SE181858C1 - - Google Patents

Description

Inventor: W Valentine Priority Requested Iran, May 29, 1957 (USA) The present invention relates to a process for the preparation of an unstable, oral administration, administrable poliomyelitis vaccine.

Polio.myelitisNirus has been brought to grow and transferred to joke pato.gen form through the use of various kanda media, sasoni kyeklingloster, rat brain, rat spinal cord and tissue cultures. In general, it can be said that the activity of the virus is stable under freezing conditions in the presence of moisture and that the stability of the activity decreases with it. 'Mande tem.- p.eratur. For example, at about 0 G, poliomyelitis virus usually dies in about 24 hours. At normal refrigeration temperature (approximately 4 ° C), the activity of the virus is less stable than under freezing conditions. Awn .om. poliomyelitis virus is stable in the frozen state, it is mainly inactivated when using a freeze-drying process.

However, there are ten possible routes of administration which can be used in the administration of immunity to poliomyelitis. The most logical and most difficult way is the natural, ie. oral administration, with the use of attenuated joke pathogenic, live virus under quantitatively and qualitatively regulated b.ing conditions. Prior to the advent of the present invention, many obstacles have been necessary to eliminate = this ultimately desired residue could have been achieved. One of the many reasons for oral administration. joke kun.nat used on a large scale in previous challenge polyn vaccination, due to the above described instability ay poliomyelitis virus.

In order to eliminate these responsibilities, a starting number of proposals have previously been made. Among other things, a number of agents for suspending air poliomyelitis virus have been proposed. Polyethylene glycol has been used to suspend viruses, after which the suspension is filled into gelatin capsules. However, it has been found here that this suspension medium does not constitute a suitable carrier, since virus loses its activity in this medium after young, after one month of storage. Attempts have been made to use glycerol, which is a choice of pharmaceutically and biologically stabilizing medium, but this attempt has been successful, which is due to the fact that the glycerol is transferred to gelatin oil in liquid form when the virus suspension is filled into ordinary gelatin capsules.

Live poliomyelitis vaccines have been administered omit by adding a suspension of poliomyelitis virus to milk after the onset of a frozen condition. Attempts have also been made in the past to add viruses in the form of a suspension in polyethylene glycol to capsules for oral administration. All of these known methods require the preparation of the preparation immediately before administration, due to the instability of the virus, whether in milk, polyethylene glycol, other media or in a dry state. Many other proposals have been made, but none of these have made it possible to produce a vaccine which is suitable for use and has shown stable efficacy.

There is therefore an initial need for a carrier or a suspension medium in which the viral activity is stable and which is suitable for encapsulation in gelatin capsules. The medium should preferably be given a dry, straw-hard powder. Prior to the present invention, no such carrier or medium has been encountered and the problem of dispensing, dispensing, storing and administering a poliomyelitis virus vaccine has greatly prevented foam stays, which would allow immunization against poliomyelitis virus.

According to the present invention, a polyneuritis vaccine is prepared which is stable and administrable on the oral route and suitable for encapsulation. This vaccine is based on stratified granulated articles of hydrated gelatin, which in an absorbed state contains a weakened, joke pathogenic poliomyelitis virus. A single invention is prepared by mixing a liquid suspension with a weakened, joke pathogenic poliomyelitis virus in a dose of culture medium with finely divided non-hydrated gelatin having a particle size corresponding to one. sieve with a mesh size of approximately 2,0-0,1 mm (10140 mesh), at a temperature between about 5 ° C and 20 ° C, the ratio weight: vol. the gelatin and viral suspension vary between 10: 1 and 1: 4, whereby the gelatin particles become hydrated and absorb viruses to form a strawable, granulated vacein.

It has been found that a suspension on the flag of the known types of poliomyelitis virus in their culture media can be combined in this way with non-hydrated gelatin to form a hydrated, strawable, encapsulating resin, particulate, granular poliomyelitis virus vaccine, the viral activity of which remains stable for a long time. . Although the vaccine according to the invention is hydrated, it has a dry appearance in the main salt because it is granular and spreadable. This vaccine enables the encapsulation of poliomyelitis virus and the long-term storage of the encapsulated vaccine without affecting the stability of its activity on illegal salt. It is therefore possible to produce an oral poliomyelitis vaccine in large quantities, which do not need to be prepared immediately before administration. The viral activity of this encapsulated poliomyelitis vaccine is stable both at room temperature (ie approximately 20 ° C) and at normal refrigeration temperature (ie approximately 4 ° C) for a period of several months.

In preparing the vaccine of the present invention, a culture medium containing a diluted poliomyelitis virus is mixed with gelatin. by stirring. During stirring, the gelatin is hydrated with the virus-containing culture medium and the virus is absorbed by the gelatin. During hydration, the vaccine develops from being a thin paste to a vaccine, which consists of individual particles, when the hydration is complete. The vaccine thus formed is spreadable and granular and contains a substantially uniform distribution of the virus throughout.

Each edge of virus culture medium can be anyandas according to the invention. Examples include chicken fetus, rat brain and spinal cord (eg frau hamsters, cotton rat, mouse and common rat) and tissue cultures. The virus is grown in any of the known media in a non-pathogenic form and the resulting suspension of virus in water is recovered together with the desired tissue, salts, antibiotics, antifungal agents and the like, and mixed with gelatin in the manner described above. The diluted joke pathogenic poliomyelitis virus used in the invention may have. one titer from 1 to 9 doses of virus per ml. In practice, it usually has a titer radian of about 3 oeh. 8. The most appropriate oral virus dose has a titer between about 4 and 6. After culturing in tissue culture medium as described in the following example (virus medium No. 1), the poliomyelitis virus usually has a titer of about 9, with the most appropriate titer for oral administration is obtained by dilution. After culturing the rat brain and spinal cord medium according to the fattening example (virus medium no. 2), the poliomyelitis virus usually had a titer between approximately 3 and 6.

Gelatin with a particle size between about 2.0 and 0.1 mm mesh size (10-140 mesh) can be used according to the invention. Gelatin with a particle size corresponding to a mesh size of approximately 0.25-0.15 mm (60100 mesh) is most obligatory. Various antibacterial and antifungal agents can be added to the virus-containing medium before it is mixed with gelatin. In the practice of the invention, it has been found appropriate to use, for example, glycerol, a 6-yard alcohol (eg sorbitol, dulcitol or mannitol), nystatin, 13-naphthol, mixtures of the methyl and propyl esters of p-hydroxybenzoic acid (e.g. for example, a mixture-flake consisting of 1-5 parts by weight of the methyl ester per part by weight of the propyl ester) and mixtures of any of these antifungal agents with virus and its culture media. For example, it may be appropriate to add nystatin or any agent having an equivalent effect to the medium used to grow the virus before incubation. After incubation and premixing with gelatin, glycerol or some equivalent agent may be added. Examples of useful antibacterial agents are penicillin, tetracycline, chlorotetracycline, streptomycin and the like. These antibacterial and antifungal agents are absorbed together with the water, virus, etc., which are present in the virus-containing medium, during the hydration of the gelatin. The resulting strawable granulated vaccine can be encapsulated in soft gelatin capsules or hard gelatin capsules using conventional apparatus and procedures. Various diluents may be added prior to encapsulation to further increase the strobility of the vaccine of the present invention. Salunda can be added, for example, starch, sasoin potato starch, milo starch (a kind of corn starch) and ordinary corn starch to facilitate encapsulation in soft capsules.

The amount of gelatin and virus suspension used can vary widely.

For example, it has been found possible to successfully prepare the vaccine of the present invention using 10 parts by weight of gelatin per part by volume of virus suspension (25-75% by volume of glycerol, preferably about 50%). On the other hand, it has been found that it is possible to prepare the vaccine with good results by using 1 part by weight of gelatin per 4 parts by volume of the virus suspension. If the amount of gelatin alias is substantially equal to the above-described ratio of 10: 1, a non-uniform distribution of virus on the gelatin particles arises, since the present water shortage is insufficient to completely wet the surface of all gelatin particles from the beginning. If, on the other hand, the amount of gelatin is collected during the above-mentioned ratio 1: 4, the composition becomes lumpy and wet and dart: 5r, answer to handle. The most suitable ratio between gelatin and poliomyelitis virus of the three types is from 1-2 parts by weight of gelatin per 3 parts by volume of the virus suspension. When a diluent is added to this composition, it can be weighted in an amount of air about 5-20% by weight, depending on the total weight of gelatin and virus suspension. It has been found to be particularly convenient to add the diluent in an amount of about 10-12% by weight.

The present process exhibits several essential features. In order first and foremost to facilitate encapsulation, the gelatin material used should have a particle size corresponding to a mesh weight of approximately 2.0-0.1 mm (10-1mesh). The best results are obtained with gelatin, which has a particle size between about 60 and 100 mesh (0.25-0.15 mm mesh). The temperature of all the constituents must be further regulated in a relatively narrow range from. about 5 ° to about 20 ° C, and the working conditions are such that a temperature from about 3 to about 5 ° C gives optimal results. Finally, it is convenient to add the gelatin in small amounts while stirring to the virus-containing medium. It is also possible to add diet to the gelatin. However, in the case of delta foliage, the diet is necessary to exercise caution by stirring slowly while stirring rapidly to avoid excessive hydration, which results in large gelatin dumps which are incompletely hydrated.

If one does not work at the above-mentioned temperatures, an unsatisfactory product is obtained. If the upper temperature limit (20 ° C) is exceeded, large, sticky, incompletely hydrated particles of gelatin are obtained by alit-rapid hydration. At these lower temperatures, the amount by weight of the large particles which are unsuitable for encapsulation will exceed the amount by weight of fine particles suitable for encapsulation. In fact, completely satisfactory results can only be achieved if a temperature of 10 ° C and below is used. Below the lower temperature limit (-5 ° G), virus-containing solids differ from the liquid phase. It is therefore convenient to work above 0 ° C. If gelatin with a. particle size, which is outside the range 2.0-0.1 nun (mesh size), is used, but it is inappropriate to encapsulate the product odor after hydration.

The invention is further illustrated by the following examples. The experiments described in these examples can of course be varied, for example, by using a chicken fetus for culturing the joke pathogenic, diluted poliomyelitis virus, which is used according to the invention.

Example 1. In the following, (1) a typical method of producing a vavnach culture medium and its use for culturing poliomyelitis virus, type I (SM stain), type lit (TN stain) or type III (Fox strain) and (2 ) a typical method of producing a medium of the mouse brain and mouse spinal cord and the use of this medium for the production of sputum poliomyelitis viruses of aroma types and strains as those indicated in (1) above. These media are referred to in the following examples as virus-containing medium no. viral medium no. 2. (1) Viral medium no. 1.

Growth, of cells from apnjure (see Virology, vol. 2, August 1956, pp. 575 and 576).

Kidneys, taken from rhesus monkeys and cynomolgus monkeys, are cut into small pieces and digested over the surface (approximately 15 hours). 4 ° C with a 0.25% solution of trypsin, after which living cells are recovered. Approximately 150,000 .mu.m of these cells per ml are suspended in a tissue culture medium consisting of: Ox blood serum ultrafiltrate15 parts by volume Hanks balanced saline solution80 Fetal extracts Penicillin - 50 units per ml of liquid 'Streptomycin'. Micrograms per ml of liquid Hanks halanated saline consists of: Nan.8.0 g KCl10.4 g CaCl2 (anhydrous) 0.2 g MgS, 0.7E1200.2 g Na2HPO4 • 2H2O 0.06 g per 1000 ml water KH2PO40, 06 g Glucose1.00 g 0.35 g The resulting cell suspension is introduced into Povitsky flasks of 1 liter each and incubated at 37 ° C. .

Cultivation of viruses.

After the sixth day, the liquid portion of the tissue culture medium is removed from each flask and replaced with 500 ml of tissue culture medium No. 199 (see 'Reagents & Media for Tissue Culture & Virus Propagation', published by Difco, A.f.s.). The contents of each flask are then inoculated with 5 ml of diluted polio-4-- - myelitis virus (see Journal of the American Medical Association, December 1, 1956, p. 1281). The inoculated medium and the cells are incubated at 370 G. The strains are examined to obtain the virulence medium 1 with respect to the activity by titration in tissue cultures. at this time, freeze or store until mixed with gelatin. (2) Virus-containing medium No. 2.

Swiss albina moss is each inoculated with 0.03 ml of diluted virus through the intracerebral canal. When the moss has become paralyzed (paralyzed), the hemispheres and spinal cord are separated from each mouse. A 20% (w / v) suspension of the recovered brain and spinal cord is then prepared in normal saline solution, which is added per ml with 50 micrograms of streptomycin and 50 units of penicillin. The virus-containing medium No. 2 thus obtained can be mixed with gelatin or frozen and stored and stored until it is to be used.

Example 2. A polyamine myelitis virus vaccine of type I (SM strain) was prepared from the following components: Gelatin (60-80 mesh, corresponding to a sieve with a mesh size of 0.25 to 0.177 mm) 50 g - Maize starch 13 g Virus-containing medium No. 137, 5 ml Glycerol37.5 nil The virus-containing medium No. 1 was mixed with the glycerol to a virus suspension. The suspension was then cooled and the gelatin was bathed in carbonated ice and ethanol before being mixed at about 4 ° C. The corn starch was finally mixed with the composition while stirring.

The thus prepared hydrated, sprinkling particulate, granulated vaccine was then encapsulated in hard gelatin capsules and the activity of the vaccine was determined. Examples of representative results obtained are given in the following table: Titers for vaccine activity Time I days 1713 27 ° C 10-4.2 10-441 + 4 ° C -4.3 -4. -4. -42 -15 ° C -4.3 -.2 'Example 3. A type I polioanyelitis virus vaccine (SM stain) was prepared in the same way, sour in Example 2 with the following components: Gelatin (60-80 mesh) 23.8 g % w / w May starch 6.8 g 10% Virus suspension (virus-containing medium no. 2-50% (volume / volume) and glycerol of pharmacopoeia quality —50% (volume / volume) 34 ml%> »All components and the apparatus used was cooled to 4 DEG C. before mixing. The gelatin was slowly hydrated with stirring at low temperature as in Example 2 and when the hydration was complete, the corn starch was added with stirring in three portions. particulate, granulated vaccine with the same effective titer as in, Example 2.

Example 4. Type II poliomyelitis virus vaccine (TN strain) with effective titer and I strobar, particulate, granular form. was prepared according to Example 2 using the following components: Gelatin (60-80 mesh) 56.3 ml 68.8% ((w / w) The hydration temperature was 40 G. When the hydration was complete, the corn starch was added in three portions while stirring.

Example 5. A poliomyelitis virus vaccine of type II (TN sludge) with effective titer and in strobar, particulate, granular form was prepared according to Example 4 with the following components: Gelatin (60-100 mesh, corresponding to a mesh size of 0.25 0.149 (mm) Pharmacopoeia-grade maize starch Virus-containing medium No. 1 Pharmacopoeia-grade glycerol After kneading in the cold (+ 4 ° C), the corn starch was mixed in until separate particles healed, ie until all lumps and similar probes were broken.

Example 6. A poliomyelitis virus vaccine of type III (Fox strain) with effective titer was prepared on the same salt as in Example 2 and using the same population cells (except for the virus), i.e. gelatin, starch, glycerol and medium No. 1 in the same amounts Temp.

Origin. activity Strengthening of virus Virus suspension (virus-containing medium no. 2-50% (v / v) and glycerol of pharmacopoeia quality —50 (v / v) 14.05 g 15.6% (w / w) 14; 05 g 15.6% ( w / w) 70 μg 50 ml 50 ml - - as in Example 2. On this salt is obtained a hydrated, sprinkling, particulate, granular vaccine which, when encapsulated in milked capsules, was found to have an activity, determined by tissue culture titration, of the same size as part according to, example 2 .recover the vaccine.

Example 7. A poliomyelitis virus vaccine of type III (Fox strain) with an effective titer was tiered in the same way and using the same constituents (fora-tom the virus itself), i.e. gelatin, glycerol, starch and medium No. 2 in the same amounts as in Example 3. In this way a hydrated, sprinkling, particulate granulated vaccine was obtained, which was encapsulated in hard capsules. In tissue culture titration, this iris vaccine has the same activity as the pure Example 3 obtained.

Claims (9)

Patent claim: A process for the preparation of a rigid, orally administrable poliomyelitis vaccine, suitable for encapsulation, characterized in that a liquid suspension of an attenuated, joke pathogenic poliomyelitis virus is mixed into its ocular medium with finely divided, joke hypoxicized gelatin having a particle size, a screen with a mesh size between about 2.0 to 04 mm (10-140 mesh), at a temperature between about -5 ° and + 20 ° C, the ratio of weight: volume between the gelatin and the virus suspension varying between. 10: 1 and 1: 4, thereby hydrating the gelatin particles, and absorbing the virus while pouring on a strOhart, granulated vaccine. 2. A process according to claim 1, wherein the components are mixed with each other at a temperature between 0 and 10 ° C, preferably between 3 and 0 ° C. 3. A method according to claim 1 or 2, characterized in that the gelatin has a particle dryer corresponding to a screen with a mesh size between approximately 0.25 and 0.15 mm (60-100 mesh). 4. A method according to any one of claims 1-3, characterized in that the components are mixed with each other with stirring. 5. A method according to any one of claims 1-3, characterized in that the gelatin is added in small portions with stirring to the liquid suspension of the virus. B. A process according to any one of claims 1-5, which may further incorporate an antifungal agent into the mixture, such as glycerol, sorbitol, mannitol, dulcitol, nystalin, methyl and propyl esters of β-hydroxybenzoic acid, finaphthol and mixtures thereof. . 6. A process according to any one of claims 1-6, characterized in that in addition a diluent is incorporated into the mixture, such as corn starch, potato starch, milo starch (a kind of corn starch), cotton flour 81, and ablininjoch corn starch, which contains 1 to 2% mineral oil. 7. A process according to any one of claims 1-7, characterized in that in addition an antibacterial agent is incorporated into the mixture, such as penicillin, streptomycin, chlorotetracycline, tetracycline and mixtures thereof. Method according to any one of claims 1-8, characterized in that the virus used has a titer frail, 1 to 9 doses of virus per ml and preferably from 3 to 9 (loser virus per Request publications: Patent filings from Sweden 123 366, 150947; France 934 928; Germany 908 90 9. Other publications: Phytopathology. 46 (1956), pp. 236--237. C. Stockholm 1962. Hung. Boktr. P. A. Norstedt & Sorier. 020089