DE1229677B - Process for the preparation of injectable emulsions containing antigens - Google Patents

Description

Process for the preparation of injectable emulsions containing antigens The invention relates to a method for producing injectable antigens containing Emulsions, especially those containing vaccines.

It is known to use vaccines in the form of water-in-oil emulsions, in which the antigen is contained in the aqueous disperse phase. Such Compositions are used in human and veterinary medicine. These emulsions are often the simple solutions of vaccines in water or suspensions of a Antigens in water are preferred, as it is possible to use the emulsions to obtain the desired to achieve an immunological effect even with smaller amounts of the antigen.

However, emulsions of this type are very highly viscous liquids; man cannot pack them in smaller containers because they are removed from the containers Causes difficulties. Emulsions of this type are usually manufactured in Syringes or sold in inserts for such syringes; but this becomes hers Use more expensive.

It has now been found that those resulting from the high viscosity Difficulties can be overcome if you take the vaccine in the form of a multiple emulsion applies. Multiple emulsions differ from the usual emulsions in that they that the droplets of the disperse phase contain smaller droplets of another phase.

This second disperse phase usually has the same composition like the coherent phase, but is separate from the latter. procedure for the production of multiple emulsions of this type are known. For this purpose, for example referred to the book by M a n e g o 1 d, "Emulsionen" (1952), p. 14, and on the book by C 1 a y t o n,)> Theory of Emulsions, "pp. 327 to 329.

According to the process of the invention, injectable emulsions are obtained, which contain an antigen.

They consist of multiple oil-water emulsions.

In these emulsions the aqueous phase is the continuous phase. An oil phase is dispersed in this. Inside the oil droplets is another one dispersed aqueous phase which contains antigens.

The injectable emulsions are produced according to the invention that a water-in-oil emulsion is emulsified into a second aqueous phase.

As an aqueous phase in which the antigen is dissolved or dispersed a physiological saline solution or other aqueous medium can be used, z. B. a phosphate buffer; Depending on the intended use, distilled can also be used Use water. The oil can be any of the therapeutically useful known per se Be mineral oils that for the production of the well-known water-in-oil emulsions used by vaccines.

Certain vegetable oils such as peanut oil can also be used.

Mineral oils are not processed in the metabolism. Using water-in-oil emulsions from vaccines can build up under the skin The vaccine builds up around the injection site, causing difficulties can. In the case of emulsions according to the invention, on the other hand, the oil is in the contiguous aqueous phase and is easily distributed throughout the body. Another The advantage of the multiple emulsions according to the invention is that one very a small amount of oil is enough to trap the antigen. The entire to be injected However, the amount corresponds to the usual injection technology.

To prepare emulsions according to the invention, 1. a Mixture of a water-in-oil emulsion containing the antigen in the aqueous phase contains, 2. an aqueous medium and 3. use an emulsifier, which under Emulsifying conditions promotes the formation of oil-in-water emulsions.

Suitable emulsifiers include nonionic polyesters and Ethers and various resins such as B. acacia resin and gum arabic.

The processes customary for this purpose can be used to prepare the emulsions be used. You can z. B. the emulsified mixture by a mechanical Pass the homogenizer through it, or you can expose it to ultrasound, or you can can generate the necessary mechanical forces by making the mixture passes through a narrow bore of a needle with the aid of a syringe; this The latter process cannot of course be carried out on a large scale, but only on a laboratory scale use. In all cases it is usually desirable to premix the ingredients, ventilation is expediently avoided.

Then you run them straight through the emulsifier. It can be use numerous commercially available mechanical emulsifying devices if one ensures that sterility is guaranteed.

If ultrasound is used for emulsification, the mixture is cooled preferably with ice to about 0 ° C and then continues a series of intermittent Ultrasound bursts interrupted by vigorous shaking. Ultrasonic with a frequency of 18,000 to 20,000 per second is well suited.

The stability of the multiple emulsions depends to some extent on the ratio of the water-in-oil emulsion to the aqueous medium.

The best results have been obtained when using this quantitative ratio within the limits of 3: 1 to 1:10, based on parts by volume. You can z. B.

Use ultrasound at a ratio of 1: 9; with mechanical Process can be in a ratio of 2: 1. The multiple emulsions thus obtained are very shelf-stable; after storage for example 3 to 4 months at elevated temperature, e.g. B. at 37 "C, there was no decrease in antibody production observed.

Such multiple emulsions are particularly stable and immunologically effective, in which the mean diameter of the oil droplets is between 10 and 50 microns, in particular between 10 and 20 microns. In such cases the mean diameter is der - droplets of the dispersed aqueous phase about 1 to 7 microns. This is special appropriate when the antigen is from toxic substances, from rickettsia or obtained from viruses.

The amount of the necessary to get the best results Emulsifier depends to a certain extent on the quantitative ratio of the water-in-oil emulsion to the aqueous phase. It is also dependent on the amount of emulsifier, which is present in the original water-in-oil emulsion.

The emulsifier should preferably be used in such amounts that the emulsifiability of the two agents is approximately equivalent. The amount of the second Emulsifier, which promotes the formation of oil-in-water emulsions, is usually about 0.25 to 5 parts by volume per 100 parts by volume of the aqueous medium.

The stability of the emulsions can be improved by adding agar will. If the emulsions are to be stored for a longer period of time, it is often advantageous to add about 1 part by weight of agar to 100 parts by volume of the aqueous medium.

The first water-in-oil emulsion containing the antigen can be produced by known methods. In many cases it is useful same or use similar mechanical homogenizers to make both emulsions to manufacture. It is advantageous in the preparation of the water-in-oil emulsion To have an above normal concentration of the antigen in the aqueous phase. on this type can change the concentration of the antigen in the final multiple emulsion can be kept at normal levels and the usual amounts used will. Since in the given case the ratio of the water-in-oil emulsion to aqueous phase is known, the appropriate concentration of the antigen in the original aqueous phase can be calculated.

The mineral oil distillate known under the trade name Drakeol 6VR is commonly used to make injectable water-in-oil emulsions. This oil is also very suitable for the multiple emulsions according to the invention to manufacture. A suitable emulsifier that prevents the formation of water-in-oil emulsions promotes, is known under the trade name Arlacel A (fatty acid ester of sorbitol anhydride). For the present purpose this emulsifier can be used in amounts of about 5 to 15 parts by volume 100 parts by volume of Ö1 are needed each time.

The method of the invention can be used in the manufacture of all kinds of antigens are used, their immunogenic effectiveness by the addition of Ö1 is increased. So the antigen can either be an allergen or toxic Substances, viruses or bacteria are obtained. Other antigens can come from others Substances are obtained, e.g. B. from mycoplasma, rickettsia or trachoma. Typical Antigens are for example the following: Bacterial antigens: Bordetella pertussis, Salmonella antigens, e.g. B. Typhoid and paratyphoid types, Shigella antigens, z. B. Shigella Dysenteriae, Brucella abortus (live and killed), enveloping substance from P. Pestis, Cholera, Chlorstridia Antigens; Mycoplasma Antigens: Contagious Cattle consumption; Rickettsia antigens: Murine typhus, Rocky Mountain spotted fever; Virus Antigens: Influenza, Measles, Adenoviruses, Poliomyelitis, Jap. B. encephalitis, Foot and mouth disease, rinderpest, Newcastle disease; Obtained from toxigens Antigens: tetanus, diphtheria, botulinus.

As is known, it is beneficial to have antigens obtained from smaller organisms originate, e.g. B. toxigens and viruses, to use together with oil. That meets also apply to the present compositions. With bacterial antigens it is sometimes beneficial to destroy the cell walls and extract the active substances and to use. According to the invention, compositions can also be prepared which contain two or more antigens in the multiple emulsions. You get in such cases a corresponding reaction of the body for each antigen. the As noted above, the multiple emulsions can be produced by microphotography controlled to ensure that the emulsions are actually multiple and that no inversion has taken place, due to which a conventional oil-in-water emulsion arises. Here you can regulate the duration and type of emulsification process, to ensure that the desired dispersion is obtained and that it is not excessive large oil droplets are created, which causes cream to form.

The stability and shelf life of multiple emulsions after The invention is described in Example 3 below. You can also use the stability Ouchterlony diffusion plates show in which the central bore an antitoxin contains and the outer bores contain emulsions according to the invention. Multiple emulsions according to the invention were tested on mice, according to a method described in the journal Annals of Allergy, 1962, 20, No. 7, 472-9. The compositions according to the invention meet the requirements of this test.

The following examples serve to illustrate the invention.

Example 1 Ovalbumin obtained by chromatographic ion exchange was purified was dissolved in 1 ml of 0.85% saline solution. Proportions of this solution were injected in 1 mol of a mixture of 9 parts of mineral oil and 1 part of emulsifier, with the help of a syringe with a needle, which has a bore of 0.5 mm would have. The mixture was repeated through the needle passed through it, always again a new solution of ovalbumin was added. After incorporating the entire aqueous solution, the emulsion was tested by placing a portion in ice water gave. It was found that a perfect water-in-oil emulsion was present.

18 mol of a normal saline solution was placed in a vial of 25 ml content done. About 0.2 ml of ethoxylated sorbitol derivative was added as an emulsifier.

The mixture was shaken, then 2 ml of the water-in-oil emulsion was added to. After closing the vial, the whole thing was shaken. This resulted in a certain dispersion of the water-in-oil emulsion to small drops, which quickly framed. Then the vial was surrounded with ice and a laboratory probe was inserted Ultrasound machine. The frequency of the ultrasound machine was 18,000 to 20,000 per second. After about 5 to 10 seconds of sonication, the vial became taken from the ice and shaken to remove any larger drops that might stick to the Surface had risen to disperse. Treatment with ultrasound and the shaking was repeated about three to five times until a milk-like product was created. It was found by microscopic observation that the desired Emulsion was formed. The multiple emulsion thus produced was a thin, easily agile one Liquid that flowed like water and easily inserted into a hypodermic syringe could be. In contrast, the original water-in-oil emulsion was a thick, viscous, cream-like liquid that flowed very slowly.

The effectiveness of the simple water-in-oil emulsion on the one hand and the multiple emulsion on the other hand was tested in experiments on mice. Ten mice each were treated in equivalent amounts so that each mouse received an injection of 2 mg ovalbumin. The formation of antibodies was determined by agglutination of tanned cells. The following table shows the geometric mean of the values obtained. Days 10 15 1 20 1 30 1 40 1 50 1 60 1 70 l 100 1 200 Water-in-oil Emulsion. . . . . . . . . 0.6 10 80 210 470 650 940 1000 790 750 Multiple emulsion. 2.3 40 150 740 1700 2800 4100 4800 1 4000 1800 The results show that when the multiple emulsion according to the invention is used, far more antibodies are produced in the mice than when the comparable water-in-oil emulsion is used.

A similar group of mice was simply injected with 2 ml of ovalbumin treated with a saline solution alone, antibodies were produced in quantities below that one unit.

Example 2 Influenza Vaccin This commercially available under the name "Muneven" distributed antigen contains three different viruses, namely A2 / Singapore / 1/57, A2 / England / 1/61 and B / England / 939/59 which were killed with formalin.

The original water-in-oil emulsion was made by dilution of a volume part of "Muneven" with 10 parts by volume of a normal saline solution. then this solution was in an equal volume of a mixture of 9 parts of mineral oil and stirred in 1 part emulsifier. A syringe was used for training, whose needle had a bore of 0.5 mm. The point of the needle became straight kept under the surface of the oil mixture.

The total amount of the saline solution was added in five portions, with the mixture was shaken vigorously between additions.

After two or three additions, the entire mixture was repeated passed through a needle with a bore of 0.5 mm. Then you gave more Saline solution and repeat the process until all of the solution in the vaccine was removed Oil was incorporated. Finally, the water-in-oil emulsion was subjected to two shocks of 5 seconds of an ultrasound with a frequency of about 20 000 per second.

The emulsion was kept in a cup cooled with ice, into which the probe of the ultrasound machine was inserted. The resulting The water-in-oil emulsion was very thick and hardly poured out.

The same procedure was used to make a second Water-in-oil emulsion with the exception that the Muneven with 3.3 parts by volume of a normal saline solution has been diluted. Also the water-in-oil emulsion obtained in this way was very viscous and could not be poured out.

1 part by volume of this water-in-oil emulsion became 2 parts by volume of one normal saline solution added, which is ethoxylated to 100 parts by volume - of the solution 1 part by volume Contained sorbitol derivative. The mixture was then shaken vigorously and at intervals with ultrasound at a frequency of 20,000 per second for a total duration of treated for about 20 seconds.

In this ultrasound treatment, too, the mixture was cooled in ice. A stable multiple emulsion with a viscosity similar to water was obtained. Microscopic examination showed that it was indeed a multiple emulsion acted and that a good distribution of the water-in-oil phase had been achieved.

The dilution of the Muneven was such that 0.2 ml of the final water-in-oil emulsion and the multiple emulsion were 75 HA units from A2 / Singapore / 1/57 and 25 HA units from A2 / England / 1 / 61- included. Two groups of six mice each were treated subcutaneously with 0.2 ml of the water-in-oil emulsion or with 0.2 ml of the multiple emulsion. The sera were examined after 21 and 35 days. They were titrated for the presence of antibodies which prevented hemagglutination by a slightly modified method from the procedure prescribed by the World Health Organization using 4 HA units of virus A2 / Singapore / 1/57. The geometric means of the values obtained are given in the table below. Days 0 0 | 21 1 35 Water-in-oil emulsion .... 18.3 13.6 31.7 Multiple emulsion ... ... 13.6 40.0 100.0 A post-death test on day 46 showed that in the mice treated with the water-in-oil emulsion, one to three large deposits of the emulsion were present under the skin. The mice treated with the multiple emulsion contained only a few small droplets, which consisted entirely of oil.

The multiple emulsion was very stable after 14 weeks of storage at 4 ° C.

Example 3 To show the stability of the multiple emulsions and to prove that the antigen preferentially remains in the disperse aqueous phase and does not migrate into the continuous aqueous phase, a number of Multiple emulsions made with bovine serum albumin. The antigen was made with fluorescein offset so that every hike of the antigen observed under an ultraviolet microscope could be. The original water-in-oil emulsion was created using a syringe prepared as described in Example 2. A saline solution was used for this of bovine serum albumin in a concentration of 10 / o in a mixture of 9 parts by volume Mineral oil and 1 part by volume of emulsifier incorporated. The -below- table contains also the proportions. The resulting thick and non-pourable water-in-oil emulsion was then converted into a multiple emulsion. 1 part by volume of the water-in-oil emulsion was mixed with 9 parts by volume of a normal saline solution, which is ethoxylated as an emulsifier Contained sorbitol derivative. The table also shows the amounts of emulsifier used specified. The cooled mixture was treated several times with ultrasonic shocks, until the desired dispersion was obtained.

In all cases the multiple emulsions are milk-like, easy flowing ones Liquids that can be easily drawn into syringes. The multiple emulsions were stored at 56 ° C for 4 months.

After storage, they were examined under an ultraviolet microscope to determine whether an antigen had migrated into the continuous aqueous phase. It has been found that the multiple emulsions are very stable, even at such high temperatures. Water- Emul Oil volume stability at 56 ° C gator O / o hiiOftVnis 1 month 2 months | 4 months 1 1: 1 NLNLNLSL 1 * 1: 1 NLNLNLNL 1 1: 3 NLNLNLNL 1 * -1-: 3 NL N: LNLNL 4 1: 1 NLNLNLNL 4 * 1: 1 NLNL - NLSL 4 1: 3 NLNLSLL 4 * 1: 3 NLNLSLL

Explanations: * These compositions contain 1 part by weight of agar to 100 parts by volume of the liquid.

N. L. no hike.

S. L. easy hike.

L. hike.

In one case the multiple emulsion was made by passing it through the mixtures through a mechanical homogenizer; after a month's storage at about 18-20 ° C in a siliconized vial these were emulsions not collapsed.

Example 4 Ovalbumin Following the procedure of Examples 1 and 2 was first made a thick water-in-oil emulsion.

A needle with a bore of 0.5 mm and repeated the treatment with ultrasound. A thick one was obtained non-pourable mass containing 20 mg ovalbumin per ml.

A solution of acacia resin was also prepared by dissolving it of 0.2 g acacia resin B.P. in 2 ml of a normal saline solution on the water bath at 56 "C. After cooling to room temperature became a such volume of the thick water-in-oil emulsion was added. The mix was shaken vigorously, cooled to 0 ° C and then repeated for 4 to 10 seconds each time treated with continuous bursts of ultrasound at a frequency of 20,000 per second. In between it was shaken again and again. After a total duration of treatment A stable multiple emulsion was created by ultrasound for 20 seconds, what microscopic examination proved it. This multiple emulsion was water-like Viscosity and showed no signs even when stored at 37 ° C for 3 to 4 months a reversal. When used by subcutaneous injection in mice, a useful antibody formation observed.

Example 5 Tetanus Vaccine The solution of a tetanus toxoid prepared with an M 100 glaxo-phosphate buffer salt solution and 0.013% sodium ethyl mercurithiosalicylate (Merthiolate) contained (200 LF units of the toxoid per milliliter). The solution was used as a source of an antigenic material. 100 ml of this solution was added dropwise added to 100 ml of a mixture of 9 parts by volume of mineral oil and 1 part by volume of emulsifier.

Mixing was done in a small funnel according to G i u s t i, the ingredients being carefully mixed with a spatula to to prevent the formation of eddies and the entry of air. Then one led this mixture through a mechanical homogenizer according to G i u 5 t i, which was operated at a speed of 2 and a Clayton setting of 2. About 50 ml of the thick, non-pourable water-in-oil emulsion was stored at 4 ° C.

A solution was also made by dissolving 2 parts by volume of ethoxylated Sorbitol derivative in 100 parts by volume of a salt solution Glaxo PBS.

This solution was sterilized and shaken while cooling to prevent precipitation. After cooling, such amounts of the merthiolate were added admitted that its final concentration was 0.013%.

80 ml of this salt solution were then mixed with 160 ml of the thick water-in-oil emulsion mixed. Shake vigorously to disperse the oil and then premix in a funnel according to G i u 5 t i, which has a speed of 2 and a Clayton setting operated by 3.5. 250 ml of an easily mobile milk-like material were obtained Liquid with a water-like viscosity. The mixture that is now 8 LF units of the toxoid each contained 0.25 ml was examined under the microscope to determine whether the desired multiple emulsion had formed.

The effectiveness of this vaccine was tested by subcutaneous injection of 25 ml each in ten guinea pigs. After 4 weeks the amount of the antitoxin was determined as follows: 1 animal. 8.0 units / ml 4 animals ............... 16.0 units / ml 3 animals ............... 32.0 units / ml 1 animal ............... 64.0 units / ml 1 animal above over 64.0 units / ml Another eight guinea pigs were treated in the same way, after the vaccine had been stored at 4 ° C for 6 months. The antitoxin levels 4 weeks after the injection, the table below shows: 1 animal. . 8.0 Units / ml 1 animal. . 16.0 units / ml 5 animals. ........ 32.0 units / ml 1 animal. .. 64.0 units / ml Example 6 Newcastle Disease Vaccine A liquid from a Glaxo laboratory tissue culture that is three times normal concentration of the Newcastle disease virus was used. 50ml of this concentrated Liquid slowly became 50 ml of a mixture of 9 parts by volume of mineral oil and 1 part by volume of emulsifier added. The mixture was then processed further as it is described in Example 5.

The milky multiple emulsion of the vaccine was water-like Viscosity and was easily drawn up through a needle in syringes.

The dilution factor of the original concentrate was 3, so that you could inject the usual amount of vaccine when using it.

Example 7 Influenza Vaccine As in Examples 5 and 6 described, worked using an aqueous influenza vaccine. This vaccine contained the following substances: A / Singapore / 1/64 with 18,000 HA units per milliliter, A / England / 1/64 with 6000 HA units per milliliter, B / England / 939/59 with 12,000 HA units per milliliter and B / Taiwan / 4/62 with 6,000 HA units each Milliliters.

With the aid of the multiple emulsion produced in this way, a sufficient Antibody formation observed. The concentration of the vaccine was such that the usual amounts could be injected.

Example 8 This example shows the formation of a multiple emulsion with 7 different antigens. The multiple emulsion was prepared according to the method of Examples 5 to 7 prepared. The antigens given in the table were mixed, using a small amount of a glaxo-phosphate buffer saline solution. Bordetella pertussis was last admitted. When using 0.25 ml, the multiple emulsion had the desired concentration of vaccines.

Components of the aqueous phase 1. Bordetella pertussis, 40 10. Organisms / ml .......................... 25.0 ml 2nd tetanus, 1000 LF units / ml .... 3.37 ml 3. Diphtheria, 6800 LF units / ml .. 1.96 ml 4. Polio I, 85 D units / ml. 100 ml. . 98.1 ml 5. Polio II, 10 D units / ml. 100 ml .. 100.0 ml 6. PolioIII, 1OD units / ml 100 ml .. 100.0 ml 7. Measles (treated with ether), 100HA units / ml. 100 ml 50.0 ml of phosphate buffer saline solution ........ 38.6 ml Total ... 417.0 ml These aqueous phase containing the antigens was prepared using a mixture of 9 parts by volume of mineral oil and 1 part by volume of emulsifier initially in a water-in-oil emulsion and then converted into a multiple emulsion using 2 parts by volume the water-in-oil emulsion per part by volume of a PBS mixture with ethoxylated sorbitol derivative. The multiple emulsion obtained was an easily mobile stable liquid, which did not separate even after prolonged storage at elevated temperatures.

Claims (1)

Claim: Process for the production of injectable emulsions, which contain antigens in a disperse aqueous phase surrounded by oil, thereby gekennzeichn et that the water-in-oil emulsion in a second aqueous phase emulsified.