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WO2000071077A2 - Adjuvant a stabilite et biocompatibilite optimisees (sba) pour accroitre la reponse immunitaire humorale et cellulaire - Google Patents

Adjuvant a stabilite et biocompatibilite optimisees (sba) pour accroitre la reponse immunitaire humorale et cellulaire Download PDF

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Publication number
WO2000071077A2
WO2000071077A2 PCT/EP2000/004644 EP0004644W WO0071077A2 WO 2000071077 A2 WO2000071077 A2 WO 2000071077A2 EP 0004644 W EP0004644 W EP 0004644W WO 0071077 A2 WO0071077 A2 WO 0071077A2
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Prior art keywords
sba
composition according
particles
oil
adjuvant
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PCT/EP2000/004644
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German (de)
English (en)
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Gmbh Pharmasol
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MÜLLER, Rainer, Helmut
Grubhofer, Nikolaus
Olbrich, Carsten
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Priority to AU58091/00A priority Critical patent/AU5809100A/en
Publication of WO2000071077A2 publication Critical patent/WO2000071077A2/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants

Definitions

  • Antigens are applied to animals and humans to produce antibodies.
  • the goals are, for example, the immunization of humans or animals to protect against diseases or the production of antibodies, which are subsequently isolated and processed into products.
  • the most common application route for antigens is parenteral administration, alternative routes are e.g. oral, nasal and topical application.
  • a common problem is that often the strength of the immune response to the administered antigen is insufficient for the intended purpose. Sometimes the problem can be solved by greatly increasing the administered dose of the antigen.
  • a serious problem, however, is that antigens are often very expensive and the dose increase leads to a corresponding increase in the price of the vaccine. These costs lead to a heavy burden on the health system, for many sections of the population - especially in the Third World - the vaccine becomes too expensive and desirable mass vaccinations cannot be carried out.
  • butyricum proposed by Freund to complete the adjuvant are also poorly tolerated. They also cause granulomas and fever as well as abscesses at the injection site [Brown, E.A. Rev Allergy 1969 23 (5): 389-400].
  • the task therefore is to find a more tolerable adjuvant, in particular from the point of view of inexpensive vaccines. make desirable worldwide vaccinations against certain diseases that can only be financed with an inexpensive vaccine and the increasing importance of immunological production techniques and products. Even with increasing awareness of the need for protection of animals and corresponding pressure from legislation, a more compatible, (ie biocompatible) equally efficient, but at the same time inexpensive adjuvant must be found. In addition, it must be physically stable so that admixing before the injection can be omitted and fluctuations in efficiency - for example due to different dispersions - are eliminated.
  • the most commonly used adjuvant approved for human use today is a fine suspension of aluminum hydroxide, the roots of which are even more archaic than those of Freund's emulsion [Glenny, A.T. et al. J. Pathol. 1926, 29 31-40]. It is based on the assumption that antigens are better recognized by the immune system if they are adsorbed on the surface of the aluminum hydroxide particles. However, it is disadvantageous that aluminum hydroxide is less efficient than Freund's adjuvant and it can also cause granulomas.
  • emulsions have recently been described which consist of more compatible materials such as, for. B. Squalan and Sqalen, [Sanchez-Pestador, L. et al. J. Immunol. 1988 141, 1720-1727, Masihi KN, Lunge, W., Bremer W., Ribi, E. Int. J. Immunopharmacol. 1986 8 (3), 339-45, Hunter RL, Bennett, B. Scand J Immunol. 1986 23 (3), 287-300, Allison AC, et al. Semin Immunol. 1990 2 (5), 369-74].
  • One of these systems is MF59 (European Patent Application 0399843A2).
  • MDP N-acetylmuramyl-L-alanyl-D-isoglutamine
  • Thr-MDP threonyl analog the MDP
  • GMDP N-acetylglucosaminyl-N-acetylmuramyl dipeptide
  • the new emulsions are by no means always tolerated without reaction, the synthetic macromolecules such as glycopeptides have so far not been shown to be superior to the mycobacteria in addition, they are far too expensive. Solid particles have approval problems. Many adjuvants show too high toxicity and thus too little biocompatibility. There are also problems with physical stability (avoidance of aggregation or coalescence). The FIA emulsion must be freshly prepared and injected, long-term storage is not possible. Sufficient physical storage stability is, however, the essential prerequisite for a marketable product - especially for pharmaceuticals.
  • GMDP had to be used in a mixture with colloidally disperse solid lipid particles in a particle size ⁇ 200 nm to increase the immunostimulating effect (US Patent Gerbu, processing number of the US Patent Office is 08 / 816,787).
  • the structure of the particles was systematically modified in studies for this invention (e.g. surfactants, stabilizers) to find lipid particles that could further increase the immunostimulating effect of GMDP. More surprising
  • lipid particles alone are just as efficient as the combination of lipid particles and GMDP (example 9).
  • the expensive GMDP can be dispensed with in the invention and a comparably high humoral immune stimulation can be achieved by an inexpensive lipid particle alone.
  • the strength of the immunostimulating effect depends on the surface properties of the particles (surfactants used, particle charge) and the particle size. If the lipid particle is negatively charged, the efficacy is approx. 1/3 of FIA; if a positively charged lipid particle is generated by adding EQ 1, the efficacy is comparable to FIA (no significant difference, t-test) (Example 9).
  • the desired potency can thus be set in a targeted manner via the composition of the lipid particles used. This avoids over-reactions of the organism.
  • SBA consist of in water or aqueous liquids or non-aqueous, e.g. oily liquids dispersed lipid particles, which can, but need not, be stabilized by surfactants or polymers. With a sufficiently high viscosity of the outer phase or fineness of the particles, a physically stable dispersion is obtained. The same applies to low-viscosity outer phases, if the particles carry a sufficiently high charge in the same direction and the sediments formed can be easily redispersed.
  • the SBA is produced by dispersion or precipitation, generally known methods described in textbooks in pharmacy and process engineering being used. During dispersing, coarsely dispersed lipids are broken up by mechanical methods. The lipids can be in the solid state (e.g. mortar mill) or in the liquid state (eg emulsification of molten lipids by stirrers). To produce the SBA dispersion, the lipids can first be comminuted and then dispersed in the outer (eg aqueous) phase or alternatively can be comminuted directly in the outer phase. For the production of SBA dispersions by dispersing tion can z. B. used include: piston-gap homogenizers (e.g.
  • APV homogenizers French Press
  • jet stream homogenistors e.g. microfluidizers
  • rotor-stator stirrers e.g. Ultraturax, Silverson homogenizers
  • static mixers on a microscale and macroscale e.g. mixers from Sulzer
  • gas jet mill e.g. gas jet mill
  • rotor-stator colloid mill e.g. mortar mill
  • SBA dispersions show sufficient long-term physical stability. Determination of the particle size with photon correlation spectroscopy (PCS) and laser diffractometry (LD) showed no or negligible particle growth over periods of 1 to 3 years (example 3).
  • PCS photon correlation spectroscopy
  • LD laser diffractometry
  • the stability of SBA dispersions is far superior to that of Freund's incomplete adjuvant (FIA) (example 1); higher stability is also shown compared to newer adjuvant emulsions (example 2).
  • FIA Freund's incomplete adjuvant
  • SBA dispersions represent a simple system. In contrast to glycopeptides, the additives used are chemically simple and robust. The use of heat in sterilization processes does not lead to chemical decomposition, the dispersion remains physically stable and particle aggregation does not occur.
  • Example 4 shows an example of the sterilization of SBA dispersions by autoclaving (121 ° C., 15 minutes, 2 bar). FIA shows phase separation under the same conditions.
  • Biocompatibility Low toxicity and good biocompatibility are essential for a widely used adjuvant.
  • SBA dispersion In a sheep study, no abnormalities were observed at the application site after application of SBA dispersion (example 6). The good tolerance is explained by the extremely low toxicity of lipids observed in vitro in cell cultures. Compared to polymers approved by the German regulatory authorities and the FDA for parenteral administration, they show a viability at least approximately 20 times higher at high particle concentrations (example 5). The good biocompatibility is attributed to the fact that body proteins generally only adsorb to a small extent on the particle surface - in contrast to other particles (Example 7). In addition, there are no proteins on the surface that promote intolerance reactions.
  • the properties of the adjuvant should be such that it can be mixed with a number of different antigens.
  • the mixture should be applied in physiological saline or other isotonized solution. Due to the reduction of the zeta potential, physiological saline leads to destabilization in dispersions and subsequent aggregation [Lucks, JS et al., Int. J. Pharm 1990 58, 229-235].
  • the SBA adjuvant should be physically stable for a sufficiently long time after being added to the antigen in isotonic solution.
  • Example 8 shows that the lipid particles according to Zumi to physiological saline are stable for over 6 hours, measurable aggregation does not occur.
  • surface properties such as loading can also be set in a targeted manner and the SBA adjuvant can thus be produced in a species-specific manner.
  • Positive and negative charges can be generated by adding appropriately charged surfactants or stabilizers.
  • the strength of the load can be adjusted via the concentration of the additive.
  • the ideal addition is charged substances, which, like cetylpyridinium chloride, are approved as preservatives for parenteral application (example 12).
  • lipids can be used to produce SBA dispersions. These are both chemically uniform lipids and their mixtures.
  • the lipids are characterized in that they are present in the end product SBA dispersion in the crystalline state (e.g. ⁇ -, ßi-modification) or in the liquid-crystalline state ( ⁇ -modification) or in their mixture.
  • liquid lipids e.g. oils, lipophilic hydrocarbons, lipophilic organic liquids such as oleyl alcohol
  • solid lipids e.g. glycerides, lipophilic hydrocarbons such as hard paraffin
  • lipids as a dispersed phase and can be used as an individual component or as a mixture: natural or synthetic triglycerides or mixtures thereof, monoglycerides and diglycerides, alone or mixtures thereof or with, for example, triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols, including their esters and ethers as well as in the form of lipid peptides, or any mixtures thereof.
  • Synthetic monoglycerides, diglycerides and triglycerides are particularly suitable as individual substances or as a mixture (eg hard fat), Imwitor 900, triglycerides (eg glycerol trilaurate, glycerol myristate, glycerol palmitate, glycerol stearate and glycerol behenate) and waxes such as cetyl palmitate and white. Also hydrocarbons, such as hard paraffin.
  • the proportion of the inner or lipid phase based on the total formulation is 0.1% to 80% (m / m) and is preferably in the range from 1% to 40% (m / m).
  • dispersion stabilizing additives e.g. Emulsifiers, in order to be able to produce stable dispersion, can be incorporated in the form of pure substances or in the form of mixtures in order to stabilize the particles.
  • the amount of such additives which can be added in relation to the total weight of the aqueous dispersion, is in the range from 0.01% to 30% and preferably in the range from 0.5% to 20%.
  • the surfactants, stabilizers and polymers which are generally known from the production of dispersions can be used to stabilize the SBA dispersions or to modify them in a targeted manner. Examples include:
  • sterically stabilizing substances such as poloxamers and poloxamines (polyoxyethylene-polyoxypropylene block copolymers), ethoxylated sorbitan fatty acid esters, especially polysorbates (eg Polysorbate 80 or Tween 80®), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and esters and ethers of sugars or of Sugar alcohols with fatty acids or fatty alcohols (eg sucrose monostearate, sucrose distearate, sucrose cocoat, sucrose stearate, sucrose dipalmitate, sucrose palmitate, sucrose laurate, sucrose octanoate, sucrose oleate).
  • poloxamers and poloxamines polyoxyethylene-polyoxypropylene block copolymers
  • ethoxylated sorbitan fatty acid esters especially polysorbates (eg Polysorbate 80 or Tween
  • charged ionic stabilizers such as diacetyl phosphates, phosphatidylglycerol, lecithins of various origins (eg egg lecithin or soy lecithin), chemically modified lecithins (eg hydrogenated lecithins), as well as phospholipids and sphingolipids, mixture of lecithins with phospholipids, sterols (eg cholesterol Cholesterol derivatives, as well as stigmasterol) and also saturated and unsaturated fatty acids, sodium cholate, sodium glycocholate, sodium taurocholate, sodium deoxycholate or their mixtures, amino acids or anti-flocculants, such as Sodium citrate, sodium pyrophosphate, sodium sorbate [Lucks, J.S.
  • Zwitterionic surfactants such as (3 - [(3-cholamidopropyl) dimethylammonio] -2-hydroxy-l-propanesulfonate) [CHAPSO], (3 - [(3-cholamidopropyl) dimethylammonio] - 1-propanesulfonate) [CHAPS] and N-dodecyl- N, N-dimethyl-3-ammoniol-propane sulfonate.
  • Cationic surfactants especially compounds used as preservatives, e.g. Benzyldimethylhexadecylammonium chloride, methylbenzethonium chloride, benzalkonium chloride, cetylpyridinium chloride.
  • cellulose ethers and cellulose esters e.g. methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose
  • polyvinyl derivatives and polyvinyl alcohol polyvinyl pyrrolidone
  • polyvinyl acetate alginates
  • polyacrylates e.g. carbopol
  • xanthans and xanthans and xanthans are preferably present in the SBA dispersion in an amount of 0.01% to 20% (m / m) and in particular in an amount of 0.05% to 10%.
  • viscosity-increasing substances are incorporated in the formulation in a similar ratio, preferably in an amount of 0.01-20% and in particular in an amount of 0.1% to 10% (m / m) and preferably in the range between 0.5% and 5%.
  • the outer phase can be water, aqueous solutions or liquids miscible with water, as well as glycerin or polyethylene glycol and oily liquids such as miglyols (medium chain triglycerides - MCT) and other oils (castor, peanut, soybean, cotton seeds -, rapeseed, linseed, olive, sunflower, and safflower oil can be used.
  • miglyols medium chain triglycerides - MCT
  • other oils castor, peanut, soybean, cotton seeds -, rapeseed, linseed, olive, sunflower, and safflower oil can be used.
  • Surfactant-free SBAs are produced by dispersing the lipid phase in an aqueous solution which contains one or more viscosity-increasing substances, either alone or in combination with other substances, and also sugars, sugar alcohols, especially glucose, mannose, trehalose, mannitol, sorbitol and others. Furthermore, it is possible to use a combination of the viscosity-increasing substances or the combination of these with sugars or sugar alcohols, or in a further combination with charge stabilizers or anti-flocculants.
  • SBA dispersions can be used as an adjuvant to many different antigens for vaccination against various diseases.
  • Glycoproteins such as Gonococcal Protein I, Brucella abortus antigen, Tetanus toxoid, Diphteria toxoid, Listeria monocytogenes, virus antigens such as Semliki Forest Virus, Enceohalomyocarditis virus, Porcine rarovirus, Pseudorabies virus, Newcastle desease virus, Bovine virus, HIV, Influenza diarrheal virus , Herpes simplex, hepatitis C, measles, parasites such as malaria, Eimeria spp.,
  • the SBA dispersions provide an adjuvant that:
  • SBA dispersions can be widely used to reduce the antigen dose and thus the costs with toxicologically acceptable excipients, since the addition of SBA at a low antigen dose achieves the same immunostimulating effect.
  • Antigens with previously insufficient antigenicity for a vaccine can be converted into an efficient vaccine by adding immune response-stimulating SBA. Due to the inexpensive manufacture of existing comparable
  • SBA dispersions are suitable as an adjuvant for vaccinations in the veterinary field, where for profitability reasons only very low-priced
  • Vaccines can be used.
  • Adjuvants used to date have focused on increasing the humoral immune response. In view of the efficiency of action of SBA dispersions, it is no longer necessary to add another adjuvant to the SBA lipid particles, or to add adjuvants such as GMDP bring no further increase in the humoral immune response. Obviously the immune system is at its maximum capacity of response, additional adjuvant can no longer bring an additional effect. Thus, additives to SBA have no advantage for the humoral response, additives as described in the Gerbu patent (patent DE 19611235 C1) are superfluous due to the surprisingly found potency of the SBA described in the invention.
  • the invention opens up the possibility of increasing the cellular immune response in the case of a second vaccination by combining SBA dispersions with other adjuvants.
  • the adjuvants can also be incorporated into the lipid particles. Incorporation is possible by incorporation in the solid particle matrix, enrichment in the interface in the case of amphiphilic adjuvants or by simple adsorption on the particle surface. Adjuvants can be incorporated during particle production or subsequently (for example in the case of incorporation, example 16).
  • adjuvants are dissolved, solubilized or dispersed in the molten lipid phase and the lipid phase containing adjuvants is then further processed.
  • these can also be dissolved in the outer phase of the SBA dispersion and then accumulate in the particle interface or by adsorption on the surface. Incorporation of adjuvants leads to prolonged release by diffusion or in the course of particle degradation by enzymes. Delayed release over a longer period increases the immune response.
  • Another attractive area of application is antibody production in animals.
  • the antibody yield can be significantly increased by adding the adjuvant.
  • Example 1 Determination of the physical stability of SBA versus Freund's Incomplete Adjuvant (FIA): An aqueous SBA dispersion was produced by high pressure homogenization at 95 ° C. from 20% beeswax, 2% Tween 80 (PCS diameter 289 nm polydispersity index 0, 101) . FIA was produced according to the method described by Freund [Freund, JJ Immunology 1948, 60, 383-398]. SBA and FIA were stored at the temperatures of the climatic zones that are used in the stability testing of drugs [EMEA guideline CPMP / QWP / 159/96, January 1998)]. The storage temperatures were: 5 ° C, 25 ° C, 40 ° C.
  • FIA The physical stability was determined by measuring the particle size with laser diffractometry, characterization parameters were the diameter 50% and the diameter 95% (50% and 95% of the particles are below the specified size, sensitive parameters for particle aggregation).
  • FIA showed significant particle growth after just a few minutes of storage - even at room temperature - so FIA is not a storage-stable adjuvant.
  • the particle sizes of SBA remain unchanged over a period of 1 year under the 3 storage conditions (Table 1).
  • Example 2 Determination of the physical stability of SBA versus squalene adjuvant: SBA was prepared as described under 1, it contained 10% cetyl palmitate and 1.2% miranol (PCS diameter 210 nm, PCS polydispersity index 0.189). Squalene adjuvant was prepared as described in European patent application 0 399 843 with filing date May 25, 1990 (adjuvant MF59). Particle size measurement was carried out using laser diffractometry, storage was carried out as in Example 1 at 3 temperatures (Table 2). In addition, an accelerated stability test was carried out, SBA and MF59 dispersions were shaken at 40 ° with a frequency of 50 Hz (Table 3). SBA shows increased stability both in normal storage and in the stress test. Table 2: Investigation of the stability of SBA (10% cetyl palmitate, 1, 2% miranol) in comparison with MF59
  • Table 3 Stability of MF 59 against SBA at 40 ° C and a shaking frequency of 50 Hz
  • Example 3 Long-term stability of SBA: SBA dispersion consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C for one year. The PCS data and laser diffractometer diameter showed little or no change (Table 4).
  • Example 4 Heat stability of SBA on autoclaving (heat, pressure) versus FIA and MF59:
  • the SBA dispersion was composed of 18% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water. Sterilization of 20 mL each was carried out in injection vials according to the standard conditions of the European Pharmacopoeia (121 ° C, 2 bar, 15 minutes). Particle size was determined using PCS and laser diffractometry (LD 95%) (PI: polydispersity index, measure for the width of the particle distribution, MW: mean from 3 measurements, staff: standard deviation, PI polydispersity index) (Table 5).
  • the FIA emulsion showed after autoclaving Phase separation, MF59 significant particle growth. SBA is physically stable and can be sterilized by autoclaving.
  • Table 5 Heat stability of SBA upon autoclaving (heat, pressure) versus FIA and MF59: The SBA dispersion was composed of 18% hard paraffin, 4% Tween 89 / Span 85, 7/3 and water. Sterilization of 20 mL each in injection vials at 121 ° C, 2 bar, 15 minutes. Particle size was determined using PCS and laser diffractometry.
  • Example 5 Physiological tolerance: To assess the tolerance, the cytotoxicity of SBA in cell cultures was determined (human granulocytes, HL60 cells). To quantify the toxicity, the viability of the cells was assessed using the MTT test [Mosmann, T., J. Immunol. Meth. 1993, 65, 55-63]. The SBA dispersion was composed of 10% cetyl palmitate, 0.5% poloxamer 188 and water. The number of cells per well was 200,000 for human granulocytes and 200,000 for HL60 cells. Incubation was for 12 hours. In SBA the viability was 80% for the granulocytes and 85% for the HL60 cells.
  • the viability of nanoparticles made of PLA was only 5%, for nanoparticles made of PLA / GA it dropped to 0%.
  • the tolerance of SBA in cell cultures is at least a factor of about 20 better than that of the FDA-approved polymers for parenteral administration.
  • Example 7 Biocompatibility - Interaction with Body Proteins:
  • the SBA dispersion was composed of 10 compritol, 2.5% Poloxamer 407 and water. Production was carried out with high pressure homogenization. The particles were incubated for 5 minutes with human plasma, then separated from the plasma and the body proteins adsorbed on the particle surface using two-dimensional polyacrylamide gel electrophoresis [Blunk, T et al. Electrophoresis 14, 1382-1387 (1993)].
  • Example 8 Stability in phosphate-buffered physiological saline (PBS): SBA composed of 20% hard paraffin, 5% Tween 80 / Span85 (7/3) and water were mixed with PBS (2 mL SBA + 2 mL saline). The physical stability in the physiological saline solution was determined as a function of time using laser diffractometry. There was no increase in particle size over 6 hours
  • Example 9 Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA: sheep were vaccinated with the strain Mycoplasma Bovis PG 45 R9.
  • the vaccine antigen was cultivated in stand culture over 72 hours under microaerophilic conditions in Hayflick medium. Inactivation was carried out by adding 0.1% ⁇ -propiolactone. The cells were separated, washed with phosphate buffer pH 7.4 and adjusted to a content of 1 x 10 10 CFU / mL. The sterility of the preparation was checked in accordance with the German Pharmacopoeia Edition 10. The dry matter determination showed a content of 1 mg / mL Mycoplasma Bovis antigen.
  • the adjuvant SBA, GMDP and FIA were mixed equally with the antigen in buffer. Injection volume was 5mL, spread over 4 injection sites.
  • composition of SBA was 4% hard paraffin, 1% EQ1 (N, N di- ( ⁇ -stearoylethyl) - N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3).
  • the composition of the GMDP adjuvant was 5% lipid and 0.5% surfactant.
  • FIA was made as in Example 1.
  • Blood was drawn on day 0 before vaccination, on day 35 and on day 63.
  • the antibodies were determined using ELISA.
  • a commercially available marked anti-IgG sheep from Sigma was used for the ELISA test.
  • SBA showed a comparable intensity of action as the combination of lipid particles with GMDP. Furthermore, SBA was comparable in effectiveness to FIA ( Figure 1). There was no significant difference in the intensity of action between the three adjuvants.
  • FIG. 1 Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA.
  • Composition of SBA was 4% hard paraffin, 1% EQ1 (N, N di- ( ⁇ -stearoylethyl) - N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3).
  • the composition of the GMDP adjuvant was 5% EQ 1 and 0.5% Montanide 888.
  • FIA according to Example 1.
  • Example 10 Effect of SBA composition on immunititer: SBA dispersions were prepared with an identical lipid but different surfactants on the surface, ie they differ in their surface properties.
  • the formulation SBA-1 consists of 4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water
  • the formulation SBA-2 contains 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3) and water.
  • the efficiency for increasing the immune titer was tested analogously to Example 9, FIA served as a comparison.
  • there are different levels of immune responses for the two SBA dispersions (Figure 2). The strength of the desired immune response can thus be adjusted by varying the surface properties (surfactants, stabilizers, charge, etc.).
  • Figure 2 Effect of SBA composition on immune 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% EQ 1 and 4% Tween 80 / Span 85 (7/3) and water.
  • Example 1 1 Storage stability of SBA-2: The SBA dispersion SBA-2 from Example 11 was stored at different temperatures and the physical stability was determined by measuring the particle size with PCS. No particle growth occurred ( Figure 3).
  • FIG. 3 Storage stability of SBA-2: The SBA dispersion SBA-2 from Example 1 1 was stored at different temperatures and the physical stability was determined by measuring the particle size with PCS.
  • SBA dispersions were prepared with surface-active, positively charged stabilizers (EQ 1 - distearoylethyldiamonium chloride, cetylpyridinium chloride) and negatively charged stabilizers (sodium lauryl sulfate, (SDS)).
  • the composition of the SBA dispersions was: SBA-EQ 1 (20% cetyl palmitate, 4% Tween 80 / Span 85 (7/3), 1% EQ1), SBA-CPC (18% lipid, 10% surfactant, 0.1 % Cetylpyridinium chloride) and SBA-SDS (20% cetyl palmitate, 1% SDS 80).
  • the zeta potential was measured in millivolts (mV) as a measure of the charge (Electrophoresis measurement), Zetasizer4 (Malvern Instruments, UK).
  • the Helmholtz-Smoluchowski equation was used to convert the electrophoretic mobility into the zeta potential.
  • the zeta potentials were +40 mV, +28 mV and -35 mV for the 3 SBA dispersions.
  • Example 13 Increased cellular immune response in booster chickens treated with GMDP-containing adjuvant (SBA) at the time of primary immunization.
  • SBA 5% EQ 1, 0.5% Montanide 888 was mixed 1: 1 with the antigen (IgG from rabbit) and injected subcutaneously.
  • SBA contained 5 ⁇ g GMDP and the immunization schedule was as follows: first immunization and two boosters on day 14 and day 28. The antibody determination took place on day 42 and the IgY titer was measured in the egg yolk. To check for a strengthened cellular immune response, antigen contact was repeated on day 100 (renewed vaccination) and the antibody determination on day 120. The results show that in the case of the first immunization (day 14 and 28) with SBA containing GMDP, a significant increase in antibody production takes place with renewed contact with the antigen: columns 2, 3 and 4, Figure 4.
  • FIG 4 Antibody production in chickens after basic immunization and renewed antigen contact on day 100. The last antibody determination of the basic immunization took place on day 42, that of the booster vaccination (day 100) on day 120.
  • the composition of the vaccines of Examples 1-5 is as follows : 1. Day 42: antigen in PBS, day 100: antigen in PBS (n / a: no adjuvant, antigen in PBS)
  • Example 14 The adsorption of GMDP on SBA particles was investigated using PCS.
  • GMDP was mixed with SBA (4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and left to stand for 30 minutes at room temperature to allow adsorption of the GMDP to the particles.
  • the final concentration was 1,435 mg / ml
  • the size increase is 3.9 nm. (PCS diameter without GMDP: 99.4 nm, standard deviation: 0.764, diameter with GMDP: 103.3 nm, standard deviation: 0.755).
  • Example 15 Modification of the particle size: The SBA dispersion with EQ1 from Example 12 (SBA-EQ 1) was produced using various production processes in order to vary the particle size. The particle size was measured using laser diffractometry (laser diffractometer LS 230, Coulter Electronics-Germany, measuring range: 40 nm - 2000 ⁇ m). The diameter 50% of the particles is given as the characterization parameter. The following manufacturing methods were used:
  • Rotor-stator dispersion The raw emulsion was prepared as described under a) and then with an Ultraturrax (type T25, Jahnke and Kunkel, Staufen, Germany) at one
  • Static mixer Lipid and aqueous surfactant solution from a) were heated to 80 ° C. and mixed in a static mixer (Sulzer, Germany). The particle size was 15.8 ⁇ m.
  • Example 16 Molecular adjuvant to increase the cellular immune response incorporated in SBA: GMDP was dissolved in Span 85 (W / O) emulsifier and cetyl palmitate was added. The mixture was melted at 70 ° C. and, after re-cooling, was ground in a mortar mill with the addition of liquid nitrogen. The ground lipid-GMDP mixture was dispersed in a 2.5 percent Tween 80 solution and predispersed with the Ultraturrax for 1 minute at 8000 rpm. This dispersion was homogenized at 4 ° C. by means of high pressure homogenization in 3 cycles at 1000 bar. The PCS diameter is 260 nm with a polydispersity index of 0.430.
  • Example 17 Molecular adjuvant for increasing the cellular immune response incorporated in the interface: Saponins are generally known for increasing the cellular immune response. The particles were produced using a rotor stator analogous to Example 15. The composition of the SBA dispersion is 5% cetyl palmitate, 0.5% saponin (Quil A saponin) and water. The saponin was dissolved in the aqueous phase, this was heated to 80 ° and the melted lipid was added. Production was carried out with an Ultraturrax, stirring at 10,000 RPM for 5 minutes. The diameter 50% determined with the laser diffractometer was 2.28 ⁇ m.
  • Example 18 Production of SBA in the presence of an amphiphilic adjuvant.
  • amphiphilic surfactant CHAPS has been described in the literature as an agent for increasing the immune response.
  • the particles consist of 5% cetyl palmitate and 0.5% CHAPS.
  • the particles were produced analogously to example 20.
  • the diameter 50%, determined by means of laser diffractometry, is 1,897 ⁇ m.
  • Example 19 Comparison of SBA versus pure molecular adjuvant: SBA dispersion No. 2 from example 10 (SBA-2) was tested in sheep (conditions as in example 9) against molecular adjuvant, ie. H. pure GMDP (N-acetylglucosaminyl-N-acetylmuramyl dipeptide). The concentration of GMDP (0.1 mg / ml) was analogous to Example 9. The in vivo testing was carried out as described in Example 9. SBA 2 shows a higher active intensity than pure GMDP (Fig. 5). Composition of SBA-2: 4% hard paraffin, 1% EQ 1 (N, N Di- (ß-
  • Example 20 Effect of charge (surface property) on the immune response (sheep study analogous to Example 9): There is no difference in the potency between the positively charged particles SBA 4 and SBA 2. EQ1 from SBA 2 can be replaced without loss of effectiveness by the toxicologically tested and approved pharmaceutical preservative cetylpyridinium chloride (SBA 4). One observes in relation to the negatively charged particles of the formulation SBA 5 a stronger effect of the positively charged particle formulations (Fig. 6).
  • the SBA formulations have the following composition: SBA 4: 4% hard paraffin, 4% Tween 80 / Span 85 (7/3), 0.5% cetylpyridinium chloride. SBA 5: 4% hard paraffin, sodium deoxycholate 0.2%, sodium cholate 0.2%, sodium oleate 1%, lipoid E80 2%. SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3). The surface charge (zeta potential) was determined in conductivity water with a conductivity of 50 ⁇ S / cm: SBA 4: +41.2 mV, SBA 2: +40.5 mV, SBA 5: -36.4 mV.
  • PCS diameter and polydispersity index (PI) were: SBA 4: 103 nm (PI 0, 1 10), SBA 5: 107 nm (PI 0, 1 15), SBA 2: 10 lnm (PI 0, 101) .
  • Example 21 Species independence of the effect (chickens): The antigen from Example 9 was mixed with SBA 1 and SBA 2 in a ratio of 1: 1 and 0.5 ml was injected per chicken. The antibody titers were determined from the hen's eggs. An ELISA test was used for quantification. In contrast to the description in Example 9, a labeled anti-IgG chicken was used in the ELISA test. The first immunization took place on day 0. A booster with the same preparations took place on day 31. Analogous to the results in example 10, the formulation SBA 2 had a stronger effect (FIG. 7).
  • composition SBA 1 4% hard paraffin and 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (PI 0, 1 12) and SBA 2: 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: lOnnm (PI 0, 101).
  • Example 22 Influence of the lipid matrix on the adjuvant effect: In the formulation SBA 2, the non-biodegradable hard paraffin was exchanged for the biodegradable glycerol tribehenate (SBA 3). The intensity of the effect does not differ; Hard paraffin can be replaced by glycerol tribehenate (Fig. 8)
  • composition SBA 2 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: 10 lnm (PI 0, 101)
  • SBA 3 4% glycerol tribehenate, 1% EQ 1 and 4 % Tween 80 / Span 85 (7/3), PCS diameter: 105nm (PI 0, 112).
  • Example 23 Formulations SBA 1 and SBA 2 were tested in comparison to aluminum hydroxide (procedure analogous to Example 9). The effect of SBA 1 and SBA 2 is the same as that of aluminum hydroxide (control: antigen in PBS). Analogous to Example 9, SBA 2 is more effective than SBA 1 (Fig. 9).
  • composition SBA 2 4% hard paraffin, 1% EQ 1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: 10 lnm (PI 0, 101)
  • SBA 1 4% hard paraffin, 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (PI 0, 112).
  • Figure 1 Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA.
  • Figure 2 Effect of SBA composition on immune titer.
  • FIG. 4 Antibody production in chickens after primary immunization and renewed antigen contact on day 100, the last antibody determination of the primary immunization took place on day 42, that of the booster vaccination (day 100) on day 120.

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Abstract

L'invention concerne un adjuvant à stabilité et biocompatibilité optimisées (SBA) utilisé pour accroître la réponse immunitaire humorale et cellulaire, cet adjuvant étant injecté avec un ou plusieurs antigènes. Ledit adjuvant est constitué de particules à base de lipides solides ou de mélanges de lipides solides. Un tel adjuvant peut être utilisé pour la production de vaccins efficaces et compatibles, pour la vaccination d'hommes et d'animaux et également pour la production d'anticorps. Grâce à la sélection de la taille des particules, de la charge des particules et des caractéristiques superficielles, il est possible de moduler de façon ciblée l'intensité de la réponse immunitaire et en outre de l'adapter spécifiquement à une espèce. Audit adjuvant (SBA) peuvent être également ajoutés d'autres adjuvants, par exemple des adjuvants moléculaires tels que le GMDP, ce qui accroît encore plus la réponse immunitaire cellulaire. Le SBA est plus performant, plus économique et plus simple à manipuler que les produits utilisés jusqu'ici et il est bien toléré in vivo.
PCT/EP2000/004644 1999-05-20 2000-05-22 Adjuvant a stabilite et biocompatibilite optimisees (sba) pour accroitre la reponse immunitaire humorale et cellulaire WO2000071077A2 (fr)

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US10369018B2 (en) 2012-04-30 2019-08-06 Ossur Hf Prosthetic device, system and method for increasing vacuum attachment

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TR200103333T2 (tr) * 1999-05-20 2002-04-22 Pharmasol Gmbh Dayanıklığı ve biyolojik uygunluğu iyileştiren yardımcı ilaç (SBA)

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US8052978B2 (en) 2004-07-22 2011-11-08 Agence National De Securite Sanitaire De L'alimentation, De L'environnement Et Du Travail Vaccine composition against Rhodococcus equi
US10369018B2 (en) 2012-04-30 2019-08-06 Ossur Hf Prosthetic device, system and method for increasing vacuum attachment

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WO2000071154A2 (fr) 2000-11-30
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