CA2373239A1 - 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) for enhancing the humoral and cellular immune response 1. Background to the Invention Antigens are administered to animals and humans to produce antibodies. The aims of doing this are, for example, the immunization of humans or animals for protection against diseases or the production of antibodies which are subsequently isolated and processed into products. Antigens are most frequently administered parenterally, with e.g. oral, nasal and topical application as alternative methods.
A frequent problem is that often the strength of the immune response to the antigen administered is not sufficient for the intended purpose . Sometimes the problem can be solved by strongly increasing the dose of the antigen administered. However, it is a serious problem that antigens are often very expensive and increasing the dose leads to a corresponding increase in the cost of the vaccine. These costs lead to a considerable strain on the health system; for many social strata - especially in the Third World - the vaccine becomes too expensive and desirable mass vaccinations cannot be carried out.
An alternative approach is the administration of antigens together with an adjuvant, which enhances the antigen effect and thus leads to a higher antibody titre. The adjuvant principle was first described by Jules Freund in 1948 [Freund, J. J. Immunology 1948, 60, 383-398] and achieved with two mineral-oil-based emulsions. Freund's Incomplete Adjuvant (FIA) is a mixture of mineral oil with mannide monooleate (Montanide, Arlacel). For application this is mixed with the antigen solution and the emulsion formed is injected. The increase in immune response thus brought about is so great, that FIA is still referred to as the "gold standard" when new adjuvants are developed and tested.

Their efficiency and quality are evaluated by comparison with FIA, with many newly developed adjuvants however remaining well below the efficiency of FIA (e. g. with 0.2, FIA = 1.0).
The addition of inactivated mycobacteria to FIA (e.g. M.
tuberculosis) further increased the immune response. This mixture is designated Freund's Complete Adjuvant (FCA). The oil emulsion according to Freund however produces inflammatory and ulcerating swellings (granulomas) at the injection site, some of which burst and turn into extensive abscesses. According to present standards, the use of the oil emulsion according to Freund for humans is therefore completely out of the question. It is sometimes used in animals, but often the animals' general condition is so strongly impaired that uneconomic losses result.
The M. tuberculosis or M. butyricum mycobacteria proposed by Freund for completion of 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 exists, to find a well-tolerated adjuvant, in particular with a view to producing cost-effective vaccines, and enabling desirable world-wide immunizations against specific diseases which can only be economically achieved with a low-cost vaccine, and the increasing significance of immunological production techniques and products. Also, with growing awareness of the need for protection of animals and corresponding pressure of legislation, a well-tolerated (i.e. biocompatible), equally efficient but at the same time low-cost adjuvant must be found.
In addition it must be physically stable, so that admixture before the injection is not necessary and variations in efficiency - e.g. caused by differently fine dispersion - are eliminated.
At present the most frequently used adjuvant licensed for human application is a fine suspension of aluminium hydroxide, whose roots go back even further than those of Freund's emulsion [Glenny A.T. et al. J. Pathol. 192, 29 31-40]. This is based on the assumption that antigens are better recognized by the immune system if they are adsorbed onto the surface of the aluminium hydroxide particles. A disadvantage is, however, that aluminium hydroxide is less efficient than Freund' s adjuvant and it can also cause granulomas.
Starting from Freund's emulsion, recently emulsions have been described, which consist of better tolerated materials, such as, for example 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 [European Patent Application 0399843A2).
Based on the suspensions according to Glenny, particles ranging from different polymers to various anorganic and inorganic particles (e. g. carbon) were used [O' Hagan, D. T. , Jeffrey, H. , Roberts M.J., McGee, J.P., Davies, S.S. Vaccine. 1991 9 (10), 68-71, Glenny, A.T. et al. J. Pathol. 1926, 29 31-40] . Disadvantages of these systems are the cytotoxicity of some polymers (e. g.
formaldehyde formation in the case of polyalkylcyanoacrylates), lack of degradation, or too slow a rate of degradation in the organism (e. g. polystyrene or polymethacrylate derivatives), insufficient biocompatibility (e.g. capsule formation in the case of PLA particles, pH shift to pH2) and inability to obtain a registration from the registration authorities (e. g. soot particles) .
An alternative way of using particular adjuvants such as oil drops or solid particles is the use of macromolecular adjuvants.
It has been shown that the mycobacteria described by Freund can be replaced by units from their capsule substance, which are better tolerated by the body. Especially described and synthesized were: MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) [Adam, A., Lederer, E., Med. Res. Rev. 1984 4, 111-152] Thr-MDP
(threor~yl ar~alog~ae of MDP) [Byars P:E, et al. Vaccine. 1987 5 (3) , 223-8] and the GMDP originating from the yoghurt bacillus (N-acetylglucosaminyl-N-acetylmuramyl-dipeptide) [Grubhofer, N.
Immunology Letters 1995 44, 19-24]. GMDP is a homologue of MDP;
an increased immune-system stimulating effect has now been demonstrated for GMDP (Patent Specification DE 19511235 Cl).
Further stages in the development of an adjuvant have included the use of molecular adjuvants in high concentration, so that the solubility was exceeded and a suspension was obtained (e.g. DDA
(dimethyldioctadecyl ammonium bromide) [Grubhofer, N. Immunology Letters 1995 44, 19-24] and to enhance the humoral response, the mixture of macromolecular adjuvants such as glycopeptides with solid particles (e. g. GMDP with colloidal particles) [Grubhofer, N. Immunology Letters 1995 44, 19-24].
Up to now however, no solution has been found whose effectiveness is equal to that of Freund' s adjuvant, and which at the same time meets the other requirements: the new emulsions are not always taken without side effects, the synthetic macromolecules such as glyco-peptides have thus far not been shown to be superior to the mycobacteria, and in addition they are much too expensive. There are registration problems with solid particles. Many adjuvants show too high a level of toxicity and thus too low a level of biocompatibility. In addition there are problems with physical stability (avoidance of aggregation and /or coalescence). Thus the FIA emulsion must be freshly produced and injected; long-term storage is not possible. Sufficient physical stability during storage is however the essential prerequisite for a marketable product - especially for drugs.
The objectives are therefore the production of a new adjuvant with:
1. sufficient physical stability 2. low cytotoxicity and sufficient biocompatibility 3. effectiveness comparable to that of FIA
4. low-cost production.

1. Description of the invention Up to now, in order to increase the immune-system stimulating effect, GMDP has had to be used in mixture with colloid-disperse solid lipid particles in a particle size < 200 nm (USA Patent Gerbu, US Patent Office processing number is 08/816,787). The structure of the particles was systematically modified in studies for this invention (e. g. surfactants, stabilizers used) to find lipid particles which could further enhance the immune-system-stimulating effect of GMDP. Surprisingly it was found at the same time that lipid particles alone are just as efficient as the combination of lipid particles and GNmP (Example 9). Thus in the invention there is no need for the expensive GMDP and a comparable level of humoral immune-system stimulation can be achieved with a low-cost lipid particle alone.
The strength of the immune-system-stimulating effect here depends on the surface properties of the particles (surfactants used, particle charge) and the particle size. With negative charge of the lipid particles the efficiency amounts to ca. 1/3 that of FIA; with production of a positively charged lipid particle by the addition of EQ1 the effectiveness is comparable to that of FIA (no significant difference, t-Test) (Example 9). The desired effectiveness can thus be adjusted via the composition of the lipid particles used. This avoids over-reaction of the organism.
From adjuvant research carried out into anorganic and polymer suspensions to date, it is known that for each antigen there is a particle size with maximum efficiency, likewise a species dependency is known. Thus the particle size of aluminium hydroxide suspensions best suited for a vaccine is empirically determined; polymer particles as adjuvants for immunization had a different effect depending on their size [Kreuter J, et al.
Vaccine. 1986, 4, 125-9]. In spite of the very different matrix material, the same was surprisingly found for the lipid particles, so that the effectiveness can be modulated by changing t he particle size antigen-specificall~.~ and species-specifically.

The lipid particle dispersions of the stable, biocompatible adjuvant (SBA) consist of lipid particles dispersed in water or aqueous liquids or non-aqueous, e.g. oily liquids, these particles possibly, but not necessarily, being stabilized by surfactants or polymers. With sufficiently high viscosity of the outer phase or fineness of the particles, a physically stable dispersion is obtained. The same also applies in the case of low-viscosity outer phases, if the particles have the same, sufficiently high, charge and thus sediments formed can easily be redispersed.
The production of the SBA takes place via dispersion techniques or precipitation, using the generally known methods described in pharmacy or process engineering textbooks. During dispersion, coarsly dispersed lipids are desintegrated by mechanical processes. The lipids can be in the solid aggregate state (e. g.
mortar mill) or in the liquid aggregate state (e. g.
emulsification of lipid melts by stirrers). To produce the SBA
dispersion the lipids can first be comminuted and then dispersed in the outer (e. g. aqueous) phase or alternatively comminuted directly in the outer phase. To produce SBA dispersions by dispersion the following can be used for example, amongst others piston-gap homogenizers (e. g. APV homogenizers, French Press), jet stream homogenizers (e. g. Microfluidizer), rotor-stator agitators (e. g. Ultraturrax, Silverson homogenizers), microscale and macroscale static mixers (e. g. mixers from the company 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 and laser diffractometry (LD) showed no, or negligible, particle growth over periods of 1 - 3 years (Example 3). The stability of SBA dispersions is far superior to that of Freund's incomplete adjuvant (FIA) (Example 1), and also shows greater stability compared with more recent adjuvant emulsions (Example 2).

_ 7 _ Stability: SBA dispersions represent a simple system. In contrast to glycopeptides, the adjuvant substances used are chemically simple in structure and robust. Application of heat in sterilization processes does not lead to any chemical decomposition, the dispersion remains physically stable and no particle aggregation occurs. An example of the sterilization of SBA dispersions by autoclaving (121°C, 15 minutes, 2 bar) is shown by Example 4. FIA shows phase separation under the same conditions.
Many vaccines are stored at a low temperature (4 - 6~C) . With optimized SBA dispersions, storage is also possible at room temperature and also at even higher temperatures, without physical destabilization occurring (Example 11). This simplifies the handling of SBA-based product, especially for hotter climatic zones (e. g. use as adjuvant in mass vaccinations in Third World countries).
Biocompatibility: Low toxicity and good biocompatibility are essential for an adjuvant with broad application. In a study with sheep, following application of SBA dispersion, nothing unusual was observed at the application site (Example 6). The fact that it was well tolerated is explained by the extremely low toxicity of lipids observed in vitro in cell cultures . Compared to the polymers accepted by the German registration authorities and the FDA for parenteral administration, they show viability that is higher by a factor of ca. 20 in the case of high particle concentrations (Example 5). The good biocompatibility is attributed to the fact that generally body proteins only adsorb to a slight extent on the particle surface - in contrast to other particles (Example 7). In addition, no proteins which stimulate intolerance reactions were detected on the surface.
For broad application of an adjuvant, for reasons of costs, the solution that presents itself is to produce an adjuvant preparation which is admixed into the antigen solution before application. Ideally the adjuvant should be produced with properties such that it can be admixed with a number of different antigens. To reduce the pain of injection, the mixture should be administered in physiological sodium chloride solution or other isotonic solution. Due to the reduction of the zeta potential, physiological sodium chloride solution leads to destabilization in dispersions and subsequent aggregation [Lucks, J.S. et al., Int. J. Pharm. 1990 58, 229-235]. The SBA adjuvant should be physically stable for a sufficiently long time after admixture with the antigen in isotonic solution. Example 8 shows that the lipid particles after admixture with physiological sodium chloride solution are stable even over 6 hours, no measurable aggregation occurs.
In addition to particle size, surface properties such as charge can also be specifically adjusted so that species-specific SBA
adjuvant can be produced in an effective strength. Positive and negative charges can be produced by the admixture of correspondingly charged surfactants or stabilizers. The strength of the charge can be adjusted via the concentration of the additive, with ideal additives being ionic substances which, like cetyl pyridinium chloride are accepted as a preservative for parenteral application (Example 12).
A large number of different lipids can be used for the production of SBA dispersions. These are both chemically homogeneous lipids and mixtures thereof. The lipids are characterized in that they are present in the SBA dispersion end product in the crystalline state (e. g. B-, iii-modification) or in the liquid-crystalline state (a-modification) or in mixtures thereof. If lipid mixtures are used, liquid lipids (e.g. oils, lipophilic hydrocarbons, and lipophilic organic liquids such as oleyl alcohol) can also be admixed with the solid lipids (e. g. glycerides, lipophilic hydrocarbons such as hard paraffin) ("lipid blends").
The following lipids are used for example as dispersed phases and can also be applied as individual components or as a mixture:
natural or synthetic triglycerides and/or mixtures thereof, _ g _ 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 thereof. Especially suitable are synthetic monoglycerides, diglycerides and triglycerides 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, for example, cetyl palmitate and white wax (DAB -German Pharmacopeia). Also hydrocarbons, such as, for example, hard paraffin.
The proportion of the inner or lipid phase relative to the whole formulation is 0.1% to 80% (m/m) and preferably lies within 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 dispersions, these can be incorporated in the form of pure substances or in the form of mixtures, to stabilize the particles.
The quantity of such additives, which can be added, in relation to the whole weighed portion of the aqueous dispersion, lies within the range 0.01% to 30%, and preferably within the range 0.5% to 20%. For stabilization of the SBA dispersions or for their specific surface modification, surfactants, stabilizers and polymers can be used, which are generally known from the production of dispersions. Examples of this are:
1. Sterically stabilizing substances such as poloxamers and poloxamines (polyoxyethylene-polyoxypropylene-block-copoly-mers), ethoxylated sorbitan fatty acid esters, especially polysorbates (e. g. Polysorbate 80/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 (e. g. sucrose mcr~cstearate, sucrose distearate, sucrose cocoate, sucrose stearate, sucrose dipalmitate, sucrose palmitate, sucrose laurate, sucrose octanoate, sucrose oleate.
2. Charged ionic stabilizers such as diacetylphosphates, phosphatidyl glycerine, lecithins of various origin (e.g. egg lecithin or soya lecithin), chemically modified lecithins (e. g.
hydrogenated lecithins), as well as phospholipids and sphingolipids, mixture of lecithins with phospholipids, sterols (e.g. cholesterol and cholesterol derivatives, as well as stigmasterol) and also saturated and unsaturated fatty acids, sodium cholate, sodium glycholate, sodium taurocholate, sodium deoxycholate or mixtures thereof, amino acids - or anti-flocculants, such as, for example, sodium citrate, sodium pyrophosphate, sodium sorbate [Lucks, J.S. et al., Int. J. Pharm.
1990 58, 229-235] . Zwitterionic surfactants such as, for example, (3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propane sulphonate) [CHAPSO] , (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate) [CHAPS], and N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulphonate. Cationic surfactants, especially compounds used as preservatives, such as, for example benzyldimethyl-hexadecylammonium chloride, methylbenzethonium chloride, benzalkonium chloride, cetyl pyridinium chloride.
3. Viscosity-increasing substances such as, for example, cellulose ethers and cellulose esters (e. g. methyl cellulose, hydoxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose), polyvinyl derivatives as well as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate, alginates, polyacrylates (e. g. Carbopol), xanthanes and pectins.
The charged stabilizers are, if necessary or desirable, preferably contained in the SBA dispersion in quantities ranging from 0.01% to 20% (m/m) and especially in a quantity of 0.05% to 10%. Viscosity-increasing substances are, if necessary or desirable, incorporated in the formulation in similar proportions, preferably in a quantity of 0.01-20%, and especially in a quantity of 0.1% to 10% (m/m) and preferably in the range between 0.5% and 5%.
As the outer phase (dispersion medium, continuous phase), water, aqueous solutions or liquids miscible with water, such as glycerine or polyethylene glycol and oily liquids such as miglycols (medium chain triglycerides - MCT) and other oils (castor, groundnut, soya, cottonseed, rape, linseed, olive, sunflower, and safflower oil) can be used.
Surfactant-free SBAs are produced by dispersion of the lipid phase in an aqueous solution, which contains one or more viscosity-increasing substances, either alone or in combination with other substances such as sugar, sugar alcohols, especially glucose, mannose, trehalose, mannitol, sorbitol and others.
Furthermore, it is possible to use a combination of the viscosity-increasing substances or a combination of these with sugars or sugar alcohols, or in a further combination with charged stabilizers or anti-flocculants.
SBA dispersions can be used as adjuvants to many different antigens for immunization against various diseases. Examples of this are:
Glycoproteins such as, for example, gonococcal protein I, brucella abortus antigen, tetanus toxoid, diphtheria toxoid, listeria monocytogenes, Virus antigens such as, for example, Semliki Forest virus, encephalomyocarditis virus, porcine rarovirus, pseudo-rabies virus, Newcastle disease virus, bovine viral diarrhoea, HIV, influenza, cytomegalovirus, herpes simplex, hepatitis C, measles, Parasites, such as malaria, eimaria spp. etc.
In summary it can be said that with the SBA dispersions an adjuvant is available that:

1. possesses sufficient physical stability to be produced as a product and especially as medication, 2. possesses low toxicity and good biocompatibility, especially if biologically degradable lipids such as glycerides are used, 3. possesses an effect comparable to that of Freund's incomplete adjuvant (FIA) and 4. can be produced cost-effectively from low-cost excipients with low-cost manufacturing methods.
SBA dispersions can be used extensively to reduce the antigen dose, and hence costs, with toxicologically -acceptable excipients, as, with the addition of SBA, the same immune-system-stimulating effect is achieved at a lower antigen dose.
Antigens which have hitherto had insufficient antigenicity for a vaccine, can be made into an efficient vaccine by the addition of immune response-stimulating SBA.
Thanks to cost-effective production with existing efficiency comparable to that of FIA, SBA dispersions are suitable as adjuvants for veterinary vaccinations, when only very low-cost vaccines can be used for reasons of profitability.
Adjuvants used hitherto have focussed on enhancing the humoral immune response. In view of the efficiency of SBA dispersions, it is no longer necessary to add a further adjuvant to the SBA
lipid particles and/or the addition of adjuvants such as GMDP
bring no further increase to the humoral immune response . Clearly with the immune system at its maximum response capacity, additional adjuvant can bring no additional effect. Thus additions to SBA bring no advantage for the humoral response, additives such as those described in Gerbu's patent (Patent Specification DE 19611235 C1), are superfluous, thanks to the surprising efficiency found for the SBA described in the invention.

Surprisingly, however, it has been ascertained that following application of GMDP in combination with SBA dispersions, in the case of subsequent reinoculation (booster), a clearly enhanced cellular immune response was achieved, than when SBA dispersion was previously used alone as an adjuvant. Thus it has been newly discovered that a combination of SBA dispersion is suitable specifically for increasing the cellular immune response in the case of reinoculation. There is an increased immune response in the case of subsequent second inoculation, if previously an additional adjuvant such as GMDP was used in mixture with SBA
dispersion (Example 13).
To produce an adjuvant with the aim of enhancing the cellular immune response it is thus advantageous to combine the SBA
dispersion with a further adjuvant. Further adjuvants for a combination are:N-acetylglycosaminyl-(i31-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine [GMDP], dimethyldioctadecylammonium bromide [DDA] , N-acetyl muramyl-L-alanyl-D-isoglutamine [MDP] , N,N Di (f3-stearoyl ethyl)-N,N-dimethyl ammonium chloride [EQ1], glycopeptides, components of the cell wall of mycobacteria, saponins, quaternary amines, such as, for example cetyl pyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate), dextrane sulphate, dextrane, 3-odesacyl-4'-monophosphoryl lipid A [MPL°], N-acetyl-L-alanyl-disoglutaminyl-L-alanin-82) - 1,2 dipalmitoyl - sn glycero-3-(hydroxy-phosphoryloxy))ethyl amide, monosodium salt [MTP-PE], granulocyte-macrophage colonies stimulating factor [GM-CSF], block copolymers, e.g. P1205, Poloxamer 401 (Pluronic L121), dimyristoyl-phosphatidyl choline [DMPC], dehydroepiandrosterone-3f3-O1-17-on [DHEA], dimyristoyl-phosphatidyl glycerol [DMPG], deoxycholic acid sodium salt, cytokines, imiquimod, DTP-GDP, saponins, 7-allyl-8-oxoguasonin, 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) , Qti.i.i A Saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN~85, Arlacel 85) , DTP-DPP, stearyl-tyrosin, N,N dioctadecyl-N', N'-bis(2hydroxyethyl) propandiamine, calcitriol.
In addition to the increase in the humoral immune response brought about by SBA dispersions, the invention opens up the possibility of enhancing the cellular immune response in the case of a second inoculation, by a combination of SBA dispersions with other adjuvants.
Instead of an admixture of adjuvants to enhance the cellular immune response, the adjuvants can also be incorporated into the lipid particles. Incorporation is possible by inclusion in the solid particle matrix, accumulation at the interface in the case of amphiphilic adjuvants or by simple adsorption onto the particle surface. The incorporation of adjuvants can take place during particle production or subsequently (e.g. in the case of incorporation, Example 16). For incorporation during production, adjuvants are dissolved in the lipid melt phase, solubilized or dispersed and the adjuvant-containing lipid phase 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 become enriched in the particle surface or by adsorption onto the surface. The incorporation of adjuvants leads to a prolonged release by diffusion or, during the course of particle degradation, by enzymes . A delayed release over a fairly long period enhances the immune response.
A further attractive area of application is antibody production in animals . The antibody yield can be distinctly increased by the addition of the adjuvant.
$xamples Example 1: Determination of the physical stability of SBA versus Freund' s Incomplete Adjuvant (FIA) : An aqueous SBA dispersion was produced by high-press~~re homogenization at 95°C from 20s beeswax, 2% Tween 80 (PCS diameter 289 nm, polydispersity index 0 .101 ) . FIA was produced according to the method described by Freund [Freund, J. J. Immunology 1948, 60, 383-398]. SBA and FIA
were stored at the temperatures of the climatic zones used for stability testing of drugs [EMEA Directive CPMP/QWP/159/96, January 1998)]. The storage temperatures were: 5°C, 25°C, 40°C.
The physical stability was determined by measurement of the particle size with laser diffractometry (LD), characterization parameters were the 50% diameter and the 95% diameter (50%/95%
of the particles are under the size indicated, sensitive parameter for particle aggregation). After a few minutes of storage - even at room temperature - FIA showed distinct particle growth; thus, FIA is not an adjuvant which is stable in storage.
On the other hand, the particle sizes of SBA remain unchanged under the 3 storage conditions over a period of 1 year (Table 1) .

Table 1: Examination of the stability of SBA (20% beeswax, 2%
Tween 80) in comparison with Freund' s incomplete adjuvant (FIA) .
LD 50% LD 95%
L fcml L~ml SBA Day 0 0.32 0.75 Day 1 0.32 0.77 Day 30 0.33 0.78 Day 365 0.33 0.79 SBA Day 0 0.31 0.73 Day 1 0.32 0.77 Day 30 0.32 0.79 Day 365 0.33 0.83 SBA Day 0 0.32 0.73 Day 1 0.32 0.77 Day 10 0.32 0.78 Day 30 0.34 0.78 FIA 5 29.54 63.05 25C minutes 30 30.29 67.39 minutes 60 38.34 75.89 minutes 120 38.85 75.91 minutes Example 2: Determination of the physical stability of SBA versus squalene adjuvant: SBA was produced as described in l, it contained 10% cetyl palmitate and 1.2% Miranol (PCS diameter 210 nm, PCS polydispersity index 0.189). Squalene adjuvant was produced as described in European Patent Application 0 399 843 with date of application 25 May 1990 (Adjuvant MF59). Particle sizes were measured by 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° at a frequency of 50 Hz (Table 3) .
SBA shows increased stability both in normal storage and also in the stress test.

Table 2: Examination of the stability of SBA (10% cetyl palmitate, 1.2% Miranol) in comparison with MF59.
LD 50$ (~m1 LD 95~ [~Cm]

SBA Day 0 0.32 0.75 Day 1 0.32 0.77 Day 30 0.33 0.78 Day 0.33 0.79 SBA Day 0 0.31 0.73 Day 1 0.32 0.77 Day 30 0.32 0.79 Day 0.33 0.83 SBA Day 0 0.32 0.73 Day 1 0.32 0.77 Day 30 0.32 0.78 Day 0.34 0.78 MF59 Day 0 0.119 0.223 Day 1 0.113 0.228 Day 2 0.111 0.231 Day 5 0.115 0.226 MF59 Day 0 0.119 0.223 Day 1 0.128 0.329 Day 2 0.130 0.480 Day 5 0.158 0.859 MF59 Day 0 0.119 0.223 Day 1 0.347 2.809 Day 2 0.463 3.563 Day 5 0.741 5.638 - 1$ -Table 3: Stability of MF 59 compared with SBA at 40°C and a vibration frequency of 50 Hz LD 5 0 % ( ~Cm ] LD 9 5 % ( ~Cm ]
SBA Day 0 0.32 0.75 Day 1 0.33 0.76 Day 2 0.3 0.81 Day 5 0.32 0.83 MF 59 Day 0 0.119 0.223 Day 1 0.597 3.848 Day 2 0.302 7.721 Day 5 0.451 8.784 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.
Day Day Day Day Day Day LD 50% (~,m] 0.35 0.36 0.35 0.35 0.35 0.39 LD 95% (hem] 0.85 0.93 0.92 0.92 0.91 1.12 averagePCS 0.316 0.327 0.337 0.336 0.346 0.365 diameter [gym]

P.I. 0.138 0.192 0.172 0.16 0.159 0.25 Example 4. Heat stability of SBA during autoclaving (heat, pressure) versus FIA and MF59: The SBA dispersion was made up of 18% hard paraffin, 4% Tween 80/Span 85 (7/3) and water.
Sterilization of 20 mL in each case was carried out in injection vials according to the standard conditions of the European Pharmacopoiea (121°C, 2 bar, 15 minutes). Particle sizes were determined using PCS and laser diffractometry (LD 95%) (P. I.:
polydispersity index, measure for the width of particle size distribution, AV: average value from 3 measurements,: St.dev.:
standard deviation, P.I. polydispersity index) (Table 5). After autoclaving the FIA emulsion showed phase separation, and MF59 showed distinct particle size growth. SBA is physically stable and can be sterilized by autoclaving.
Table 5: Heat stability of SBA during autoclaving (heat, pressure) versus FIA and MF59: The SBA dispersion was made up of 18% hard paraffin, 4% Tween 80/Span 85 (7/3) and water.
Sterilization of 20 mL in each case at 121°C, 2 bar, 15 minutes.
Particle sizes were determined using PCS and laser diffractometry.
PCS P.I. beforeLD 95% PCS P.I. afterLD 95%

diameter sterilization[N,m] beforediameter sterilization[~.m]
after [nm] before sterilization[nm] after sterilization sterilization sterilization SBA 101 0.101 0.15 103 0.109 0.154 100 0.11 0.15 101 0.111 0.155 104 0.112 0.152 101 0.12 0.154 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 246 0.121 0.223 859 0.321 5.052 234 0.103 0.223 912 0.305 5.056 254 0.114 0.222 899 0.389 5.055 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 tolerability: to assess tolerability, the cytotoxicity of SBA was determined in cell cultures (human granulocytes, HL60 cells). To quantify the toxicity, the viability of the cells was determined with the MTT test [Mosmann, T., J. Immunol. Meth. 1993, 65, 55-63]. The SBA dispersion was made up of 10°s cetyl palmitate, 0 . 5 o poloxamer 188 and water. The cell number per well amounted to 200,000 in the case of human granulocytes and 200,000 in the case of HL60 cells. Incubation took place for 12 hours. In the case of SBA viability amounted to 80 % in the case of the granulocytes and 85 % in the case of the HL60 cells. In the case of nanoparticles of PLA, viability was only 5s, whilst for nanoparticles of PLA/GA it dropped to 0%. SBA
tolerability is at least a factor of 20 better in the cell cultures than that of the polymers approved by the FDA for parenteral application.
Example 6: Tolerance after parenteral application: Aqueous SBA
dispersion was used, with the composition: 5% hard paraffin, 5%
Tween 80/Span 85 (7/3) and water. Parenteral injection into sheep (n - 30) was carried out, the injection site was the side thoracic wall, the injection volume was 5 mL divided into 4 sides of injection. The sheep showed nothing unusual, either at the injection site, nor in their behaviour.
Example 7 : Biocompatibility - interaction with body proteins : The SBA dispersion was made up of 10% Compritol, 2.5% Poloxamer 407 and water. Production was carried out with high-pressure homogenization. The particles were incubated with human plasma for 5 minutes, then separated from the plasma and the body proteins adsorbed onto the particle surface were determined with two-dimensional polyacrylamide gel electrophoresis [Blank, T. et al. Electrophoresis 14, 1382-1387 (1993)]. In the case of comparable particle surface areas, a very small protein quantity was adsorbed onto SBA, with 96.41 cpm (counts per minute) in comparison with emulsions (comparable values: 472 cpm on emulsion, 390 cpm on polystyrene particles [Harnisch, S. et al.
Electrophoresis 1998, 19, 349-354, Blank, 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 sodium chloride solution ( PBS ) : SBA made up of 2 0 % hard paraf f in, 5 Tween 80/Span 85 (7/3) and water was mixed with PBS (2 mL SBA +
2 mL salt solution). The physical stability in the physiological sodium chloride solution was determined with laser diffractometry as a function of time. Over 6 hours there was no increase in particle size (90% and 95% diameters, Table 6) (St.dev.: standard deviation) .

Table 6: Stability in phosphate-buffered physiological sodium chloride solution ( PBS ) . SBA ( 2 0 % hard paraf f in, 5 % Tween 8 0 / Span 85 (7/3)) was mixed with PBS (2 mL SBA + 2 mL salt solution).
Determination of the physical stability in the physiological sodium chloride solution with laser diffractometry as a function of time.
LD90% St. LD95% St.

[~.m] dev. [gym] dev.

SBA 0.233 0.01 0.304 0.012 SBA/PBS 0.189 0.011 0.224 0.01 1+1 minutes SBA/PBS 0.202 0.009 0.24 0.007 1+1 6 hours Example 9: Adjuvant effect in comparison to molecular adjuvant (GMDP - N-acetyl glucosaminyl-N-acetyl muramyl-dipeptide) and FIA: sheep were inoculated with the strain Mycoplasma Bovis PG
45 R9. Cultivation of the inoculation antigen took place in culture over 72 hours under microaerophilic conditions in Hayflick's medium. Deactivation was achieved by the addition of 0.1% f3-propiolacton. The cells were separated, washed with phosphate buffer pH 7.4 and adjusted to a content of 1 x 101° CFU/mL. The sterility of the preparation was tested in accordance with the German Pharmacopoiea Edition 10. Dry mass determination showed a content of 1 mg/mL Mycoplasma Bovis antigen. The adjuvant SBA, GMDP and FIA was mixed in equal parts with the antigen in buffer. The injection volume was 5 mL, divided to 4 sides of injection.
The SBA composition was 4% hard paraffin, 1% EQ1 (N,N di-(i3-stearoyl ethyl)-N,N-dimethyl ammonium chloride) and 4% Tween 80/Span 85 (7/3) . The composition of the GMDP adjuvant was 5%
lipid and 0.5% surfactant. FIA was produced as in Example 1.
Blood samples were taken on Day 0 before inoculation, on Day 35 and on Day 63. Determination of the antibodies was carried out with ELISA. For the ELISA test, a commercial marked Anti-IgG-Sheep from Sigma was used.
SBA showed an intensity of effectiveness comparable with that of the combination of lipid particles with GMDP. Furthermore, SBA
was of an efficiency comparable with that of FIA (Figure 1).
There was no significant difference in intensity of effectiveness between the three adjuvants.
Figure 1: Adjuvant effect in comparison with molecular adjuvant (GMDP - N-acetyl glucosaminyl-N-acetyl muramyl-dipeptide) and FIA. The SBA composition was 4% hard paraffin, 1% EQ1 (N,N di- (f3-stearoyl ethyl)-N,N-dimethyl ammonium chloride) and 4% Tween 80/Span 85 (7/3) and water. The composition of the GMDP adjuvant was 5% EQ1 and 0.5% Montanide 888. FIA in accordance with Example 1.
Example 10. Effect of SBA composition on antibody titre: SBA
dispersions were produced from identical lipid, but different surfactants on the surface, i.e. 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% EQl and 4% Tween 80/Span 85 (7/3) and water. The efficiency of the increase in the antibody titre was tested analogously to Example 9, with FIA used as a comparison. Depending on the surface properties, different levels of immune response resulted for the two SBA dispersions (Figure 2). The strength of the desired immune response can thus be adjusted by variation of the surface properties (surfactants, stabilizers, charge etc.).
Figure 2: Effect of SBA composition on antibody titre:
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 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 determined by measuring the particle size with PCS. No particle growth occurred (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 determined by measuring the particle size with PCS.
Example 12: Surface modification of SBA dispersions: For modification of the surface charge, SBA dispersions were produced with interacial active positively charged stabilizers (EQ1 -distearoyl ethyl diammonium chloride, cetyl pyridinium chloride) and negatively charged stabilizers (sodium lauryl sulphate, (SDS)). The composition of the SBA dispersions was: 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 for the charge, the zeta potential was measured in millivolts (mV) (Electrophoresis measurement), Zetasizer 4 (Malvern Instruments, UK). Conversion of electrophoretic mobility into zeta potential took place using the Helmholtz-Smoluchowski equation. The zeta potentials amounted to +40 mV, +28 mV and -35 mV for the 3 SBA
dispersions.
Example 13: Increased cellular immune response in the case of booster injection, in hens treated with GMDP-containing adjuvant (SBA) at the time of the initial immunization. SBA {5% EQ1, 0.5%
Montanide 888) was mixed 1:1 with the antigen (IgG from rabbits) and injected subcutaneously. SBA contained 5 ~Cg GMDP and the immunization schedule was as follows: initial immunization and two booster injections on Day 14 and Day 28. Antibody determination was carried out on Day 42 and the IgY titre in the egg yolk was measured. To investigate any enhanced cellular immune response, on Day 100 a new antigen contact (a further booster injection) took place, and on Day 120 the antibody determination. The results show that in the case of the initial immunization (Days 14 and 28) with GMDP-containing SBA with renewed antigen contact, strongly increased antibody production takes place. Columns 2, 3 and 4, Figure 4.
Figure 4: Antibody production in hens following basic immunization and renewed antigen contact on Day 100. The final antibody determination of the basic immunization took place on Day 42, that of the booster injection (Day 100) on Day 120. The composition of the vaccines of Examples 1-5 is as follows:
1. Day 42, Antigen in PBS, Day 100: antigen in PBS (n.a.:
no adjuvant, antigen in PBS).

2. 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 uvant) adj 4. Day 42, Antigen in SBA with GMDP, Day 100: antigen in PBS

5. Day 42, Antigen in FCA, Day 100: antigen PBS
in 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 at room temperature for 30 minutes, to enable adsorption of GMDP onto the particles. The end concentration amounted to 1.435 mg/ml. The growth in size amounted to 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 EQ1 from Example 12 (SBA-EQ1) was produced using various manufacturing processes, to vary the particle size. The particle size was measured using laser diffractometry (laser diffractometer LS 230, Coulter Electronics Germany, measurement range: 40 nm - 2000 ~,m). The diameter of 50~ of the particles is given as a characterization parameter. The following production methods were used:

a) High-pressure homogenization: The lipid was melted, poured into the aqueous surfactant solution, dispersed with a stirrer and the raw emulsion obtained was homogenized at 80°C with a high-pressure homogenizer (Micron LAB40, APV
Gaulin Homogeniser GmbH, Germany). Homogenization parameters were 500 bar pressure, 3 homogenization cycles.
The 50% particle diameter amounted to 0.15 ~,m.
b) The raw emulsion was produced as described in a) and homogenized with a Microfluidizer (Device type 110-Y, Microfluidix Inc., USA).
Homogenization parameters were 700 bar, 10 minutes circulation time. The average particle diameter amounted to 0.452 ~Cm.
c) Rotor-stator dispersion: the raw emulsion was produced as described in a) and then dispersed with an Ultraturrax (Type T25, Jahnke and Kunkel, Staufen, Germany) at a rate of 10,000 rpm for 1 minute and 10 minutes, dispersion .
temperature 80°C. The particle diameters amounted to 7.5 and 1. 2 ~Cm .
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 amounted to 15.8 ~,m.
e) Gas-jet mill: Behenic acid triglyceride was air-jet milled (Jetmill, Mosokawa Alpine AG) and then dispersed by stirring in the aqueous surfactant solution at room temperature. The 50% particle diameter amounted to 37.03 ~.m .
f) Mortar mill: the coarsely pulverized lipid was ground in a mortar mill, with the addition of liquid nitrogen for 3 minutes and 15 minutes (Retsch mortar mill, Retsch, Germany). The lipid was dispersed in water as in e). The average particle size was 40 Vim.

Example 16: Molecular adjuvant to enhance 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 cooling again, ground in the mortar mill with the addition of liquid nitrogen. The ground lipid-GMDP mixture was dispersed in a 2.5% Tween 80 solution and pre-dispersed with the Ultraturrax for 1 minute at 8000 rpm. This dispersion was homogenized at 4°C using high-pressure homogenization in 3 cycles at 1000 bar. The PCS diameter amounts to 260 nm with a polydispersity index of 0.430.
Example 17: Molecular adjuvant for increasing the cellular immune response incorporated in the interfacial surface: saponins are generally known to increase the cellular immune response.
Production of the particles took place with a rotor-stator analogously 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°C and the lipid melt added. Production took place with an Ultraturrax, stirring at 10,000 rpm for 5 minutes. The 50%
diameter determined with laser diffractometry amounted to 2.28 Vim.
Example 18: Production of SBA in the presence of an amphiphilic adjuvant. The amphiphilic surfactant CHAPS is described in the literature as a means of enhancing the immune response. The particles consist of 5% cetyl palmitate and 0.5% CHAPS.
Production of the particles was carried out analogously to Example 20. The 50% diameter determined by laser diffractometry amounts to 1.897 ~Cm.
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) in comparison with molecular adjuvant, i.e. pure GMDP (N-acetyl glycosaminyl-N-acetyl muramyl-dipeptide). The concentration of GMDP (0.1 mg/ml) was analogous to Example 9. The in-vivo testir~g took place as described in Example 9. SBA 2 shows a higher intensity of effectiveness than pure GMDP (Figure 5).
Composition of SBA-2: 4% hard paraffin, 1% EQl (N,N di-(f3-stearoyl ethyl)- N,N-dimethyl ammonium 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): Between the positively charged particles SBA 4 and SBA 2 no difference in strength of effectiveness can be detected. EQ1 from SBA 2 can be replaced without any loss in effectiveness by cetyl pyridinium chloride which has been toxicologically examined and approved as a pharmaceutical preservative (SBA 4). In contrast to the negatively charged particles of the formulation SBA 5, the positively charged particle formulations are observed to have a stronger effect (Figure 6).
The SBA formulations have 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, sodium deoxycholate 0.2%, sodium cholate 0.2%, sodium oleate 1%, lipoid E80 2%. SBA 2: 4%
hard paraffin, 1% EQ1 and 4% Tween 80/Span 85 (7/3). The surface charges (zeta potential) were determined in conductive water with a conductivity of 50~,5/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. 0.110), SBA 5: 107 nm (P. I.
0.115), SBA 2: 101 nm (P. I. 0.101).
Example 21: Species-independence of the effect (hens): The antigen from Example 9 was mixed with SBA 1 and SBA 2 in the ratio 1:1 and injected 0.5 ml per hen. The antibody titres were determined from the hens' eggs. An ELISA test was used for quantification. In contrast to the description in Example 9, a marked Anti-IgG-chicken was used in the ELISA test. The first immunization took place on Day 0. A booster with the same preparations was given on Day 31. Analogously to the results in Example 10, the formulation SBA 2 proved to have a stronger effect (Figure 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 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 the lipid matrix on the adjuvant effect:
in the case of the formulation SBA 2, the non-biodegradable hard paraffin was exchanged for biodegradable glycerol tribehenate (SBA 3). The intensity of effect does not differ; hard paraffin can be replaced by glycerol tribehenate (Figure 8).
Composition of SBA 2: 4% hard paraffin, 1% EQl and 4% Tween 80/Span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101); 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: The formulations SBA 1 and SBA 2 were tested in comparison with aluminium hydroxide (procedure analogous to Example 9 ) . The effect of SBA 1 and SBA 2 is identical to the effect of aluminium hydroxide (control: antigen in PBS). As in Example 9, SBA 2 has a stronger effect than SBA 1 (Figure 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); SBA 1: 4%
hard paraffin, 4% Tween 80/Span 85 (7/3), PCS diameter: 107 nm (P. I. 0.112).
List of figures:
Figure 1: Adjuvant effect in comparison with molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetyl muramyl-dipeptide) and FIA.
Figure 2: Effect of the SBA composition on antibody titre.

Figure 3: Storage stability of SBA-2.
Figure 4: Antibody production in hens following 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 injection (Day 100) on Day 120.
Figure 5: Adjuvant effect of SBA in comparison with molecular adjuvant GMDP (Example 19) Figure 6: Effect of different particle charge (Example 20) Figure 7: Adjuvant effect in hens (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 aluminium hydroxide (Example 23) [Key to figures]
Elisa Einheiten . Elisa units Tag . Day Lipid partikel mit GMDP . Lipid particles with GIMP

PCS Durchmesser . PCS diameter Infusionsflasche . Infusion bottle Ampulle . Ampoule k.A. . n.a. [no antibodies]

1g Y Titer gegen Kaninchen 1gG . 1g Y titre compared with rabbit 1gG

Antikorperbestimmung . Antibody determination Aluminiumhydroxid . Aluminium hydroxide

Claims (24)

Claims

1. Agent for enhancing the immune response in the case of vaccinations of humans and animals and for increasing the yield of antibody production, characterized in that this envolves a mixture of lipid particles which are solid at room temperature (= 20°C) with antigen, which lipid particles are admixed in a characteristic optimum dose for the subject to be immunized, and optimum particle size, particle charge and surface properties (stabilizing surfactant layer).

2. Agent according to Claim 1, with the particles being within the range 10-1000 nm.

3. Agent according to Claim 1, with the particles being within the range 1-10 µm.

4. Agent according to Claim 1, with the particles being within the range 10-200 µm, and especially within the range 10-100 µm.

5. Agent according to Claim 1, with the particles being within the range 200-1000 µm, and especially within the range 200-500 µm.

6. Agent according to Claims 1-5, characterized in that the lipids used for production of the lipid particles are 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 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 thereof.
Especially suitable are synthetic monoglycerides, diglycerides and triglycerides 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, for example, cetyl palmitate and white wax (DAB), with these being used individually or in mixtures, also hydrocarbons, such as, for example, hard paraffin.

7. Agent according to Claim 6, characterized in that liquid lipids such as liquid glycerides (miglycols), groundnut oil, castor oil, soya oil, cottonseed oil, rape oil, linseed oil, olive oil, sunflower oil, and safflower oil can be admixed to the solid lipids used to produce the lipid particles, with the particles thus produced being solid at room temperature (20°).

8. Agent according to Claims 1-7, characterized in that they are positively charged.

9. Agent according to Claim 8, characterized in that, to produce the positive charge during production of the lipid particles the substances benzyl dimethyl hexadecyl ammonium chloride, methyl benzethonium chloride, benzalkonium chloride, cetyl pyridinium chloride, N,N Di(.beta.-stearoyl ethyl)-N,N-dimethyl ammonium chloride, and dimethyl dioctadecyl ammonium bromide can be used individually or in mixtures.

10. Agent according to Claims 1-7, characterized in that they are negatively charged.

11. Agent according to Claim 10, characterized in that to produce the negative charge during production of the lipid particles, the following compounds: diacetylphosphates, phosphatidyl glycerin, lecithins of various origin (e.g.
egg lecithin or soya lecithin), chemically modified lecithins (e.g. hydrogenated lecithins), as well as phospholipids and sphingolipids, mixture of lecithins with phospholipids, sterols (e.g. cholesterol and cholesterol derivatives, as well as stigmasterol) and also saturated and unsaturated fatty acids, sodium cholate, sodium glycholate, sodium taurocholate, and sodium deoxycholate are added individually or in mixture.

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

13. Agent according to Claim 12, characterized in that for their production, during 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. saccharose monostearate), can be added individually or in mixture with one another.

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

15. Agent according to Claim 14, characterized in that it contains molecular adjuvants such as N-acetyl glycosaminyl-(.beta.1-4)-N-acetyl muramyl-L-alanyl-D-isoglutamine [GMDP], dimethyl dioctadecyl ammonium bromide [DDA], N-acetyl muramyl-L-alanyl-D-isoglutamine [MDP], N,N Di(.beta.-stearoyl ethyl)-N,N-dimethyl ammonium chloride [EQ1], glycopeptides, components of the cell wall of mycobacteria, saponins, quaternary amines, such as, for example cetyl pyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane sulphonate), dextrane sulphate, dextrane, 3-odesacyl-4' -monophosphoryl lipid A [MPL®], N-acetyl-L-alanyl-disoglutaminyl-L-alanin-82) - 1,2 dipalmitoyl - sn glycero-3-(hydroxy-phosphoryloxy))ethyl amide, monosodium salt [MTP-PE], granulocyte-macrophage colonies stimulating factor [GM-CSF], block copolymers, e.g. P1205, Poloxamer 401 (Pluronic L121), dimyristoyl-phosphatidyl choline [DMPC], dehydroepiandrosterone-3.beta.-01-17-on [DHEA], dimyristoyl-phosphatidyl glycerol [DMPG], deoxycholic acid-sodium salt, cytokines, imiquimod, DTP-GDP, saponins, 7-allyl-8-oxoguasonin, 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, Arlacel 85), DTP-DPP, stearyl-tyrosin, N,N dioctadecyl-N' ,N' -bis(2hydroxyethyl) propandiamine, calcitriol.

16. Agent according to Claim 14, characterized in that these are particular adjuvants such as e.g. aluminium hydroxide, polymer particles, liposomes.

17. Agent according to Claim 14, characterized in that the substances enhancing the effectiveness of adjuvants are added to them, from the group of bivalent transitional metal ions, quaternary amines, dextrane, dextrane sulphate, vitamin E derivatives such as vitamin E phosphate and vitamin E hemisuccinate, and isoprinosine.

18. Agent according to Claims 1- 17, characterized in that the lipid particles are produced by comminution of lipids in the solid aggregate state, e.g. mortar mill, gas-jet mill, electro-sputtering, high-pressure homogenization, microfluidization.

19. Agent according to Claims 1- 17, characterized in that the lipid particles are produced by comminution of lipids in the molten state during dispersion in an outer phase (e.g.

high-speed stirrers, microscale and macroscale static mixers, rotor-stator mills, colloid mills, high-pressure homogenization, microfluidization, spray processes such as spray drying and electro-spray processes), with the outer phase being able to be liquid (water, organic liquids, oils or mixtures thereof) or gaseous (air, nitrogen, noble gas).

20. Agent according to Claims 1- 19, characterized in that the lipid particles and possibly 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 (miglycols, groundnut oil, castor oil, soya oil, cottonseed oil, rape oil, linseed oil, olive oil, sunflower oil, safflower oil and other oils or mixtures thereof.

21. Agent according to Claim 20, characterized in that, e.g. to the outer phase, viscosity-increasing substances are added .

22. Agent according to Claims 1- 19, characterized in that the lipid particles and possibly the additional adjuvant are present in dry form, e.g. as lyophilisate, spray-dried product, solid dispersion, pellet or tablet.

-34(a)-

23. Agent for enhancing the immune response in the case of vaccinations of humans and animals and for increasing the yield of antibodies in immunology and antibody production, characterized in that this involves lipid microparticles which are solid at room temperature (= 20°C) and have a size within the range 1-1000 µm, which are applied in a characteristic optimum dose for the subject to be immunized, and optimum particle size, particle charge and surface properties (stabilizing surfactant layer), simply being admixed to the antigen solution.

24. Agent for enhancing the immune response in the case of vaccinations of humans and animals and for increasing the yield of antibodies in immunology and antibody production, characterized in that this involves lipid particles which are solid at room temperature (= 20°C) prepared from a mixture of solid and liquid lipds, which lipid particles are applied in a characteristic optimum dose for the subject to be immunized, and optimum particle size, particle charge and surface properties (stabilizing surfactant layer), simply being admixed to the antigen solution.