DE1960714B2 - Vaccines from a multiple emulsion - Google Patents

Description

There are also known from GB-PS 8 88 180 vaccines which consist of an oil-in-water emulsion, in the continuous phase of which the active ingredient is suspended or dissolved.

In general, oil-containing vaccines in the form of water-in-oil emulsions have the advantage that they do Delay progressive release of the antigens into the organism. In contrast, they also have sewing parts, which sometimes make their use impossible because, on the one hand, the injection of larger oil-containing ones calls for Quantities of reactions in the organism that can have serious consequences, and on the other hand the volume of antigen-containing solution or suspension that can be administered is proportional what limits the use of oil as an excipient for certain vaccines, which only in considerable Concentration can be established excludes.

Surprisingly, it has been found that vaccines in which the active ingredient is not only in the Water-in-oil emulsion in the discontinuous phase according to DT-AS 12 29 677 or only in the continuous aqueous phase of an oil-in-water emulsion according to GB-PS 8 88 180, but both in the dispersed aqueous phase and in the continuous aqueous phase of a multiple emulsion exists, differ considerably in terms of the protective effect and an immunological effect which is greater than the sum of the effect that can be achieved with the known emulsions. Apparently This is not just a combination of the well-known delayed effects of oil-in-water emulsions with a reinforcing effect through the water-in of the importance that one wants to give to the delaying effect.

The technical superiority of a vaccine according to the invention results from the following test report.

Test report

IZs were made containing antigens of various kinds Vaccine preparation made of the following composition:

150 ml aqueous solution of staphylococcal ana

toxin,
150 ml aqueous solution of streptococcal ana

toxin,
200 ml aqueous solution of Welchia anatoxin

(Gas fire anatoxin),
33 ml of a solution of 150 g Pasteurella multo

cida. Type B concentrate per liter of water.
50 ml of a solution of 150 g of llschcriehia coli

(Serotypes 09, 08, 0115, 0117) concentrate

1 water each.

The following vaccines were made from this:
Vaccine I

Multiple emulsion according to the invention with an emulsified primary phase of 100 ml of vaccine

55

reiting, 24 ml emulsifier (sorbitol monooleate) and 276 ml Vasclincöl and a secondary aqueous continuous phase of 800 ml vaccine preparation and 20 ml emulsifier (polyoxyethylene (20) sorbitol monooleate).

Vaccine II

Multiple emulsion analogous to DT-AS 12 29 677 with an emulsified primary phase from 100 ml vaccine preparation, 24 ml of emulsifier (sorbitol monooleate) and 276 ml of petroleum jelly and a secondary aqueous continuous phase of 800 ml of physiological salt solution and 20 ml of emulsifier (polyoxyethylene (20) sorbitol monooleate). More than 100 ml of the vaccine preparation cannot be emulsified in the continuous phase.

Vaccine III

Multiple emulsion analogous to GB-PS 8 88 180 with an emulsified primary phase from 100 m! more physiological Saline solution, 24 ml of emulsifier (sorbitoimonooleate) and 276 ml of petroleum jelly and a secondary aqueous continuous phase of 800 ml vaccine preparation and 20 ml emulsifier (polyoxyethylene (20) sorbitol monooleate).

Rabbits of 2 to 3 kg body weight were given 2 ml of vaccines I, II or III subcutaneously twice vaccinated every 28 days. 15 days after the second vaccination, the antibody titre and the Protection indices (for Pasteurella and E. coli) in the serum of the vaccinated animals. The values given in the table below were obtained.

1 mpf fabric component

Titer
Vaccine I

Protective in the serum

index at

Vaccine Vaccine

Il ul

Welchia antitoxin λ 4th I.E. 0 1. E. 2.5 I. E. Welchia antitoxin β 66 1. E. 5 I.E. 20th I. E. Welchia antitoxin e 3 I. E. 0 I.E. 2 I.E. Anti-streptolysin 0 20th I.E. 2 1. E. 15th I.E. Anti-staphylolysin " 2.9 I. E. 0 1. E. 2 I. E. Anti pasteurella 10 2 5 activity Anti-escherichia coli 2 0 ο (O117) activity

The table shows that vaccines prepared in a known manner from Mehrfsch emulsions have a significantly lower protective effect both individually and with regard to the added protection values, compared to the vaccine according to the invention.

The oil phase of the vaccines according to the invention is a oil or oil mixture of oils suitable for introduction into a living organism, for example from a mineral oil such as paraffin oil, a vegetable oil such as olive oil, peanut oil or nut oil, or one animal oil, such as lanolin, the oil in question preferably being slightly oxidizable or antioxidant, such as vitamin E.

To produce the water-in-oil emulsion can use any emulsifier that is medically or pharmacologically acceptable, in particular a propylene glycol fatty acid ester, sorbitol monooleate, Mannitol monooleate or propylene glycol monooleate or a propylene glycol fatty acid ester mixture.

The water-in-oil emulsion, which is in the oil phase in Solution is brought, can in a weight concentration corresponding to the required H LB value (Balance of hydrophilic substance / lipophilic substance) are present, urr to obtain a stable emulsion, which depends on the selected oil.

The water-in-oil emulsion is generally used with 10 to 40% aqueous phase and 90 to 60% oil phase produced.

ίο To produce the oil-in-water emulsion that the Dispersion of the water-in-oil emulsion in a continuous aqueous phase is used an emulsifier of the oil-in-water type, which with a Drug or a veterinary medicinal preparation is compatible. For example, you can have one Polyoxyethylene acetyl ether, polyoxyethylene sorbitol tristearate or use polyoxyethylene sorbitol monooleate.

This oil-in-water emulsifier is used in continuous aqueous phase in a weight concentration corresponding to the necessary HLB ratio dissolved, obtaining a stable emulsion of this type depending on the oil chosen.

For the preparation of the oil-in-water emulsion, a ratio of 3 to is generally used 8 parts of the aqueous phase to one part of the water-in-oil emulsion.

In order to obtain adequate stability of the multiple emulsion, it is desirable to have the type and select the concentration of emulsifiers with regard to the nature of the different phases for emulsification. This means that according to the invention, the H LB ratio of the emulsifier from 4 to 8 at Water-in-oil emulsions and from 10 to 15 for the oil-in-water emulsion for the presence of other components of the aqueous phase can be selected.

The aqueous phase can, for example, consist of a physiological saline solution which contains the active ingredients contains, consist. Active ingredients are, for example, living microorganisms that are weakened or avirulent Belong to inactivated microorganisms. Extracts of such microorganisms or also from microorganisms whose toxins are modified by chemicals.

So the vaccines according to the invention in the aqueous solutions inactivated bacteria such as E. Coli, Brucella and Salmonella, inactivated viruses such as Newcastle virus and measles virus, or inactivated Toxins such as tetanus toxin, welchiatoxin, staphylococcus toxin, or suspensions of Contain protozoa or leptospira.

According to the invention, it is not necessary that the discontinuous aqueous phase and the continuous aqueous phase contain the same active substances. It is clear that according to the Invention in these two phases different active substances or identical active substances from different ones Can bring concentrations.

To produce the preparations according to the invention, the aqueous phase (s) is (are) first produced if the continuous aqueous phase differs from the discontinuous aqueous phase. Then take the emulsification of the discontinuous-6 _S loan aqueous phase in the oil phase before, in which the water-in-oil emulsifier has been dissolved. The preparation of this emulsion is carried out according to the usual methods for the preparation of emulsions.

The oil-in-water emulsifier is then poured into the The oil phase consists of 600 ml of vaseline oil (medicinal continuous aqueous phase, and book quality), which contains 5% sorbitol monostearate. produces the double final emulsion, whereby the This water-in-oil emulsion is vigorous

Water-in-oil emulsion that has been previously produced homogenize the two ingredients produced. was, emulsified in the continuous aqueous phase 5 2.5% polyoxyethylene-sorbilol-mono becomes. stearate derivative to the 3 liters of inactivated virus

The following examples show several suspensions of Newcastle disease at 37 "C under types of management of the invention explained in more detail. added vigorous stirring.

To make the antigen multiple emulsion,

B e i s ρ i e 1 1 i °, the obtained water-in-oil-

Emulsion with the A. antigen suspension. who manufacture the poly a brucellosis vaccine oxyethylene sorbitol monostearate as an emulsifier

For the production of a brucellosis vaccine according to the hold, vigorously mixed.
The invention is initially a water-in-oil emulsion

by means of an aqueous phase of 300 ml of a suspension 15 B e i s ρ i e 1 3

sion of inactivated Bruceila abortus with a concentration of 400 billion germs per ml. Production of a typhoid vaccine for cats
represents.

The oil phase is made from 700 ml of vaseline oil (Pharmacopoeia for the production of a typhoid vaccine for cats

quality), the 8 percent by weight sorbitol 20 according to the invention is initially a water inmonooleate contains. Oil emulsion with the aid of an aqueous phase of 350 ml

The water-in-oil emulsion is prepared by means of a strong inactivated virus suspension. The oil phase Homogenization of the two components made. consists of 650 ml of good quality peanut oil with 8% 300 ml of the same suspension of sorbitol monooleate are then added.

Brucella abortus, which had previously been used, 25 The water-in-oil emulsion is obtained by vigorously homogenizing the two components with 2 percent by weight polyoxyethylene sorbitol.
oleate derivative, which has an aqueous phase accordingly. This emulsion is inactivated in 5 liters

an emulsifier with an HLB ratio of 15 results in a virus suspension containing 2% polyoxyethylene sorbitol. The mixing takes place at 37 ⁰ C with zu- monooleate, introduced,
addition of 2000 ml sterile physiological serum. 30 The mixture obtained is treated with a Tumix

To the multiple emulsion according to the invention to mixers emulsified and homogenized. For large-scale maintenance, If the emulsions mentioned at the beginning are in a container, a colloid mill is used. wrote water-in-oil emulsion with the aqueous one

Suspension of Brucella abortus and the emulsifier Example 4

vigorously mixed. The mixing ratio is 35 "," ■ ", 1. · ,,

1 liter of oil emulsion to 2 liters of aqueous solution. Man making a Welch.a-Vacc.ne

This gives a stable oil-in-water emulsion, which is excellent for the production of a Welchia vaccine Represents brucellosis vaccine. you first apply a water-in-oil emulsion

Using an aqueous phase of 300 ml concentrated

B e i s ρ i e 1 2 4 ° Welchia anatoxin. The oil phase is from 700 ml

_TJ . ". ....,. Vaseline oil (pharmacopoeia quality) with 7% sorbitol mono-

Production of a virus _{vaccine oleate produced as} an emulsifier.

Newcastle Disease _The Waters_; _n _oil emulsion is vigorous

To prepare an inactivated virus vaccine, homogenize the two components obtained.
the Newcastle disease according to the invention is 45 Then this emulsion in 4 liters of an aqueous first a water-in-oil emulsion with the help of a phase consisting of 49% phosphate buffer solution, 49% aqueous phase of 400 ml by ß-propiolactone in - Concentrated Welchia anatoxin solution and 2% poly-activated virus suspension. oxyethylene sorbitol monooleate introduced.

Claims (2)

Patent claims: 1. Vaccines from a multiple emulsion of the Water-in-oil type, which itself is in a continuous aqueous phase as an oil-in-water emulsion is dispersed, the physiologically active ingredients are contained in the aqueous phase, which is dispersed in the water-in-oil emulsion, characterized in that the physiologically active ingredients also in the continuous aqueous phase of the oil-in-water emulsion are included. 2. Vaccines according to claim 1, characterized in that the volume of the aqueous phase in of the discontinuous water-in-oil emulsion is 10 to 40% and per volume of the oil-in-water emulsion 3 to 8 volumes of the continuous aqueous phase are included. Oil emulsion before but there is an additional one Effect on. The vaccines according to the invention from a multiple emulsion of the water-in-oil type, which is itself dispersed in a continuous aqueous phase as an oil-in-water emulsion, the physiologically active ingredients being contained in the aqueous phase, those in the water -in-oil emulsion, are characterized in that the _: Etiologically ίο active ingredients also in the continuous aqueous Phase of the oil-in-water emulsion are included. The vaccines of the invention are special Easily fluid because the continuous phase is an aqueous phase and the amount of oil is less than with conventional ones Is oil-in-water emulsions. It follows that it is an easily injectable liquid and that the active ingredients, namely the antigens contained in the continuous aqueous phase, very are quickly released into the organism, whereby an immediate effect occurs. In addition, the existence of a second aqueous phase, which is itself in dispersion, enables although it is surrounded by an oily phase, it is a major retarding agent in the release of the active substance to the organism. A single injection prevents the body from building up a higher level of antibodies a relatively long one. Period evoked. In addition, the oily phase has that used The invention relates to vaccines from a multiple emulsion of the water-in-oil type, which itself in a continuous phase is dispersed as an oil-in-water emulsion, with the physiologically active ingredients contained in the aqueous phase, which is in the 3 °, the benefit of an adjuvant effect, both Water-in-oil emulsion is dispersed. compared to the discontinuous aqueous It is already known from DT-AS 12 29 677 to produce such vaccines in which the active ingredient is (the antigen) is in a water-in-oil emulsion which, emulsified in a second aqueous phase, can be injected with ease as a normal one is present. Vaccine in the aqueous phase, but with all the advantages, be especially with regard to the adjuvant effect, a vaccine in an oil phase. The proportions of the active substances introduced into the discontinuous aqueous phase and into the continuous aqueous phase will hang to a fairly large extent Phase existing antigens, as well as against the antigens in the continuous aqueous phase.
So you get a vaccine with the same