BE704138A - - Google Patents

Description

  

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    .Antigen adjuvant compositions ".-
The present invention relates to novel compositions containing a dispersed aqueous phase containing an antigenic substance and a continuous or dispersed oily phase, said compositions each containing at least 0.15% by volume of hydrated reaction products of a metal cation. physiologically acceptable and of a physlologically acceptable fatty acid having 12 to 24 carbon atoms and, if desired, a conventional emulsifying agent *

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   An antigenic substance has heretofore been incorporated into adjuvant compositions in the form of water-in-mineral oil emulsions which are variations of the well-known Freund's adjuvants,

   but using antigens without tuberculosis bacteria in the aqueous phase, as (was the case in the usual Freund's adjuvant compositions,
Problems are posed by the presence of two constituents which are not considered desirable from a medical point of view. One of them is the mineral oil which is not metabolized by the tissues of the organism when an emulsion is injected and the other is the emulsifying agent, mannide monooleate, which can cause tissue irritation, which sometimes tends to promote caroinogenesis and sterile abscess formation.



   U.S. Patent No. 3,149,036 to Woodhour et al. describes a modified emulsion in which the mineral oil is replaced by a triglyceride oil, such as araohide oil, and in which dry or non-hydrated aluminum salts of fatty acids are used. The same emulsifying agent, mannide monooldate, is however retained.

   In fact, as the Applicant has discovered, mannide monooleate is by far the most undesirable constituent; and since glyceride oils are somewhat preferable to mineral oils, the major drawback is

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 EMI3.1
 VOII \ t 1 \ by 'le 1,1 (11100 leH 1.0 (le 111: 11111 itle dextuurn Ol1Clll'O. Andl will note {Ille u: 11:

   Ion G () t.fn8.i..tana 3.ItC01H; 1t31.aa tEttlYt311t8 de Freund ('t.,' I'n 1 t: t ct "lll: v'.t7ns do tvnUttlOilrr les / I! Ulr .1.tln:.; Are from: ypP. ElJu-dww-huiJ 0 '- tien j>; Jv <fiu-duiifi-tiuile-duiis e / JU. Ct The iiiivciition is usni reltttive If a pr <' clùé , czirac- ûthis. et ,, ce "1.! 'we nti lise salts dt ahtminiulI1 hydrlitl8 d; raci-, IL% !, rrls cat., me ai ,, ent stuôilisl, nt to stabilize (1o.11191.0 n8 t - '' ituSlttll ^ t'lli.Qi These fouinions can 6Lre Lrut.5i'oinifos eh ('l! Iuln'l (' tIS cau-d, tr, s-hul1c-ùülS-e & U pur .ri t. ti 011 'SIVItV (.' lii.lE, 'dui, s a phase has <mouse upréc the addition of a brent, "1..ulcdl'i ... nt .. conve-': Ji.'le lu ie; uai <Lit4n 5uppl, l, mcutLiirer. t3ee> sein Ll'lJ1Uluin1ul :! .iy <ir ;,; <;: from aciuen c: rú: J.

   Else! 1i..rt, i il jr i, init.iLI11,! Cut:; tl'3.: Ii.h.tuG.'Ili, de Jels d 'f, lUiuil: 1ur :. Îltdl'FatZ; l d'udue n'JD, la zee (inde addition of salt for T'û: lBl "ort.eri 1 t.111a1U1. Water- in-oil on tJI1Ü: 1i (m elJu-d :. n5-h1! il-ù:!} n-çH.lU: i'ut h .. i h- ce: 3ui: 'c, For 1'.d: 1Nl de cor.:..odi tl, these two ûyya: a dt. '., il.- s4à.onr will be tl.j "I'fSâ G.-n [71'L3I3 ÙLJIW read description by the abbreviations 3It and litJl respectively, ellt. 1.o1" nr' ] I '011 \ mlnj onne the' 'HtUl3ion Sil upriis ni, forlmtaoll, to produce a 0.llIu1- airin MHH, we can use in this ncconde 5tiÀt) u d', 'n <ul- si.fie : 3.irt: a type cl8ss1 nUe of agent <'). Ulil'int cxjuible ¯de f01 "l!, ErUos Pulsions oil-ùfllJ5-l3liU, ph1'Ce rat, yrGiruc-;" rr1, i, tfl : .l..OS1 EH is read phiA5e ti: 3'L'rSt tt dWlD the enu fear tartc.er ure 1lhulnion EI #.

   The ucenta flôJulsifilJl: tl1 c.rectt rzs-

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 ticks include polyoxyethylenesorbitan monooleate, EHE emulsions have more favorable viscosities and also give improved immunological results compared to incomplete Freund and Woodhour emulsions, more particularly in that when antigens are present in the aqueous phase (s) , the formation of antibodies takes place more quickly. In other words, a suitable antibody titer can be reached within days of injection.



   It is often convenient to use more than one mole of stearic acid per polyvalent cation in the preparation of the EH emulsion and then to add an additional amount of polyvalent cation or a conventional emulsifying agent to the second aqueous phase beforehand. to form the EHE emulsion.



   For best results, it is preferable to introduce at least the major part of the antigenic substance with the aqueous phase in the formation of the EH emulsion before emulsifying the emulsion again to produce an EH emulsion. EHE emulsion. If an EHE emulsion is prepared without an antigenic substance, an antigenic substance is simply added to the continuous aqueous phase of the EHE emulsion.



  The results are clearly internal to those obtained by the EHE emulsions according to the invention, in which the antigenic substance is introduced into the aqueous phase with which the EH emulsion is formed. It doesn't mean that

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 the present invention eliminates the addition of antigenic material at both times, but there should be at least a substantial amount of antigenic material in the initial EH emulsion.



   Although not ordinarily preferred, there are certain advantages in a preformed EHE emulsion which can be sold as a stock emulsion to which an immunologist can add the antigenic substance he desires to use.



   The polyvalent metal cations which form the fatty acids can be cations of any physiologically acceptable polyvalent metal.



  Aluminum is preferred, but satisfactory results can be obtained with the ferric ion. It is understood that the basic hydrated fatty acid salts are not necessarily unique pure chemical compounds. As is known, fatty acid aluminum bases, such as aluminum stearates, can be produced in molar ratios which are not whole numbers. Thus, for example, if the ratio is a little greater than 1, it is possible to have mixtures of monostearate and aluminum distearate. This is simply stated in order to demonstrate that the invention is by no means limited to a single, precisely defined, ohimic compound.

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   The particular fatty acids of 12 to 24 carbon atoms to be used according to the invention are not critical and the invention is not limited to the use of a particular acid or mixture of acids. However, because of their easy preparation and their excellent physiological acceptance, palmitic and stearic acids are the preferred acids, especially stearic acid.



   In addition to the stearic and palmitic acids which are preferred, there may be mentioned, by way of example, other fatty acids such as lauric, myriatic, arachidic, behenic, lignoceric, ricinoleic, oleic, eruole, linoleic acids. and the like.



   Physiologically safe glyceride oils are not / more critical, and any acceptable oil can be used, such as peanut oil, peanut oil, soybean oil, cottonseed oil, mars oil, ohaulmoogra oil, olive oil, sesame oil, and coconut oil. Light mineral oils can also be used to form the EHE emulsions of the invention, and they also form part of the invention. In many particular designs a particular oil is used, namely, peanut oil which gives excellent results.



   However, the invention is in no way limited to its uti. lization and any physiological oil can be used.

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 logically acceptable. Of course, mixtures are also suitable.



     @ In addition to the characteristic polyvalent metals, aluminum and iron mentioned above, there may be mentioned other physiologically acceptable metals, such as magnesium, cerium, zinc, lanthanum, bismuth, etc.



   Antigenic substances can be of the most varied types. By way of examples, mention may be made of those derived from viruses, such as antigens of various types of foot-and-mouth disease, etc. Antigens of riokettsiae, bacteria, such as the tetanus toxoid, and of poly- aaooharidea, such as those derived from pneumococci and the like. Tumor antigens are also useful.



    It will be noted that the antigenic substance of pathogers must be a form in which it does not induce mania. serious clinical temptations of the disease the anti-gene material can be killed, attenuated, or otherwise rendered sufficiently benign for practical immunological use.



  The present invention is directed to the preparation of improved EH and EHE emulsions, which are carriers for the slow release of antigenic substances, and an advantage of the invention is that any of the known types of the antigenic substances can be used. Other antigenic substances can be allergens, such as pollens, dust, pellets, extracts thereof, for example the filaments of tex-

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 tiles, house dust, pollen extracts, grass pollens, etc. Some of the antigens fall under the category of poisons, venoms produced by insects or reptiles, such as for example, bee venoms and wasps, poisonous aerpens,

   scorpions and lactrodectus maotans. Another antigenic substance is provided by the stabilizing or preservative substances and an advantage of the present invention is that these acceptable substances do not destroy the stability of the resulting EH or EHE emulsions. As is known, most antigenic substances are proteinaceous in nature, but, of course, there is no limitation here; for example, the polysaccharides of pneumococci and certain other antigenic substances of the nucleic acid type are also covered by the invention and can be incorporated into the EHE emulsions according to the invention.



   The amount of antigenic substance is also not original, but it should, of course, be sufficient to induce a suitable production of antibodies and this amount will be referred to as "amount effective for the production of antibodies". The amounts effective with the various antigenic substances are well known and it is known that in many cases the adjuvants allow a reduction in the dose or a longer duration of the protective levels of the antibody or both. Often the benefits of EHE emulsions do not lie in changes

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 radicals in effective amounts, but in faster antibody development and more desirable physical characteristics.

   To these advantages of EHE emulsions, the process of the invention adds its harmless physiological characteristics.



   An advantage of the present invention is that the amount of hydrated basic salt of a polyvalent fatty acid metal is not critical. However, of course, there must be an amount sufficient to stabilize the emulsions and, in general, the lower limit is 0.15% w / v, calculated on the total volume of the emulsions. Amounts of 0.5% and more give even superior stability and are therefore preferred. Higher amounts of the fatty acid polyvalent metal salt can be used and there is no critical upper limit.

   Of course, however, the amounts need not be so enormous that an emulsion with good fluidity does not result. This is just a practical problem and is not a critical limitation to the process of the present invention. invention.



   Most of the emulsions prepared according to the invention exhibit varying degrees of thixotropy. This is sometimes an advantage because the emulsion does not always have to be in a very fluid state.

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 since, by the effect of thixotropy, injection through a hypodermic syringe causes a temporary lowering of viscosity.



   The hydrated basic fatty acid polyvalent metal salt can be added in a preformed state or the fatty acid can be dissolved in the oil, if necessary with slight heating, and a soluble salt of the metal added, not exerting. no toxic effect, for example, an chloride, nitrate or sulfate, in the aqueous phase usually with a pH adjustment between 4 and 10, which produces considerable amounts of hydroxide, and the formation of the hydrous salt of polyvalent metal of the fatty acid during emulsification of the initial EH emulsion.

   Of course, if the fatty acid salt is to be formed in situ, the fatty acid can also be introduced into the aqueous phase in the form of a salt with a physiologically acceptable cation, such as for example a sodium salt, and one can then add a soluble salt of the polyvalent metal, for example aluminum sulphate. An advantage of the invention is that the process for preparing the EHE emulsion in two stages is not original and this results in simple practical conditions of operation.



   The ratios of the volume of the dispersed aqueous phase (VE1) to the volume of the oily phase (VH) and to the volume of the continuous aqueous phase (VE2) in

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 EHE emulsions prepared according to the invention are not extremely critical and can be varied over a considerable range while maintaining good activity. (In the discussion which follows it should be borne in mind that during the emulsification of the EH emulsion formed first in the continuous aqueous phase there may be substantial migration of the dispersed aqueous phase to the water phase. 'interior of the aqueous phase continues so that, although the initial phase ratio VE1 / VH / VE2 is 25:25:

  50, the final emulsion can have a phase ratio of 10:25:65). In general, the percentage by volume of oil can vary from 5 to 60% with retention of good builder activity, a preferred range being 20 to 30%. If oil contents of less than 5 are used, the volume of adjuvant emulsion necessary for a good immunological response becomes excessively large for injection. If oil contents higher than 60% are used, the viscosity of the final emulsion becomes much too high for injection.



   In the case of EH exhaustions, the VE1 / VH ratio can vary between about 5:95 and 60:40 or more. The ratio is not critical for the use of emulsions as adjuvants but it is simply there. a problem of a practical nature. For very low ratios, aqueous preparations of very economical antigen

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        These may be necessary to provide sufficient antigen mass in a reasonable injection volume. For very high VE1 / VH ratios, the viscosity of the emulsion becomes too high for easy injection. The present invention is not limited to a particular type of agitation.



  However, the Applicant has discovered that the use of ultrasonic agitation gives excellent results in the second stage and is found to be very satisfactory.



   The invention is hereinafter described with reference to a number of typical illustrative examples. Due to the thixotropic nature of most emulsions, it is desirable to characterize viscosities. by a needle viscosity test in which the flow behavior of the emulsions is compared in a manner closely related to what occurs in actual use when the emulsions are injected. The expression “needle viscosity” is understood to denote the viscosity determined by this test, which is carried out as follows:

   
A standard 5 ml hypodermic syringe is fitted with a needle No. 25 for EHE emulsions (or 20 for an EH emulsion), 2.54 m long, filled with the liquid to be tested and fixed in a vertical position with the 'needle down. We place a weight of 2kg on the plunger of the syringe and we measure the time necessary to expel

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 lml of liquid.

   Under these conditions, with needle n 25, it takes 2.6 seconds for 1 ml of water, while it takes 140-150 seconds for 1 ml of Freund's classic incomplete adjuvant, a water-in-oil emulsion containing 50 parts of aqueous phase emulsified in a mixture of 45 parts of Drakeol 6VR mineral oil and 5 parts of mannide monooleate. With the # 20 needle, water escapes too quickly for an accurate measurement, while Freund's incomplete adjuvant requires 2 seconds per ml.



   The following examples illustrate the present invention without, however, limiting its scope.



   EXAMPLE Preparation of an adjuvant composition by the melon process of the invention
The present example illustrates the preparation of a stable adjuvant composition into which the hydrated reaction products of the polyvalent cation and the fatty acid are forced in situ.
A composition is prepared containing the following ingredients!

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 peanut oil 49% by volume stearic acid 1,% w / v aluminum sulfate ootadeoahydrate 0.6% w / v aqueous glycine solution (0.3M) 48.2% by volume.

   aqueous sodium hydroxide solution (6N) 1.2% by volume, Stearic acid is dissolved in peanut oil by heating to 50 ° C., while stirring with a magnetic bar stirrer, and the resulting solution is poured into a Waring mixer. The aluminum sulfate octadecahydrate is dissolved in the aqueous glycine solution with stirring at room temperature.



   The pH of the solution is then adjusted to 7.2 with 6N aqueous sodium hydroxide solution, and the resulting suspension & oily phase are slowly added to the mixer.
Waring.



   The table below represents the properties of the composition prepared above TABLE *. droplets stable in water at ambient temperature yes negative electrical conductivity percentage of aqueous phase produced at 1780 rpm for 45 minutes 0 viscosity * 30 seconds

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   -). the viscosity is related to the time required to expel 1.0 ml of emulsion from a 5 ml syringe through a 2.54 nm hypodermic needle No. 20 using a force of 2 kg.



   EXAMPLE 2 Preparation of an adjuvant composition by the prooédé of the invention
This example illustrates the formation of the hydrated reaction products in the aqueous phase prior to emulsification.



   A composition is prepared containing the following ingredients! peanut oil 50.0% by volume stearic acid 0.98% by volume aluminum sulphate octadecahydrate 0.57% deionized water 46.87% by volume aqueous sodium hydroxide solution (6N) 1.58% by volume Dissolved aluminum sulphate ootadéoahydrate in half the amount of deionized water and the pH is adjusted to 7.08 with aqueous sodium hydroxide solution, then the stearic acid is suspended in the remaining amount of water deionized.

   The suspension of aluminum hydroxide is then added dropwise in a suspension of stearic acid, and the resulting suspension is added dropwise to the peanut oil previously placed in a mixture.

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 geur Waring,
The table below gives the properties of the composition prepared as above,
TABLE II droplets stable in water yes at ambient temperature negative electrical conductivity percentage of aqueous phase produced at 1780 rpm for 45 minutes 0 viscosity 14 seconds
EXAMPLE Use of the hydrated reaction products of the oation,

  magnesium
The present example illustrates the preparation of a stable adjuvant composition using the hydrated reactants of the magnesium cation.



   A composition is prepared comprising the following ingredients: peanut oil 48.0% by volume stearic acid 2.0% w / v magnesium chloride hexahydrate 0.8% w / v aqueous glycine solution ( 0.3 M) 47.4% by volume aqueous sodium hydroxide solution (6N) 1.8% by volume

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 Stearic acid is dissolved in peanut oil by heating to 50 ° C. while stirring with a magnetic bar stirrer and then the resulting solution is poured into a Waring mixer. The magnesium chloride hexahydrate is dissolved in the aqueous / glycine solution and the pH is adjusted to 7.0 with the aqueous sodium hydroxide solution. The resulting suspension is slowly added to the oil phase in the Waring mixer.



   The following table gives the properties of the composition prepared above:
TABLE III droplets stable in water yes at ambient temperature negative electrical conductivity percentage of aqueous phase produced at 1780 rpm for 45 minutes 0 viscosity 28 seconds
EXAMPLE 4 Use of the hydrated reaction products of lauric acid
The present example illustrates the preparation of a stable adjuvant composition using the hydrated reaction products of lauric acid.



   A composition is prepared comprising the following ingredients!

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 peanut oil 48% by volume lauric acid 2.0% by weight / volume aluminum sulphate ootadaoahydrate 11.1% by weight / volume aqueous glycine solution (0.3 M) 46.5% by volume aqueous sodium hydroxide solution ( 6N) 2.4% by volume Lauric acid was dissolved in peanut oil by heating to 50 ° C while stirring with a magnetic bar stirrer, and the resulting solution was poured into a Waring mixer. aluminum sulfate octa-deoahydrate in aqueous glycine solution with stirring at room temperature and the pH is adjusted to 7.1 with 6N aqueous sodium hydroxide solution.

   The resulting slurry is then slowly added to the oil phase in the Waring mixer.



   The table below gives the properties of the composition prepared above.
TABLE IV droplets stable in water at ambient temperature yes negative electrical conductivity percentage of aqueous phase produced at 1780 rpm for 45 minutes 0 viscosity n seconds
EXAMPLE Use of the hydrated reaction products of the ceric cation
This example illustrates the preparation

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 tion of a stable adjuvant composition using the hydrated reaction products of the oeric cation.



   A composition comprising the following ingredients is prepared peanut oil 49% by volume stearic acid 1.0% by weight / volume oeric ammonium sulfate dihydrate Ce (SO4) 2 [(NH4) 2SO4] 22H2O 1.2% by weight / volume aqueous glycine solution (0.3 M) 47% by volume aqueous sodium hydroxide solution (6N) 1.8% by volume
Stearic acid is dissolved in peanut oil by heating to 50 ° C. while stirring with a magnetic bar stirrer and the resulting solution is poured into a Waring mixer.

   The ammonium ceric sulfate dihydrate is then dissolved in aqueous glycine solution with stirring at room temperature and the pH is adjusted to 7.2 with 6N sodium hydroxide solution. The resulting slurry is then slowly added to the oily phase in the Marins mixer,
The table below gives the properties of the composition prepared above:

   

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   TABLE V stable droplets in water yes ambient temperature negative electrical conductivity percentage of aqueous phase produced 0 to 1780 rpm for 45 minutes viscosity 14 seconds
EXAMPLS 6 Preparation of an adjuvant composition according to the invention containing tetanus toxoid in the dispersed aqueous phase
This example illustrates the preparation of a stable adjuvant composition in which the dispersed aqueous phase contains the tetanus toxid.



   A composition is prepared comprising the following ingredients: peanut oil 48.0% by volume stearic acid 2.0% by weight / volume aluminum sulphate ootadeoahydrate 1.1% by weight / volume aqueous solution of glyoine (0.3 M) amounting to 43 Lf units per ml of tetanus toxoid 46.6% by volume aqueous sodium hydroxide solution (6N) 2.3% by volume

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 Stearic acid is dissolved in peanut oil and heating to 50 c, while stirring with a magnetic bar stirrer and the resulting solution is poured into a Waring mixer,

     The aluminum sulfate octadecahydrate is then dissolved in the aqueous glycine solution with stirring at room temperature and the pH is adjusted to 7.0 with 6N sodium hydroxide solution. The resulting solution is then added to the oil phase in the Waring mixer.



   The following table represents the properties of the composition prepared above TABLE VI droplets stable in water yes at room temperature negative electrical conductivity percentage of aqueous phase produced at 1780 rpm for 45 minutes 0 viscosity 30 seconds
EXAMPLE 7 Use of the hydrated reaction products of the ferric cation
The present example illustrates the preparation of a stable adjuvant composition using the hydrated reaction products of the ferric cation.



   A composition is prepared comprising the following ingredients!

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 peanut oil 48% by volume stearic acid 2.0% w / v ferric chloride hexahydrate 1.0% w / v deionized water @ 46.4% by volume aqueous soda (6N) 2.6% by volume On The stearic acid is dissolved in peanut oil by heating to 50 ° C. while stirring with a magnetic bar stirrer, and the resulting solution is poured into a Waring mixer. The ferric chloride hexahydrate is then dissolved in the aqueous glycine solution with stirring at room temperature and the pH is adjusted to 6.8 with 6N sodium hydroxide solution.

   The suspension is then slowly added. resultant mixture to the oily phase in the Waring mixer.



   The following table indicates the properties of the composition prepared above TABLE VII droplets stable in water yes at room temperature negative electrical conductivity percentage of aqueous phase produced 0 to 1780 rpm for 45 minutes viscosity 18 seconds

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   EXEMPT Use of the hydrated reaction products of oleic acid
The present example illustrates the preparation of a stable adjuvant composition using the hydrated reaction products of oleic acid.



   A composition is prepared comprising the following ingredients peanut oil 48.0% by volume oleic acid 2.0% by volume aluminum sulfate ootadecahydrate 1.1% w / v aqueous solution of glyoin (0.3 M) 46 , 6% by volume aqueous sodium hydroxide solution (6N) 2.3% by volume The oleic acid is dissolved in peanut oil by heating at 50 ° C. while stirring with a magnetic bar stirrer and the resulting solution is poured in in a Waring blender. The ootadecahydrate aluminum sulphate is then dissolved in the aqueous glycine solution with stirring at room temperature and the pH is adjusted to 7.1 with 6N aqueous sodium hydroxide solution.

   The resulting suspension is then added to the oily phase in the Waring mixer,
The following table represents the properties of the composition prepared above t

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TABLE VIII stable droplets in water yes at ambient temperature o negative electrical conductivity percentage of aqueous phase produced 0 to 1780 rpm for 45 minutes viscosity 16 seconds
EXAMPLE 9 Use of other sources of metal cation
The present example shows that the ootadecahydrate aluminum sulphate can advantageously be replaced by the hexahydrate aluminum chloride.



   A composition is prepared comprising the following ingredients peanut oil 49% by volume stearic acid 1% by weight / volume aluminum chloride hexahydrate 0.3% by weight / volume aqueous solution of glycine (0.3 M) 48.4 % by volume aqueous sodium hydroxide solution (6N) 1.3% by volume Stearic acid is dissolved in peanut oil by heating to 50 ° C., stirring with a magnetic bar stirrer, and the resulting solution is poured in in a Waring blender. The coated aluminum chloride hexahydrate was dissolved in the aqueous glycine solution with stirring at room temperature and the pH was adjusted to 7.3 with

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 6N aqueous sodium hydroxide solution.

   The resulting slurry is then slowly added to the oil phase in the Waring mixer.



   The following table represents the properties of the composition prepared above:
TABLE IX stable droplets in water yes at ambient temperature negative electrical conductivity percentage of aqueous phase produced 0 to 1780 revolutions / minute for 45 minutes viscosity 25 seconds
EXAMPLE 10 Preparation of an adjuvant composition according to the invention
The present example illustrates the preparation of a stable adjuvant composition in which the aqueous phase is an isotonic phosphate buffered solution,
A composition is prepared comprising the following ingredients:

     peanut oil 49 @ by volume stearic acid 1.0% w / v aluminum sulphate ootadeoahydrate 0.5% w / v

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 isotonic phosphate buffered solution 47.3% by weight / @ volume aqueous sodium hydroxide solution (6N) 2.2% by volume
Stearic acid is dissolved in peanut oil by heating to 50 ° C., stirring with a magnetic bar stirrer, and the resulting solution is poured into a Waring mixer. The aluminum sulfate octadecahydrate is then dissolved in the isotonic phosphate buffered solution with stirring at room temperature and the pH is adjusted to 7.0 with the 6N aqueous sodium hydroxide solution.



   The following table represents the properties of the composition prepared above t
TABLE X stable droplets in water yes at ambient temperature negative electrical conductivity percentage of aqueous phase produced 0 to 1780 rpm for 45 minutes viscosity 31 seconds
EXAMPLE 11 Preparation of an adjuvant composition according to the invention
This example illustrates the preparation

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 tion of a stable adjuvant composition in which the continuous oily phase is soybean oil.



   A composition comprising the following ingredients is prepared: soybean oil 48% by volume stearic acid 2.0% by weight / volume aluminum sulphate ootadéoahydrate 1.1% by weight / volume aqueous glycine solution (0.3 M) 46 , 4% by volume Aqueous solution of sodium hydroxide (6N) 2.5% by volume Stearic acid is dissolved in soybean oil by heating to 50 ° C., stirring with a magnetic bar stirrer and the resulting solution is poured in In a Waring mixer, the ootadéoahydrate aluminum sulfate is then dissolved in the aqueous glycine solution with stirring at room temperature, and the pH is adjusted to 7.0 with the 6N aqueous sodium hydroxide solution.



  The resulting slurry is then slowly added to the oil phase in the Waring mixer.



   The following table represents the properties of the composition prepared above:

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TABLE XI stable droplets in water yes at room temperature negative electrical conductivity percentage of aqueous phase produced 0 to 1780 rpm for 45 minutes viscosity 19 shakes.



   EXAMPLE 12 Use of other sources of metal cation
The present example illustrates the use of aluminum gel to provide the metal cation in the preparation of the adjuvant composition according to the present invention.



   A composition is prepared comprising the following ingredients peanut oil 49% by volume stearic acid 1% by weight / volume glycine 1.1% by weight / volume Suspension containing 0.46g of aluminum gel per 100ml ( prepared by neutralizing the aluminum and potassium sulphate at pH 7 with sodium hydroxide and washing the precipitate with deionized water until the absence of the sulphate ion) 48.8% by volume aqueous solution of solution (6N) 0, 1% by volume

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 The stearic acid was dissolved in araohide oil by heating to 50 ° C. while stirring with a magnetic bar stirrer, and the resulting solution was poured into a Waring mixer.

   Glycine is added to the alum gel suspension and the pH is adjusted to 7.0 with 6N aqueous sodium hydroxide solution. The resulting suspension is then slowly added to the oil phase in the Waring mixer.



   The following table represents the properties of the composition prepared above:
TABLE XII - stable droplets in water yes at ambient temperature or negative electrical wavelength percentage of aqueous phase produced 0 1780 rpm for 45 minutes viscosity 8 seconds
EXAMPLE 13 Preparation of an EHE emulsion using an olassic emulsifying agent
The present example illustrates the formation of a bimultiple emulsion as described by Seifriz, Journal of Physical Chemistry, Vol. 29, page 738, (1925).

   Further, the example shows that the novel adjuvant composition according to the present invention can be emulsified in an aqueous medium without destruction of the initial water-in-oil emulsion, which results in the formation of water-in-oil. bimultiple water-in-oil-in-water emulsion. -

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A composition is prepared comprising the following ingredients Emulsion of Example 1 15% by volume Tween 80 1% by volume isotonic phosphate buffered solution 84% by volume Tween 80 is dissolved in the isotonic phosphate buffered solution with stirring at room temperature with a magnetic bar stirrer.

   The composition prepared as described in Example 1 is then added to the saline solution and the stylet of an ultrasonic generator is inserted 5 mm into the mixture. The mixture is stirred for 5 seconds, repeating stirring four times,
A water-in-oil-in-water multiple emulsion forms. The formation of said water-in-oil-in-water multi-emulsion is confirmed by phase contrast microscopy and interference microscopy.



   EXAMPLE 14
 EMI30.1
 Preparation of an EX ± peanut oil emulsion without conventional emulating agent
The present example shows that it is possible to prepare stable bimultiple active adjuvant emulsions according to the invention in which hydrated aluminum monostearate is used as the sole stabilizing agent.

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   to 1.5 weight per volume.



   In 10ml of glyoine buffer (0.3 M glycine solution at pH 7 containing 0.01% merthiolate), 0.484 g of ootadéoahydrate aluminum sulfate is dissolved and the pH is adjusted to 9.0 with 0.6 ml of 6N aqueous sodium hydroxide solution. 0.88 ml of tetanus toxoid (630 Lf units per ml) is then added and the resulting suspension is diluted to 25 ml with the glycine buffer. In 24 ml of refined araohide oil USP, 1.242 g of stearic acid is dissolved by heating to 60 with stirring. The aqueous phase is added dropwise to the oil phase at 25 ° C. with good stirring to form a water-in-oil emulsion.

   This emulsion is added to 50ml of an aluminum hydroxide suspension prepared by dissolving 0.968g of ootadeaahydrate aluminum sulfate in the 0.3M glycine buffer, adjusting the pH to 9.0 by 1.2ml of 6 N soda and diluting to 50 ml with the glycine buffer. The mixture is stirred vigorously with the stylet of an ultrasonic generator for a total of 10 seconds.



  A two-fold water-in-oil-in-water emulsion was formed containing 6 Mf units of tetanus toxoid per ml. Most of the oil droplets have diameters in the range of 1 to 9 microns and at least 80% of these # contain water droplets. By oentrifugation for 45 minutes at 300 G, no oil layer separation was detected.

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   A first group of mice are immunized with a single subcutaneous dose of 0.5 ml of this emulsion, while the mice of a second group are immunized with a single subutaneous dose of 0.5 ml of aqueous toxoid (6 Lf units per ml). A third group does not receive an injection. After 14 days, all mice received varying doses of tetanus toxin by intramuscular injection as shown in Table XIII.



     TABLE XIII
Survivors / total animals (4 days after toxin)
 EMI32.1
 
<tb> Dose <SEP> of
<tb>
<tb> toxin
<tb>
<tb>
<tb>
<tb> (g) <SEP> 200 <SEP> 100 <SEP> 50 <SEP> 25 <SEP> 12.5 <SEP> 0.1 <SEP> 0.01 <SEP> 0.001 <SEP> 0.0001
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> toxolde
<tb>
<tb>
<tb> aqueous <SEP> 0/10 <SEP> 0/10 <SEP> 2/10
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> emulsion
<tb>
<tb>
<tb> from <SEP> the example-
<tb>
<tb>
<tb> ple <SEP> 1 <SEP> 1/10 <SEP> 2/10 <SEP> 5/10
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> not <SEP> of in-
<tb>
<tb>
<tb> jection <SEP> 0/10 <SEP> 0/10 <SEP> 10/10 <SEP> 10/10
<tb>
 
EXAMPLE 15 EHE emulsion of peanut oil containing no conventional emulsifying agent
An emulsion similar to that of Example 14 is prepared,

     except that in place of 0.88ml of tetanus toxoid, 5.25ml of influenza vaccine, Taiwan aouche in 0.3 M glycine (3280 units agglutinating

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 chicken cells per ml). The final emulsion is a water-in-oil-in-water emulsion containing 200 CCA units per ml, Most of the oil droplet diameter is in the range of 3 to 12 microns and at least 90%. of these contain internal water droplets.



   Twelve mice in a first group each received a daily dose of 0.5 ml of this emulsion, while 12 mice in a second group received one dose! Taiwan aqueous vaccine subcutaneously containing 200 CCA units per ml. After 4 weeks, the mice are bled and the antibody is assayed in the sera by the technique of Salk, J.



   Immunology, 49, 87-98 ('1944). The HI titre of the sera of mice receiving the emulsion of Example 2 is 1280 while that of the mice receiving the aqueous vaccine is 40.



   EXAMPLE 16
Preparation of an EHE emulsion of mineral oil containing no conventional emulsifying agent
An emulsion similar to that of Example 14 was prepared except that 5.25 ml of Taiwan strain influenza vaccine in 0.3 M glycine buffer (3280 units agglutinating chicken cells per ml) was used. ) instead of 0.88ml of tetanus toxoid, and 24ml of mineral oil (Drakcol 6 VR) instead of 24ml of USP peanut oil



   The final emulsion is a water-in-oil-in-water emulsion

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 containing 200 CCA units per ml. Most of the droplet diameters are in the range of 3 to 9 microns and at least 80% of these contain internal water droplets. The "needle viscosity" (see above) is 6 seconds per ml, instead of 2.6 seconds per ml for water. Centrifugation of the emulsion for 45 minutes at 3000 does not produce a layer of water. The detectable oil and foaming can be completely removed by gently stirring.



   A test for the activity of an etteo- adjuvant, killed according to the method of Example 15 gives an HI titre of 1280 o in the mice receiving the emulsion of Example 16 instead of 20 in the mice receiving the vaccine. aqueous.



    EXAMPLE 1? Preparation of an EHE emulsion of mineral oil containing no conventional emulsifying adjuvant
The present example shows that emulsions of stable adjuvants can be prepared in which the aluminum / stearic acid molar ratio is
1: 3.



   In 10 ml of glycine buffer (0.3 M glycine solution, pH 7, containing 0.01% merthiolate), 0.484 g of octadecahydate aluminum sulphate is dissolved and the pH is adjusted. to 9.0 with 0.61 ml of 6N sodium hydroxide solution. Then, 5.25ml of influenza vaocin from

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 strain Taiwan in 0.3N glycine buffer and the resulting suspension diluted to 25ml with glycine buffer.



  1.242g of stearic acid are dissolved in 24ml of light mineral oil (Drakéol 6VR) by heating to 60 ° C. with stirring.



  The aqueous phase is added dropwise to the oil phase with good stirring to form a water-in-oil emulsion.



  This emulsion is added to 50 ml of 0.3 M glycine-sodium hydroxide buffer at pH 9 and the mixture is stirred for 10 seconds with the stylet of an ultrasonic stirrer. A two-fold water-in-oil-in-water emulsion is formed containing 200 CCA units / ml Taiwan influenza antigen, having a pH of 8.4, and a "needle viscosity" of 3. , 6 seconds / ml. By centrifugation for 45 minutes at 300 ° C., no oil layer is formed.



   The adjuvant potency test according to Example 15 gave an HI titre of 1280 for the emulsion and 20 for the aqueous vacoin.



   EXAMPLE 18 Preparation of an EHE emulsion of mineral oil containing no conventional emulsifying agent
The present example illustrates the production of stable emulsions with hydrated ferric salts of stearic acid. The process illustrates the operation with light mineral oil, but the same EHE emulsions can be prepared with the glyceride oils of the previous examples as a substitute.

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 glove lightly oiled with an equal volume of glyoeride oil, As this example relates to a process for preparing the emulsion, the antigen is not deodorized, but of course, an antigen can be added as described in the previous examples.



   In 10 ml of glycine buffer (0.3M glycine solution, pH 7, containing 0.01% merthiolate) 0.234g of FeCl3 is dissolved and the pH is adjusted to 9.0 with 0.6ml of solution. aqueous 6N soda. This suspension is diluted to 25 ml with the glycine buffer. In 24ml of light mineral oil, 1.242 g of stearic acid is dissolved by heating at 60 C with stirring, The aqueous phase is added dropwise to the oily phase at 24 C with good stirring to form a water-in-emulsion. oil.

   This emulsion is added to 50 ml of a suspension prepared by dissolving 0.46 g of FeCl 3 in the 0.3 M glycine buffer, adjusting the pH to 9 with 1.2 ml of 6N aqueous sodium hydroxide solution. and then diluting to 50ml of a suspension prepared by dissolving 0.468g of FeCl3 in 0.3M glycine buffer, adjusting the pH to 9 with 1.2ml of 6N aqueous sodium hydroxide solution and then diluting to 50ml with glycine buffer. The mixture is stirred vigorously with the stylet of an ultrasonic generator for a total of 10 seconds.

   A bimultiple water-in-oil-in-water emulsion forms, most of the diameter of the oil droplets forming

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 found in the range of 1 to 10 microns and 70% less of these contain internal water droplets.



  By centrifugation for 45 minutes at 300 G, no separate oil layer was detected.

 

Claims (1)

CLAIMS 1.- Adjuvant composition comprising a water-in-oil or water-in-oil-in-water emulsion, the oil being a physiologically acceptable oil, characterized in that said emulsion comprises at least 0.15% in weight by volume of a hydrated salt of a physiologically acceptable polyvalent metal and of a physiologically acceptable fatty acid of 12 to 24 carbon atoms and an antigenic substance present in sufficient quantity to produce anticorpa formation satisfactory from an immunological point of view. 2.- A composition according to claim 1, characterized in that the water-in-oil-in-water emulsion also contains 0.1 to 4% of an emulsifying agent of the type which promotes the formation of an oil emulsion. -in-water. 3.- Composition according to either of claims 1 and 2, characterized in that the poly- valent metal cation is aluminum and the fatty acid is stearic acid, the aluminum molar ratio. / stearic acid being 1: 1. 4.- Composition according to either of claims 1 and 2, characterized in that the oil is peanut oil or a mineral oil. 5.- Composition according to either of claims 1 and 2, characterized in that the antigenic substance is an inactivated influenza virus antigen.