US 3531565 A
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UnitedStates Patent O STABLE ADJUVANT ,EMULSION COMPUSITIONS COMPRISING HYDRATED SALTS F A POLY- VALENT METALLIC CATION AND A HIGHER FATIY ACID Richard Lansing Webb, Darien, Edward Strazdins, Starnford, Conn., and Victor Jack Cabasso, Moraga, Calif., assignors to American Cyanamid Company, Stamford, Conn., a corporation of Maine Continuation-impart of application Ser. No. 654,987,
July 27, 1967. This application Sept. 25, 1969, Ser. No. 861,131
Int. Cl. C12k 5 00 U.S. Cl. 424-92 6 Claims ABSTRACT 0F THE DISCLOSURE Adjuvant emulsions of the water-in-oilinwater type, particularly those containing antigenic material in one or both water phases, are formed without special emulsfying agents by using only polyvalent metallic cations, such as aluminum, in the form of hydrated salts of fatty acids to form and stabilize the emulsions. The oil phase is a physiologically acceptable oil, preferably a triglyceride oil.
CROSS REFERENCES TO RELATE APPLICATIONS This application is a continuationinpart of our copending application Ser. No. 654,987, led July 27, 1967, now abandoned.
BACKGROUND OF THE INVENTION 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 constituents which were considered medically not desirable. One was that the mineral oil is not metabolized by the body tissues after an emulsion is injected and also that the emulsifying agent, mannide monooleate, could cause tissue irritation, which sometimes tended to favor carcinogenesis.
In U.S. Pat. 3,149,036, to Woodhour et al., a modied 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 disadvantage 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.
An improvement was made on the incomplete Freund composition and the Woodhour emulsions and is described and claimed in the co-pending application of Strazdins and Webb, Ser. No. 447,558, filed Apr. 12, 1965, now Pat. No. 3,399,263, Aug. 27, 1968, and assigned to the assignee of the present application. This application describes a process in which hydrated aluminum salts of fatty acids are the stabilizing agent for stabilizing the ice water-in-oil emulsions, and when this type of emulsion is used, no conventional emulsifying agent is required. There is also described in the application a waterin-oil-in-water emulsion. 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, it was thought necessary to use in this second emulsifying step a conventional type of emulsifying agent which is capable of making oi1-in-water emulsions, because essentially the WO is the disperse phase in water to form a WOW. Typical emulsifying agents described 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. The WOW emulsions of the application, however, still used an emulsifying agent of the sorbitan oleate type; and while this was far less undesirable than the mannide monooleate, nevertheless, even these emulsifying agents are not considered medically ideal, and although the WOW emulsions of the Strazdins and Webb patent represented a great advance and were not medically unacceptable, they still fell somewhat short of the ideal,
SUMMARY OF THE INVENTION The present invention, which may be considered as a specific improvement on one type of emulsion described in the Strazdins and Webb application, produces WOW emulsions with either physiologically acceptable glyceride oils, which is preferred, or with light mineral oils which are not medically condemned and which, although less ideal, are considered acceptable. The WOW emulsions produced by the present invention do not require any added emulsifying agents of the usual types in the second step of making the emulsion. It is stabilized by additional amounts of the hydrated fatty acid salt of the polyvalent cation, such as aluminum or iron which can be added or produced in stages if desired. It is not absolutely necessary physically to add further portions of the salt because it has been found, to everyones surprise, that if, initially, there is enough hydrated basic aluminum stearate or other polyvalent cation salt of fatty acids, the second step of emulsifying the initial WO emulsion with aqueous continuous phase to form a WOW emulsion proceeds. The emulsion has satisfactory stability. The mechanism appears to involve, at least as one factor, a wandering of some of the fatty acid salt from the interfaces between the disperse water globules and the oil in the WO which is produced as the first step and becomes present in sufficient amounts in the interface between the disperse globules of WO emulsion and the continuous water phase in the WOW emulsion. This surprising result was demonstrated by incorporating a small amount of a ferro cyanide in the aqueous phase of the WO emulsion and a small amount of a ferric salt in the aqueous material which formed the continuous phase in the WOW emulsion. The intense color developed showed that there was substantial Wandering or migration of the fatty acid salt, basic aluminum stearate in the test. It is not known why this unexpected result occurred, and the invention is therefore not intended to be limited to any theory of why a portion of the hydrated aluminum salt wanders or migrates from the interfaces in the WO emulsion to the interfaces in the WOW emulsion. Therefore, it is not desired to limit the invention to any particular theory of why the proven wandering or migration takes place. No attempt was made to determine absolute accurate quantitative figures on the amount of migration or Wandering of the basic aluminum stearate, but a semiquantitative estimate can be made that the migration in the test was quite substantial, being of the order of 50%. This percentage may vary somewhat with the intensity of agitation used in producing the WOW emulsions of the present invention and appears to be in no sense critical. While it is definitely known that the unexpected migration of the basic aluminum stearate of the fatty acid salt takes place, it is theoretically possible that some other factors may be playing a part in the very stable WOW emulsions produced by the present invention. Accordingly, it is not desired to limit the invention to a theory that the only factor involved is the migration of the basic fatty acid salt.
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 to the second aqueous phase before forming the WOW emulsion.
The reasons for the markedly increased rate of antibody formations, which are an important factor of the WOW emulsions of the present invention and of the Strazdins and Webb application, have not been deter.- mined, and the invention is therefore not intended to be limited to any theory of why this advantageous practical result occurs. For best results it is, however, preferable to introduce at least a major part of the antigenic material 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 emulsion. The results are markedly inferior to the WOW emulsions of the present invention and the Strazdins and Webb application, 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 acceptable 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 distearate. However, it is customary to refer to the compounds as basic salts, and the above statement is merely made to emphasize that the invention is in no sense critically limited to an exact single chemical compound.
The particular fatty acids from 12 to 24 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.
The physiologically acceptable glyceride oils are also not critical, and any acceptable oil may be used, such as peanut oil, satflower 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 physiologically acceptable may Ibe employed. Obviously, of course, mixtures are suitable. In fact, most of the glyceride oils are mixtures of a number of glycerides, with the possible exception of olive oil.
In addition to the typical polyvalent metals aluminum and iron, referred to above, other physiologically acceptable metals are magnesium, cerium, zinc, lanthanum, bismuth, etc.
In addition to the preferred stearic and palmitic acids, other fatty acids may be mentioned as illustrative, such as lauric, myristic, arachidic, behenic, lignoceric, ricinoleic, oleic, erucic, linoleic, and the like.
The antigenic materials may be of most varied character. Illustrative examples are those derived from virus, such as inuenza virus antigens and antigens of various types of foot and mouth disease, etc. Antigens from rickettsia; bacteria, such as tetanus toxoid; 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 suciently harmless for practical immunological use. The present invention is directed to the improved 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 category of poisons or venoms from insects or reptiles, such as for example the poisons of bees and wasps, cobra venom, antivenins such as those from scorpions, and Lactrodectus mactams. 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. 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 sucient of the metal salt to stabilize the emulsions, and in general the lower limit is 0.05% /weight/volume, w./v., based on the total volume of the emulsion. 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 emulsion 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 that the emulsion always be in thinly uid state since 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, usually with pH adjustment to between 4 and l0, producing considerable amounts of hydroxide which may be present in the desired amounts in the aqueous material which is emulsified 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 as a salt with the physiologically acceptable cation, such as for example a sodium salt. 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 Vwl) to the volume of the oil phase (designated V) and to the volume of continuous aqueous phase (designated Vw2) in the 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-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 Vwl/Vo/Vwg, the final emulsion might have phase ratios of 10/ 25/ 65.) The ranges for the three phases of the final product after wandering or migration fall within the area shown on the drawing in a three-phase diagram. In general it will be seen that the volume percent of oil can be varied from 5% to 65% ywith retention of good adjuvant activity. An oil content of 20% to 30% represents a preferred range. If oil contents below 5% are employed, the volume of adjuvant emulsion that is required for a good immunological response becomes very large for injection. If oil contents greater than 60% are employed, the viscosity of the nal emulsion becomes inconveniently high for 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. Other forms of agitation, such as mechanical homogenizers or emulsifiers, are quite useful. In general these represent agitation methods in which the emulsion is passed through quite small spaces and, of course, results in a very considerable shearing effect. For small scale preparation it is even possible to repeatedly pass the mixture through a narrow bore, hollow needle, but of course on a large scale this is less economical.
BRIEF DESCRIPTION OF THE DRAWING The drawing shows a three-phase diagram with a triangular area which includes the practically useful ranges of the different phases ofthe emulsion.
DESCRIPTION OF THE PREFERRED EMBODIMENTS As can be seen on the drawing, the ranges of the phases, Vw1/V0/Vw2, are represented by the triangular area on the three-phase diagram. The extreme points are 5% Vw1/5% Vo/90% Vwz; 5% Vivi/60% Ifo/35% Vwz; and finally, 33% Vall/32% V0/35% Vwg.
The invention will also 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 compares the flow behavior of the emulsions in a manner more closely related to those occurring in actual use when the emulsions are injected. The term needle viscosity will be understood to mean viscosity determined by this test, which is as follows:
A standard 5 ml. hypodermic syringe is fitted with a 1" long 25-gauge needle, 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, 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 Freunds adjuvant, a waterin-oil emulsion containing 50 parts of aqueous phase, emulsified in a mixture of parts of Krakeol 6 VR mineral oil and 5 parts of mannide monooleate.
yIt will be noted that viscosities are compared in terms of the well-known incomplete Freund adjuvant. It will also be noted that each example is given a title which sets forth the oil used, the antigen, and the mole ratio of the particular polyvalent cation to stearic acid. In the case of aluminum stearate, stearic acid will be abbreviated SA.
Example 1.-Peanut oiltetanus toxoid-Al/SA=1 This example illustrates that stable, active, bimultiple adjuvant emulsions of this invention can be prepared in which 1.5% w./v. aluminum monostearate in hydrated form is employed as the sole stabilizing agent.
In 10 rnl. 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 rnl. with glycine buffer. In 24 ml. of refined peanut oil U.S.P. 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 25 C. with good agitation to form a water-in-oil emulsion. This emulsion is added to 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 80% of these contain inner water droplets. On centrifugation for 45 minutes at 300X 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 14 days, all mice were challenged with varying doses of tetanus toxin by intramuscular injection as shown in Table I.
TABLE I. -SURVIVORS/TOTAL ANIMALS (4 DAYS AFTER TOXIN) Example 2.-Peanut oil-inlluenza virus-Al/SA=1 An emulsion similar to Example 1 is prepared except that 5.25 ml. of Taiwan strain iniluenza vaccine in 0.3 M glycine butler (3820 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 m1. Most of the oil droplet diameters are in the range of 3-12A microns and at least 90% of these contain inner water droplets.
Twelve mice in one group were ea-ch 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, I. Immunology, 49, 87-98 (1944). The HI titer from the pooled sera of mice receiving the emulsion of Example 2 was 1280v while that of the mice receiving the aqueous vaccine was 40.
Example 3.-Mineral oil-influenza virus-Al/SA=1 An emulsion similar to Example 1 is prepared except that 5.25 ml. of Taiwan strain inuenza vaccine in 0.3 M glycine buier (3820 chick cell agglutinating units per ml.) is used in place of 0.88` ml. of tetanus toxoid, and .24 ml. of mineral oil (Drakeol 6i VR) is used in place of 24 ml. of peanut oil U.S.P. The nal emulsion is a water-in-oil-in-Water emulsion containing 2.00 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. Centrifugation of the emulsion for 45 minutes at 300X 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 2 gave an HI titer of 1280 in the mice receiving the emulsion of Example 3 compared to a titer of 20 in the mice receiving the aqueous vaccine.
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 Tai-wan strain inuenza 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 glycinesodium hydroxide buffer and treated for 10 seconds with the probe of an ultrasonic generator. A bimultiple waterin-oil-in-water emulsion containing 200 CCA units/ ml. of Taiwan iniluenza antigen is formed with a pH of 8.4, and a needle viscosity of 3.6 sec./ ml. On centrifugation for 45 minutes at 300X g, no oil layer is formed.
A test of adjuvant activity according to Example 2 gave an HI titer of 1280 for the emulsion and for the aqueous vaccine.
Example 5.-Mineral oil-no antigenFe+3/SA=1 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 lwith 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 Fe'Cls 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 24 C. with good agitation to form a water-in-oil emulsion. This emulsion is added to 50 ml. of a suspension which is prepared by dissolving 0.468 g. of FeCl3 in 0.3 M glycine buier, 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-inoil-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 centrifugation for 45 minutes at 300X g no separate oil layer was detected.
Example 6 The procedure of Example 5 is repeated using a mechanical homogenizer instead of an ultrasonic generator for the linal step. A stable emulsion is produced having the same range of oil droplet diameters.
What is claimed is:
1. A syringeable, thixotropic adjuvant vaccine composition, formed -by vigorous agitation, consisting of a bimultiple water-in-oil-in-water emulsion, wherein the volume percent ratios of dispersed aqueous phase of oil phase to volume of continuous aqueous phase falls within the triangular area of a three-phase diagram of the drawing having apices 515:90, 5:60:35, and 32:33:35, respectively, said continuous aqueous phase being inert or containing minor and insubstantial amounts of antigen, said oil phase being a physiologically acceptable glyceride or light mineral oil in the form of droplets, the droplets having diameters in the range of 1 to 12 microns, at least 70% of the oil droplets containing inner water droplets constituting the disperse aqueous phase, and which inner water droplet disperse aqueous phase contains the major or substantial part of the antibody-forming antigenic material, the composition having at least 0.05% w./V. of hydrated aluminum stearate or hydrated ferric stearate constituting the effective emulsifier and emulsion stabilizer, the composition being substantially free from an effective amount of added emulsifying agents and the composi tion containing antigenic material at least in the dispersed aqueous phase, the amount of antigenic material being sufficient to constitute an effective amount for producing immunologically satisfactory antibody formation.
2. A composition according to claim 1 in which the stearate is hydrated aluminum stearate.
3. A composition according to claim 2 in which the amount of hydrated aluminum stearate is at least 0.25%.
4. A composition according to claim 3 in which the molar ratio of aluminium to stearic acid is approximately 1:1.
l5. A composition according to claim 3 in which the oil phase is peanut oil.
6. A composition according to claim 5 in which the antigenic substance is tetanus toxoid.
References Cited UNITED STATES PATENTS 2,487,600' 11/ 1949I Schneiderwirth 25 2 3 16 2,507,193 5/ 1950 `Buckwalter 424-271 2,529,461 11/1950 Schneiderwirth 252-310 2,661,315 12/1953 Jurist et al 424-271 2,734,844 2/ 1956 Zigler 424-271 XR 3,083,142 2/1963 Howell et al. 424-89 XR 3,096,249 7/1963 Prigal 42.4--7
(Other references on following page) 9 10 UNITED STATES PATENTS 1945, A Simple Rapid Technic of Preparing Water-in- 3149036 y9/1964 Woodho t 1 424.89 Oil Emulsions of Penicillin, Drugs and Biologics (1945). 3 244172 4/1966 Brown lrff 128 218 Brown: Ann. Allergy 2o: 126-132, 3995400, 539-541, 3,399,263 8/1968 Strazdins et aL 424 88 The New Vitellinic or Shielded Type of Injection- 3,;419660i 12/1968 Lannon 424 271 COrrObOrati'on 0f Vitellinic Eifect by Immunologic 5 Methods (1962). FOREIGN PATENTS 1,353,460 ,1/1964 Frane SHEP K. ROSE, Primary Examiner 648,053 11/ 1964 Belglum. US. CL X R OTHER REFERENCES 10 424-88, 89,91
Freund et al.: Science 101 (2627): 46S-469, May 4,