IL28639A - Antigen-adjuvant compositions comprising hydrated salts of polyvalent metals and fatty acids - Google Patents

Description

ANTIGEN- ADJUVANT COMPOSITIONS COMPRISING HYDRATED SALTS OF POLYVALENT METALS AND FATTY ACIDS w o*n a o» »3on y"oa nam - ni3 'Tssan i3r3 PATENTS AND DESIGNS ORDINANCE SPE C I FIC A TI ON ANTIGEN- ADJUVANT COMPOSITION We. AMERICAN CYANAMID COMPANY, a corporation organized and existing under the laws of the State of Maine, United States of America, and having its executive offices at Wayne, New Jersey, United States of America DO HEREBY DECLARE the nature of this invention and in what manner the same is to be performed, to be particularly ascertained in and by the following statement :- This invention relates to novel compositions comprising a disperse aqueous phase containing an antigenic substance and a continuous or a disperse oil phase, said composition having admixed therein at least 0.15 by volume of the hydrated reaction products of a physio-logically acceptable metallic cation and a physiologically acceptable fatty acid having 12 to 24 carbon atoms and if desired a conventional emulsifying agent.

Antigenic material has been incorporated into adjuvant compositions in the form of water-in-mineral oil emulsions which are modifications of the well-known Freund adjuvants but using antigens without tuberculosis bacteria in the aqueous phase, as was the case with the customary Freund adjuvant compositions .

Problems arose because there were two constit-uents which were considered medically not desirable.

One was the mineral oil which is not metabolized by the body tissues after an emulsion is injected and the other was the emulsifying agent, mannide monooleate, which could cause tissue irritation, which sometimes tended to favor carcinogenesis and sterile abscess formation.

In U.S. Patent 3,149,036, to Woodhour et al., a modified emulsion is described in which the mineral oil is replaced by a triglyceride oil, such as peanut oil, and dried or unhydrated aluminum salts of fatty acids were used. The same emulsifying agent, mannide monooleate, was, however, retained. Actually, as has been found out, the mannide monooleate was by far the most undesirable constituent; and while glyceride oils are somewhat preferable to mineral oils, the major dis- advantage caused by the mannide monooleate still remained . It should be noted that both in the incomplete ' Freund adjuvant compositions and in the Woodhour emulsions, the emulsions were water-in-oil and not water-in-oil-in-water .

This invention also relates to a process in which hydrated aluminum salts of fatty acids are the •stabilizing agent for stabilizing the water-in-oil emulsions . These emulsions may be changed into water- in-oil-in-water emulsions by suitable agitation in an aqueous phase following the addition of a conventional emulsifying agent or additional amounts of the hydrated aluminum salts of f tty acids . On the other hand if, initially, there is enough hydrated aluminum salts of fatty acid present, the second addition of the salt to convert the water-in-oil emulsion to the water-in-' V-' < For convenience and brevity throughout this specification, these two types will be abbreviated WO and WOW respectively. In emulsifying the WO emulsion, after it is formed, to produce a WOW, one may use in this second emulsifying step a conventional type of emulsifying agent which is capable of making oil-in-water emulsions, because essentially the WO is the disperse phase in water to form a WOW. Typical emulsifying agents include polyoxyethylene sorbitan monooleate.

The WOW emulsions have more favorable viscosities and also showed improved immunological results over the incomplete Freund and Woodhour emulsions more particularly in the characteristic that when antigens were present in the aqueous phase or phases, antibody formation proceeded more rapidly. In other words, an adequate antibody titer could be reached in fewer days after injection.

It is often convenient to use more than one mole of stearic acid per mole of polyvalent cation in the preparation of the WO emulsion and then to add additional polyvalent cation or a conventional emulsifying agent to the second aqueous phase before forming the WOW emulsion.

For best results it is preferable to introduce at least a major part of the antigenic material with the aqueous phase in the formation of the WO emulsion before the emulsion is further emulsified to produce a WOW emulsion. If a WOW emulsion is prepared with no antigenic material, an antigenic material is simply added to the continuous water phase of the WOW i emulsion. The results are markedly inferior to the WOW emulsions of the present invention, in which the antigenic material is introduced into the aqueous phase with which the WO emulsion is formed. This is not to say that the present invention precludes antigenic material being added at both times, but there must be at least substantial antigenic material in the initial WO emulsion.

Although ordinarily not preferred, there are some advantages in a preformed WOW emulsion which can be marketed as a stock item to which an immunologist can add the antigenic material he wishes to use.

The polyvalent metal cations which form the fatty acid salts may be of any physiologically accept-able polyvalent metal. Aluminum is preferred, but satisfactory results can be obtained with ferric iron. It should be understood that the hydrated basic fatty acid salts are not necessarily pure single chemical compounds. As is well known, basic aluminum fatty acid salts, such as aluminum stearates, can be produced with molar ratios which are not integral. Thus, for example, if the ratio is somewhat greater than one, there may well be mixtures of some aluminum monostearate and some aluminum di-stearate . The above statement is merely made to empha-size that the invention is in no sense critically limited to an exact single chemical compound.

The particular fatty acids from 12 to 2k carbon atoms to be used are not critical, and the invention is not limited to the use of any particular one or a particular mixture. However, because of the ready availability and excellent physiological acceptance, palmitic and stearic acids are the preferred ones, especially stearic acid.

In addition to the preferred stearic and palmitic acids, other fatty acids may be mentioned as illustrative, such as lauric, myristic, arachidic, be-henic, lignoceric, ricinoleic, oleic, erucic, linoleic, and the like.

The physiologically acceptable glyceride oils are also not critical, and any acceptable oil may be used, such as peanut oil, safflo er oil, soy bean oil, cottonseed oil, corn oil, chaulmoogra oil, olive oil, sesame oil, and coconut oil. Light mineral oils may also be used in forming the WOW emulsions of the present invention and are, therefore, included in the invention. In many specific descriptions a particular illustrative oil, namely peanut oil, will be used, and it produces excellent results. The invention is in no sense limited to its use, and any of the other oils which are physio-logically acceptable may be employed. Obviously, of course, mixtures are suitable.

In addition to the typical polyvalent metals aluminum and iron, referred to above, other physiologically acceptable metals are magnesium, cerium, zinc, lanthanum, bismuth, etc.

The antigenic materials may be of most varied character. Illustrative examples are those derived from viruses, such as influenza virus antigens and antigens of various types of foot and mouth disease, etc. Anti-gents from rickettsial from bacteria, such as tetanus toxoid, and polysaccharides, such as those derived from pneumococci, and the like. Tumor antigens are also useful. It should be noted that the antigenic material from pathogens should be in a form in which it will not cause clinically serious manifestations of the disease. The antigenic material may be killed, attenuated, or otherwise rendered sufficiently harmless for practical immunological use. The present invention is directed to improved WO and WOW emulsions, which are carriers for the slow release of antigenic material, and it is an advantage that any of the known types of antigenic material may be used. Other antigenic material may be allergens, such as pollens, dust, danders, extracts of the same, for example rag weed, house dust, pollen extracts, grass pollens, etc. Some of the antigens fall within the categor of poisons of venoms from insects or reptiles, such as for example the venoms of bees and wasps, poisonous snakes, scorpions, and Lactrodectus mactans. Often antigenic material is provided with stabilizing or preservative substances, and it is an advantage of the present invention that such acceptable materials do not destroy the stability of the resulting WO or WOW emulsions. As is well known, most of the antigenic material is proteinaceous in character but is, of course, not limited; for example the polysaccharides of the pneumococci and some other antigenic material of nucleic acid type is also included and can be incorporated in the WOW emulsions of the present invention.

The amount of antigenic material is also not critical, but it must of course be sufficient to produce adequate antibody production, and this amount will be referred to as "an effective amount for antibody production." The effective amounts with the various antigenic materials are well known and it is known that in many cases adjuvants permit a reduction in dosage or longer duration of protective antibody levels or both. The advantages of WOW emulsions often do not lie in drastically changed effective amounts but in the more rapid development of antibodies and in more desirable physical characteristics. To these advantages of WOW emulsions the present invention adds its safer physiological characteristics.

It is an advantage of the present invention that the amount of hydrated polyvalent basic fatty acid salt is not critical. However, of course, there must be a sufficient amount to stabilize the emulsions, and in general the lower limit is w/v, based on the total volume of the emulsions. Larger amounts from 0.5$ up show even higher stability and are therefore preferred. Larger amounts of the hydrated polyvalent metal fatty acid salt may be used, and there is no critical upper limit. Obviously, of course, the amounts must not be so enormous that a good fluid emul-sion will not result. This is purely a practical matter and does not constitute any critical limitation of the present invention.

For the most part, the emulsions of the present invention exhibit varying degrees of thixotropy. This is sometimes an advantage as it is not necessary 1 that the emulsion always be in thinly fluid state since I injection through a hypodermic syringe results in temporary lowered viscosity by the thixotropic effect.

The hydrated basic polyvalent cation fatty acid salt may be added in preformed state or the fatty acid may be dissolved in the oil, if necessary with slight warming, and a soluble salt of the metal, which exerts no toxicity, for example a chloride, nitrate or a sulfate, may be added to the aqueous phase usually with pH adjustment to between 4 and 10, producing con-j sierable amounts of hydroxide, and the hydrated polyvalent cation fatty acid salt during emulsification to form the initial WO emulsion. Obviously, of course, if the fatty acid salt is to be formed in situ, the fatty acid may also be introduced into the aqueous phase as a salt with a physiologically accaptable cation, such as for example a sodium salt and a soluble salt of the polyvalent metal, e.g. aluminum sulfate, 1 may then be added. It is an advantage of the invention that the procedure by which the WOW emulsion is made in two steps is not critical, and simple, practical operating conditions result.

The ratios of the volume of the disperse aqueous phase (designated Vwx ) to the volume of the oil phase (designated Vo) and to the volume of continuous aqueous phase (designated Vw2) in the WOW emulsions of this invention are not extremely critical and may be varied over a considerable range with retention of good activity. (In the following discussion it must be kept in mind that during emulsification of the first- V 1 formed WO emulsion in the continuous aqueous phase, a considerable wandering or migration of disperse aqueous phase to the continuous aqueous phase may occur so that while the initial phase ratios employed might be 25/25/50 for the final emulsion might have phase ratios of 10/25/65.) In general the volume percent of oil can be varied from to 60$ with retention of good adjuvant activity with 20 to 30$ being the preferred range. If oil contents below $ are employed, the volume of adjuvant emulsion that is required for a good immunological response becomes inordinately large for injection. If oil contents greater than 60$ are employed, the viscosity of the final emulsion becomes inconveniently high for in-jection.

In the case of W0 emulsions, the ratio of Vwi to Vo may vary from a.bout 5/95 up to 60/40 or higher. The ratio is not critical to the operability of the emulsions as adjuvants but is merely a matter of practicality. For very low ratios of V i to Vo, highly concentrated aqueous antigen preparations may be required to provide sufficient antigenic mass in a reasonable injection volume. For very high .ratios of Vwi to Vo, emulsion viscosity becomes too high for easy injection. The present invention is not limited to any particular type of agitation. However, it has been found that the use of ultrasonic agitation gives excellent results in the second step and is very satisfactory.

The invention will be described in conjunction with a number of specific examples which are typical illustrations . Because of the thixotropic nature of most of. the emulsions, it is desirable to characterize the viscosities by a needle viscosity test which com-pares the flow behavior of the emulsions in a manner closely related to that occurring in actual use when the emulsions are injected. The term "needle viscosity" will be understood to mean visfiosity determined by this test, which is as follows: A standard 5 ml. hypodermic syringe is fitted with a 1" long 25-gauge needle for WOW emulsions (or gauge for WO emulsions), filled with the fluid to be tested and clamped in a vertical position with the needle downward. A 2 kg. weight is placed on the syringe plunger and the time to expel 1 ml . of fluid is recorded. Under these conditions with the 25 gauge needle, 2 .6 seconds are required to expel 1 ml . of water, while 140-150 seconds are required to expel 1 ml. of the classical incomplete Freund's adjuvant, a water-in-oil emulsion dontaining 50 parts of aqueous phase, emulsified in a mixture of 45 parts of Drakeol 6 VR mineral oil and parts of mannide monooleate .

With the 20 gauge needle, water is expelled too rapidly for accurate measurement while incomplete Freund's adjuvant requires 2 seconds per ml .

EXAMPLE 1 Utilization Of The Inventive Concept In Preparing An Adjuvant Composition This example demonstrates a stable adjuvant composition in which the hydrated reaction products were formed in situ: A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 9 Stearic acid 1.0 Aluminum sulfate octadecahydrate 0.6 Aqueous glycine solution (0.3 M) 48.2 Aqueous sodium hydroxide solution (6N) 1.2 The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender. The aluminum sulfate octadecahydrate was dissolved in the aqueous glycine solution by stirring at room temperature; the pH of the solution was then adjusted to 7.2 with the aqueous 6N sodium hydroxide solution, and the resulting suspension was added slowly to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: Table 1 Stable droplets in water at room temperature Yes Electrical conductivity Negative Percent aqueous phase produced at 1,780 rpm for 4-5 minutes 0 Viscosity* 39 seconds *Viscosity refers to the time required to expel 1.0 ml. of emulsion from a 5 ml. syringe through a 1",20 gauge hypodermic needle using a force of 2 kilograms.

EXAMPLE 2 Utilization Of The Inventive Concept In Preparing An Adjuvant Composition This example demonstrates the formation of the hydrated reaction products in the aqueous phase prior to emulsification.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 50.0 Stearic acid Ο.98 Aluminum sulfate octadecahydrate 0.57 Deionized water 46.87 Aqueous sodium hydroxide solution (6N) I.58 The aluminum sulfate octadecahydrate was dissolved in one-half of the quantity of the deionized water, and the pH was adjusted to 7.08 with the aqueous sodium hydroxide solution. The stearic acid was then sus-pended in the remaining quantity of deionized water.

The aluminum hydroxide suspension was then added in a dropwise fashion to the stearic acid suspension, and the resulting suspension was added in a dropwise fashion to the peanut oil which had been previously placed in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: EXAMPLE 3 Utilization Of The Hydrated Reaction Products Of The Magnesium Cation This example demonstrates at stable adjuvant composition utilizing the hydrated reaction products of a magnesium cation.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 48.0 Stearic acid 2.0 Magnesium chloride hexahydrate 0.8 Aqueous glycine solution (0.3 M) 47.4 Aqueous sodium hydroxide solution (6N) 1.8 The stearic acid was dissolved in peanut oil by warming to 50°C., while stirring with a magnetic stirring bar, and the resulting solution was then poured into a Waring blender. The magnesium chloride hexahydrate was dissolved in the aqueous glycine solution and the pH was adjusted to 7.0 with the aqueous sodium hydroxide solution. The resulting suspension was slowly added to the oil phase in the Waring blender.

The following table represents the proper-ties of the composition hereinabove prepared: EXAMPLE 4 Utilization Of The Hydrated Reaction Products of Laurie AcTd This example demonstrates a stable adjuvant composition utilizing the hydrated reaction products of lauric acid.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 8 Lauric acid 2.0 Aluminum sulfate Aqueous glycine solution (0.3 M) 46.5 Aqueous sodium hydroxide solution (6N) 2.4 The lauric acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender. The aluminum sulfate octadecahydrate was dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.1 with the aqueous 6 sodium hydroxide solution. The resulting suspension was then added slowly to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: EXAMPLE 5 Utilization Of The Hydrated Reaction Products Of A Ceric Cation This example demonstrates a stable adjuvant composition utilizing the hydrated reaction products of a ceric cation.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 9 Stearic acid 1.0 Ceric ammonium sulfate dihydrate Ce(S04)2[(NH4)aS04]22H20 1.2 Aqueous glycine solution (0.3 M) 47 Aqueous sodium hydroxide solution (6N) 1.8 The stearic acid was dissolved in the peanut oil by warming to 50°C.i while stirring with a magnetic stirring bar and the resulting solution was poured into a Waring blender. The ceric ammonium sulfate dihydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.2 with the aqueous 6 sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender .

The following table represents the properties of the composition hereinabove prepared: Table 5 Stable droplets in water at room temperature Yes Electrical Conductivity Negative Percent aqueous phase produced at 1,780 rpm for 4-5 minutes 0 Viscosity 14 seconds' EXAMPLE 6 Utilization Of The Inventive Concept In Preparing An Adjuvant Composition Containing Tetanus Toxoid In The Disperse Aqueous Phase* This example demonstrates a stable adjuvant composition in which the disperse aqueous phase contains tetanus toxoid.

A composition consisting of the following ingredients was prepared: Percent by volume Peanut oil 48.0 Stearic acid 2.0 Aluminum sulfate octadecahydrate 1.1 Aqueous glycine solution (0.3 M) containing 43 Lf units per ml . of tetanus toxoid 46.6 Aqueous sodium hydroxide (6N) 2.3 The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender. The aluminum sulfate octadecahydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.0 with the aqueous 6N sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following represents the properties of the composition hereinabove prepared: EXAMPLE 7 Utilization Of The Hydrated Reaction Products Of A Ferric Catio This example demonstrates a stable adjuvant composition utilizing the hydrated reaction products of a ferric cation.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 48 Stearic acid 2.0 Ferric chloride hexahydrate l.o Deionized water 46.4 Aqueous sodium hydroxide (6N) 2.6 The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stir-ring bar, and the resulting solution was poured into a Waring blender. The ferric chloride hexahydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 6.8 with the aqueous 6N sodium hydroxide solution.

The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: Table 7 Stable droplets in water at room temperature Yes Electrical conductivity Negative Percent aqueous phase produced at 1,780 rpm for 5 minutes 0 Viscosity 18 seconds EXAMPLE 8 Utilization Of The Hydrated Reaction Products Of Oleic Acid This example demonstrates a stable adjuvant composition utilizing the hydrated reaction products of oleic acid.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 48.0 Oleic acid 2.0 Aluminum sulfate octadecahydrate 1.1 Aqueous glycine solution (0.3M) 46.6 Aqueous sodium hydroxide solution (6N) 2.? The oleic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar and the resulting solution was poured into a Waring blender. The aluminum sulfate octadecahydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.1 with the aqueous 6N sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: EXAMPLE 9 Utilization Of The Other Sources Of The Metallic Cation This example demonstrates that aluminum chloride hexahydrate can be successfully substituted for aluminum sulfate octadecahydrate .

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 49 Stearic acid 1 Aluminum chloride hexahydrate 0.3 Aqueous glycine solution (0.3 M) 48.4 Aqueous sodium hydroxide The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender. The aluminum chloride hexahydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.3 with the aqueous 6N sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: EXAMPLE 10 Utilization Of The Inventive Concept In Preparing An Adjuvant Composition This example demonstrates a stable adjuvant composition in which the aqueous phase is phosphate-buffered saline.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 49 Stearic acid 1.0 Aluminum sulfate octadecahydrate 0.5 Phosphate-buffered saline Aqueous sodium hydroxide solution (6N) 2.2 The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender . The aluminum sulfate octadecahydrate was then dissolved in the phosphate-buffered saline by stirring at room temperature, and the pH was adjusted to 7.0 with the aqueous 6 sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: EXAMPLE 11 Utilization Of The Inventive Concept In Preparing An Adjuvant Composition This example demonstrates a stable adjuvant composition in which the continuous oil phase is soya bean oil .

A composition consisting of the following ingredients was prepared: Percent by Volume Soya bean oil 48 Stearic acid 2.0 Aluminum sulfate octadecahydrate 1.1 Aqueous glycine solution (0.3 M) 46.4 Aqueous sodium hydroxide solution (6N) 2.5 The stearic acid was dissolved in the soya bean oil by warming to 50°C, while stirring with a magnetic stirring bar, and the resulting solution was poured into a Waring blender. The aluminum sulfate octadecahydrate was then dissolved in the aqueous glycine solution by stirring at room temperature, and the pH was adjusted to 7.0 with the aqueous 6N sodium hydroxide solution The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: Table 11 Stable droplets in water at room temperature Yes Electrical conductivity Negative Percent aqueous phase produced at 1,780 rpm for 4-5 minutes 0 Viscosity 19 seconds EXAMPLE 12 Utilization Of Other Sources Of A Metallic Cation This example demonstrates the use of alum gel to provide the metallic cation of the adjuvant composition of the present invention.

A composition consisting of the following ingredients was prepared: Percent by Volume Peanut oil 9 Stearic acid 1 Glycine 1.1 Suspension containing 0.46 grams of alum gel per 100 ml. (prepared by neutralizing potassium aluminum sulfate to pH 7 with sodium hydroxide and washing the precipitate with deionized water until free from sulfate ion.) 48.8 Aqueous sodium hydroxide solution (6N) 0.1 The stearic acid was dissolved in the peanut oil by warming to 50°C, while stirring with a magnetic stir-ring bar, and the resulting solution was poured into a Waring blender. The glycine was added to the alum gel suspension and the pH was adjusted to 7-0 with the aqueous 6N sodium hydroxide solution. The resulting suspension was then slowly added to the oil phase in the Waring blender.

The following table represents the properties of the composition hereinabove prepared: Table 12 Stable droplets in water at room temperature Yes Electrical conductivity Negative Percent aqueous phase produced at 1 ,780 rpm for - minutes 0 Viscosity 8 seconds EXAMPLE 13 WOW Emulsion Using Conventional Emulsifying Agent This example demonstrates the formation of a bimultiple emulsion as described by Seifriz, Journal of Physical Chemistry, volume 29 , page 738, ( 1925 ) . Fur-thermore, the example demonstrates that the novel adjuvant composition of the present invention may be emulsified in an aqueous media without destruction of the original water-in-oil emulsion, resulting in the formation of the bimultiple water-in-oil-in-water emulsion.

A composition consisting of the following ingredients was prepared: Percent by Volume Emulsion of Example 1 15 Tween 80 1 Phosphate-buffered saline 84 The Tween 80 was dissolved in the phosphate-buffered saline by stirring at room temperature with a magnetic stirring bar. The composition prepared in the manner set forth in Example 1 was then added to the saline solution and the probe of an ultrasonic generator was inserted to a depth of 5 nun. in the mixture. The mixture was ultrasonically agitated for 5 seconds, repeating the agitation for a total of four times.

A multiple water-in-oil-in-water emulsion was formed. The formation of said multiple water-in-oil-in-water emulsion was confirmed by phase contrast and interference microscopy.

EXAMPLE 14 Peanut Oil WOW Emulsion Containing No Conventional Emulsifying Agent This example illustrates that stable, active, bimultiple adjuvant emulsions of this invention can be prepared in which 1.5 W aluminum monostearate in hy- V drated form is employed as the sole stabilizing agent.

In 10 ml. of glycine buffer (0.3 M glycine solution, pH 7> containing 0.01$ merthiolate) is dissolved 0.484 g. of aluminum sulfate octadecahydrate and the pH is adjusted to 9.0 with 0.6 ml. aqueous 6 N sodium hydroxide solution. Then 0.88 ml. of tetanus toxoid (630 Lf units per ml.) is added and the resulting suspension diluted to 25 ml. with glycine buffer. In 24 ml. of refined peanut oil USP is dissolved 1.242 g. of stearic acid by warming to 60° with stirring. The aqueous phase is added dropwise to the oil phase at 2 °C. with good agitation to form a water-in-oil emulsion. This emulsion is added to 50 ml. of aluminum hydroxide suspension, which is prepared by dissolving 0.968 g. of aluminum sulfate octadecahydrate in 0.3 M glycine buffer, adjusting the pH to 9·0 with 1.2 ml. of 6 N sodium hydroxide and diluting to 50 ml. with glycine buffer. The mixture is agitated vigorously with the probe of an ultrasonic generator for a total of 10 seconds. A bimultiple water-in-oil-in-water emulsion containing 6 Lf . units of tetanus toxoid per ml. is formed. Most of the oil droplet diameters are in the range of 1 - 9 microns and at least Qo > of these contain inner water droplets. On centri-fugation for 45 minutes at 300 x g., no separate oil layer was detected.

Mice in one group were immunized with a single subcutaneous 0 .5 ml. dose of this emulsion, while those in a second group were immunized with a single subcutaneous 0 .5 ml. dose of aqueous toxoid (6 Lf . units/ml.). A third group received no injection. After 1 days, all mice were challenged with varying doses of tetanus toxin by intramuscular injection as shown in Table 13 .

TABLE 13 Survivors/Total Animals ( 4 days after Toxin) Dose of Toxin g.) 200 100 50 25 12 .5 0 .1 0 .01 0 .001 0 .0001 Aqueous Toxoid 0/10 0/10 2/10 Emulsion No injec-tion 0/10 0/10 10/10 10/10 EXAMPLE 15 Peanut Oil WOW Containing Ho Conventional Emulsifying Agent An emulsion similar to Example 14 is prepared except that 5 .25 ml. of Taiwan strain influenza vaccine in 0.3 M glycine buffer (3280 chick cell agglutinating units per ml.) is used in place of 0.88 ml. of tetanus toxoid. The final emulsion is a water-in-oil-in-water emulsion containing 200 CCA units per ml. Most of the oil droplet diameters are in the range of 3 - 12 microns and at least 90 of these contain inner water droplets.

Twelve mice in one group were each given one subcutaneous 0.5 ml. dose of this emulsion, while 12 mice in a second group were each given one subcutaneous dose of aqueous Taiwan vaccine containing 200 CCA units per ml. After four weeks, mice were bled and sera were tested for antibody titer by the technique of Salk, J. Immunology, 49, 87-98 ( 1944 ) · The HI titer from the pooled sera of mice receiving the emulsion of Example 2 was 1280 while that of the mice receiving the aqueous vaccine was 40.

EXAMPLE 16 Mineral Oil WOW Emulsion Containing No Conventional Emulsifying Agent : An emulsion similar zo Example 14 is prepared except that 5 .25 ml. of Taiwan strain influenza vaccine in 0 .3 glycine buffer (3280 chick cell agglutinating units per ml.) is used in place of 0.88 ml. of tetanus toxoid, and 24 ml. of mineral oil (Drakeol 6 VR) is used in place of 24 ml. of peanut oil U.S. P. The final emulsion is a water-in-oil-in-water emulsion containing 200 CCA units per ml. Most of the oil droplet diameters are in the range of 3 - 9 microns and at least 80$ of these contain inner water droplets. The "needle viscosity" (see above) is 6 seconds per ml. compared to a value of 2.6 sec. per ml. for water. Centrifugetion of the emulsion for 5 minutes at 300 x g. does not produce a detectable oil layer and the creaming which occurs can be completely reversed by gentle shaking.

A test of adjuvant activity made according to the procedure of Example 15 gave an HI titer of 1280 in the mice receiving the emulsion of Example 16 compared to a titer of 20 in the mice receiving the aqueous vaccine .

EXAMPLE 17 Mineral Oil WOW Emulsion Containing No Conventional Emulsif ing Agent This example illustrates that stable, active, . adjuvant emulsions of this invention can be prepared in which the molar ratio of aluminum to stearic acid is 1:3.

In 10 ml. of glycine buffer (0.3 M glycine solution, pH 7, containing 0.01$ merthiolate) is dissolved 0.484 g. of aluminum sulfate octadecahydrate and the pH is adjusted to 9·0 with 0.6 ml. of aqueous 6 N sodium hydroxide solution. Then 5.25 ml. of Taiwan strain influenza vaccine in 0.3 M glycine buffer is added and the resulting suspension diluted to 25 ml. with glycine buffer. In 24 ml. of light mineral oil (Drakeol 6 VR) is dissolved 1.242 g. of stearic acid by warming to 60° with stirring. The aqueous phase is added dropwise to the oil phase with good agitation to form a water-in-oil emulsion. This emulsion is added to 50 ml. of 0.3 M pH 9 glycine- sodium hydroxide buffer and treated for 10 seconds with the probe of an ultrasonic generator. A bimultiple water-in-oil-in-water emulsion containing 200 CCA units/ml. of Taiwan influenza antigen is formed with a pH of 8.4, and a "needle viscosity" of J.6 sec . ml. On centrifugation for 45 minutes at 300 xg., no oil layer is formed.

A test of adjuvant activity according to Ex-ample 15 gave an HI titer of 1280 for the emulsion and 20 for the aqueous vaccine .

EXAMPLE 18 Mineral Oil WOW Emulsion Containing No Conventional Emulsifying Agent This example illustrates the production of stable emulsions with hydrated salts of ferric iron and stearic acid. The procedure illustrates an operation with light mineral oil, but the same WOW emulsions can be prepared with the glyceride oils of the preceding examples by the substitution of an equal volume for the light mineral oil. As this example relates to a process of making the emulsion, no antigen is described, but of course an antigen may be added as described in the preceding examples .

In 10 ml. of glycine buffer (0.3 M glycine solution, pH 7> containing 0.01 merthiolate) is dissolved 0.234 g. of FeCl-j and the pH is adjusted to 9.0 with 0.6 ml. of aqueous 6 N sodium hydroxide solution. This suspension is diluted to 25 ml. with glycine buffer. In 24 ml. of light mineral oil is dissolved 1.242 g. of stearic acid by warming to 60° with stirring. The aqueous phase is added dropwise to the oil phase at 2 °C. with good agitation to form a water-ln-oil emulsion. This emulsion is added to 50 ml. of a suspension which is prepared by dissolving 0.468 g. of FeCl^ in 0.3 M glycine buffer, adjusting the pH to 9 with 1.2 ml. of 6 N sodium hydroxide and then diluting to 50 ml. with glycine "buffer. The mixture is agitated vigorously with the probe of an ultrasonic generator for a total of 10 seconds. A bimultiple water-in-oil-in-water emulsion is formed. Most of the oil droplet diameters are in the range of 1 - 10 microns and at least 70 of these contain inner aqueous water droplets . On centrifuga-tion for 5 minutes at 300 xg. no separate oil layer was detected.

Claims (4)

Having now particularly described and ascertained the nature our said invention and in what manner the same is to be performJBj we declare that what we claim is:

1. An adjuvant composition comprising a vrater-in-oil or a water-in-oil-in-water emulsion, the 011 being a physiologically acceptable oil, characterized by having at least 0.15$ w/v of a hydrated salt of a physiologically acceptable polyvalent metal and a physiologically acceptable fatty acid having from 12 to 2k carbon atoms and an antigenic material present in a sufficient amount for producing immunologically satisfactory antibody formation.

2. A modification of Claim 1, characterized by the fact that the water-in-oil-in-water emulsion also comprises .1 - of a conventional emulsifying agent of the type which favors formation of oil-in-water emulsions .

3. A composition according to Claims 1 and 2, wherein the polyvalent metallic cation is aluminum and the fatty acid is stearic acid, in molar ratio of aluminum of stearic acid of 1:1.

4. A composition according tb Claims 1 and 2, wherein the oil is peanut oil or mineral oil. 5· A composition according to Claims 1 and 2, wherein the antigenic material is inactiviated influenza virus antigen. DATED the day of September,