate and glycerol behenate), Waxes, for example, cetyl palmitate and white wax and (DAB), which are used individually or as a mixture, and also, for example, the preparation, characterized in that hydrocarbons, such as light paraffins. A liquid lipid according to claim 6, for example liquid glycerides (miglycol), peanut oil, castor oil, soybean oil, cottonseed oil, seed oil, linseed oil, olive oil, sunflower oil and safflower oil are used to prepare lipid particles. Formulated to a solid lipid, wherein the particles produced are solid at room temperature (20 ° C.). The formulation according to any one of claims 1 to 7, characterized in that it is positively charged. The method of claim 8, in order to generate a positive charge during the preparation of the lipid particles, benzyl dimethyl hexadecyl ammonium chloride, methyl benzetonium chloride, benzalkonium chloride, cetyl pyridinium chloride, N, N-di- (β-stearo) Formulations characterized in that one ethyl) -N, N-dimethyl ammonium chloride and dimethyl dioctadecyl ammonium bromide materials can be used individually or in mixtures. The formulation according to any one of claims 1 to 7, characterized by being negatively charged. 11. The method of claim 10, wherein diacetylphosphate, phosphatidyl glycerine, lecithin of various origins (e.g. egg lecithin or soy lecithin), chemically modified lecithins (e.g. hydrogenated lecithin) are used to produce negative charges during the preparation of the lipid particles. Phospholipids and sphingolipids, mixtures of lecithin and phospholipids, sterols (eg cholesterol and cholesterol derivatives, and stigmasterol), and saturated and unsaturated fatty acids, sodium cholate, sodium glycolate, sodium taurocholate and sodium deoxycholate Wherein the agents are added individually or in mixtures. 8. The formulation according to any one of claims 1 to 7, characterized by no charge or only slight charge. 13. The process according to claim 12, in order to produce them during the preparation of the lipid particles, polyethylene glycol-sorbitan fatty acid esters (especially twin series such as Tween 80), polyoxyethylene polyoxypropylene copolymers (especially poloxamer series and Poloxamine series), ethoxylated mono- and di-glycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and esters and ethers of sugars or sugar alcohols with fatty or fatty alcohols (e.g., saccharose monostearate Non-chargeable substances such as) may be added individually or in a mixture with each other. 14. The formulation according to any one of claims 1 to 13, wherein at least one adjuvant is added to them. The method of claim 14, wherein N-acetyl-glycosaminoyl- (β1-4) -N-acetyl muramil-L-alanyl-D-isoglutamine [GMDP], dimethyl dioctadecyl ammonium bromide [DDA], N Of -acetyl muramil-L-alanyl-D-isoglutamine [MDP], N, N-di- (β-stearoyl ethyl) -N, N-dimethyl ammonium chloride [EQ1], glycopeptide, mycobacteria Cell wall components, saponins, quaternary amines such as cetyl pyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3-[(3-colamidopropyl) -dimethylammonio ] -1-propane sulfonate), dextran sulfate, dextran, 3-odesyl-4'-monophosphoryl lipid A [MPL ], N-acetyl-L-alanyl-disoglutaminyl-L-alanine-82) -1,2 palmitoyl-glycero-3- (hydroxy-phosphoryloxy) ethyl amide, monosodium salt [MTP -PE], granulocyte-macrophage colony stimulating factor [GM-CSF], block copolymers such as P1205, poloxamer 401 (Pluronic L121), dimyristoyl-phosphatidyl choline [DMPC], de Hydroepiandrosterone-3β-01-17-one [DHEA], dimyristoyl-phosphatidyl glycerol [DMPG], deoxycholate sodium salt, cytokine, imiquimod, DTP-GDP, saponin, 7-allyl-8 -Oxoguasonine, 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, for example Polysorbate 80 (Twin 80), quill A saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN 85, Arlacel 85), DTP-DPP, stearyl-tyrosine, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine, calcitriol and the like Formulation characterized in that. 15. The formulation of claim 14, wherein these are specific adjuvants such as, for example, aluminum hydroxide, polymer particles, liposomes. 15. The method of claim 14, wherein the divalent transition metal ion, quaternary amine, dextran, dextran sulfate, vitamin E derivatives such as vitamin E phosphate and vitamin E hemisuccinate, and isopranosine are selected. , Adding a substance that enhances the efficacy of the adjuvant. The lipid particle according to any one of claims 1 to 17, wherein the lipid is pulverized, for example, by grinding the lipid into a solid aggregate state by a mortar grinder, a gas jet grinder, an electron-sputtering method, a high pressure homogenization method, or a microfluidization method. Formulations characterized in that the preparation. 18. The method of any of claims 1 to 17, wherein the lipids are ground in the molten state during dispersion in the external phase (e.g., high speed stirrers, micro and large scale static mixers, rotor-stator grinders, colloid grinders). , High pressure homogenization methods, microfluidization methods, spraying processes, such as spray drying and electron spraying processes, to produce lipid particles, wherein the external phase is liquid (water, organic liquid, oil or mixtures thereof) or gaseous (air, Nitrogen, noble gas). 20. The process according to any of claims 1 to 19, wherein the lipid particles and possibly additional adjuvant are aqueous phases such as water, isotonic sugar solution and isotonic sodium chloride solution, PEG 400 Or non-aqueous liquids such as 600, organic liquids such as oils (miglycol, peanut oil, castor oil, soybean oil, cottonseed oil, seed oil, linseed oil, olive oil, sunflower oil and safflower oil and other oils) or mixtures thereof. Formulations characterized in that. The formulation according to claim 20, wherein a substance which increases the viscosity, for example, is added to the outer phase. 20. The process according to any one of the preceding claims, wherein the lipid particles and possibly additional adjuvant are in anhydrous form, such as lyophilisate, spray dried product, solid dispersant, pellet or tablet form. Formulation characterized in that.