DE2309329C2 - Use of ethyleneimine solutions as inactivating agents in the manufacture of vaccines - Google Patents

Description

The present invention relates to the use of neutralized ethyleneimine solutions for inactivating insectious virus and bacterial suspensions for the manufacture of vaccines.

It is known that for the production of the Above-mentioned pharmaceutical preparations inakiivierungsformen or means required that on the one hand the Destroy the infectiousness of the viruses or bacteria to be used safely, but on the other hand their antigenic Leave properties completely intact. In order to obtain safely inactivated preparations, this process must be carried out in one First-order reactions proceed, so that the time of complete loss of infectivity is certain can certainly expand. This is the case with many common inactivation methods (heat, UV light) or Inactivating agents (formaldehyde, hydrogen peroxide, 0-propiolactone) are not the case. Other known Substances that do not have this disadvantage are chemically unstable, such as. B. the acylthylenimines (German Patent 10 41 648) or require a longer exposure time and / or higher concentrations, such as. B. Ethyläthylenimin (DE-OS 19 24 303).

It has been found that the use of neutralized ethyleneimine solutions for inactivation of infectious virus and bacterial suspensions for the production of vaccines, whereby the ethylene imine ■)? solutions the suspensions up to a final concentration of ethyleneimine of 0.01 to 04% (vol / vol) be added, is superior to the use of known inactivating agents.

Surprisingly, the ethyleneimine does not show the above-mentioned disadvantages of the previously known Inactivating agent, ethyleneimine is very technical Easily accessible, very stable and easy to store, and allows a quick reaction in a first-order reaction and safe inactivation of infectious microorganisms without impairing their antigenicity or immunogenicity. Due to the short response time at a comparatively low concentration, the inactivation is extremely gentle on the inactivating material. The use of ethyleneimine as an inactivating agent in the manufacture of immunizing substances for pharmaceutical preparations is therefore an enrichment of pharmacy.

Ethylenimine is added to the virus or bacterial suspensions to be inactivated up to final concentrations of 0.01-0.5 (vol / vol) added. Depending on the test conditions, the reaction time is up to a few hours two days. You work at temperatures between 0

and 45 ⁰ C, preferably between 20 and 37 "C,

The virus and bacterial strains inactivated according to the invention with ethyleneimine can be used for prophylaxis and treatment of viral and bacterial infections in both human and veterinary medicine be used.

The virus or bacterial antigens obtained after the use according to the invention can after usual methods and formulated as a solution, syrup, emulsion, suspension, spray, ointment, paste, cream, Lotion, aerosol, tablets, etc. can be administered.

The formulations are produced in a manner known per se, e.g. by diluting the virus or bacterial preparations inactivated according to the invention with suitable solvents and / or inert, non-toxic, solid, semi-solid or liquid Carriers, optionally with the use of emulsifiers and / or dispersants, sprays and propellants and using suitable stabilizers.

Such solvents, carriers, emulsifiers or dispersants are mentioned here:

Water, non-toxic organic solvents or Diluents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g. peanut / sesame oil), alcohols (e.g. Ethyl alcohol, glycerine) glycols, e.g. B. propylene glycol, Polyethylene glycol) and water; solid carriers, such as z. B. natural rock flour (e.g. highly dispersed silicic acid, silicates), sugar (e.g. cane, milk and Glucose); Aluminum hydroxide, polysaccharides (dextrans, agar); Emulsifiers such as nonionic and anionic emulsifiers (e.g. polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, alkyl sulfonates and aryl sulfonates); Dispersants (e.g. Lignin, sulphite waste liquors, methyl cellulose, starch and polyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc, stearic acid and sodium lauryl sulphate).

The following stabilizers are listed:

Amino acids, sugars, proteins, polysaccharides, polyalkylene glycols. These stabilizers can be used in aqueous solution as well as in the lyophilized state can be added.

The virus or bacterial strains inactivated according to the invention or those obtained from them Medicines can be used in the usual way, e.g. B. oral, intramuscular, intraperitoneal. Subcutaneously, locally, especially on all mucous membranes of the human and animal body.

example 1

To a solution of foot and mouth disease (FMD) virus, type C, pH 8.0, in the form of the centrifuged Culture fluid of an infected culture of permanent calf testicle cells, a 2% (vol / vol) neutralized solution of ethyleneimine up to a final concentration of 0.03% (vol / vol). That The mixture was kept at 37 ° C. with stirring. Samples were taken from it after certain times. The inactivation reaction was stopped by adding 10 percent by volume of a 20% sodium thiosulfate solution. The samples taken were then undiluted and diluted in steps of 10 each 10 tissue culture tubes with primary calf kidney cells inoculated in an amount vg / i 0.1 ml. The one with it The virus titers determined showed a decrease with increasing time, that of a first-order reaction corresponded. After 3 hours of exposure, the inoculation of 0.5 ml was no longer infectious verifiable.

The figure shows the course of the inactivation of FMD virus, type C at 37 ° C and pH 8.0. On the The ordinate becomes the logarithm of the infectious units, the abscissa the exposure time in hours specified. Curve 1 shows the course of the inactivation at a final concentration of 0.03% (vol / vol).

A virus solution treated in this way for 4 hours was applied in an amount of 0.1 ml to 20 3-4 day old baby mice intraperitoneally and in an amount of 1.5 ml of 5 Guinea pigs inoculated subcutaneously: all animals showed no signs of disease 3 weeks after the In the serum of all guinea pigs, vaccination was able to neutralize antibodies against the untreated one infectious parent virus can be detected.

The virus solution inactivated in this way showed an equally high complement-binding capacity as that which did not treated, active virus stock solution.

Example 2

In parallel and in direct comparison to that in the example 1, the following substances and concentrations were used instead of 0.03% (vol / vol) ethylene used:

a) 0.05% (vol / vol)

b) 0.05% (vol / vol)

c) 0.05% (vol / vol)

d) 0.05% (vol / vol)

e) 0.05% (Vol / Vol)

Ethylene imine

(see figure, curve 2)

Ethylethyleneimine

(see figure, curve 3)

N-acetylethyleneimine

(see figure, curve 4)

2,2'-dimethylethyleneimine

(see figure, curve 5)

Isopropylethyleneimine

(see figure, curve 6).

The illustration shows the inactivation process. In the case of ethyleneimine (curve 2) there was none after 1.5 hours Infectious virus could be detected more, with the other substances this condition was only after 7-8 Hours or 11 hours.

Example 3

The procedure described in Example 1 was conducted at a reaction temperature of 22 ⁰ C instead of 37 ° C repeated. With the passage of time, there was also a linear decrease in infectivity. After 16 hours, no more infectious virus was detectable.

Example 4

The procedure described in Example 1 was carried out using an FMD virus solution of the type Oi carried out. After a reaction time of 4 hours, no more infectivity could be determined.

Example 5

FMD virus solutions of type C and type Oi were each prepared according to the method described in Example 1 Inactivated for 5 or 6 hours. With the help of the well-known polyethylene glycol process, it became Virus concentrates are produced, of which 1 ml is inoculated intramuscularly to each piglet susceptible to S became. After no Showed symptoms of illness were each the same Parts of the inactivated virus concentrates mixed with an adjuvant and injected intramuscularly into 20 pigs injected

The animals all produced neutralizing antibodies

against both active virus types. 8 weeks after the

ίο all 10 out of 10 animals were vaccinated against the Infection with active FMD virus type C immune to infection with active FMD virus type Oi were immune to 9 out of 10 animals, one animal became ill, however, much milder than the controls without inactivation.

Example 6

The procedure described in Example 1 was used to inactivate the infectious bovine virus

} ° Rhinotracbcitis (IBR) - a representative of the deoxyribonucleic acid containing virus - used instead of FMD virus which contains ribonucleic acid. Here, too, a linear decrease in infectivity titer was found, so that after a reaction time of 6 hours

- ⁵ no more infectivity could be detected. In each case 2 ml of a virus solution treated for 6 hours were injected intramuscularly into 10 guinea pigs. 3 weeks after the vaccination, neutralizing antibodies against the IBR virus were detectable in the serum of these animals.

Example 7

The procedure was analogous to a bacteria suspension of Pasteurella haemolitica with about 10 ° bacteria / ml the method described in Example 1 ethyleneimine up to a final concentration of 0.05% (vol / vol) given. After 14 hours at 37 ° C., the bacterial mass was centrifuged off and stored in sterile, phosphate-buffered Saline resuspended.

A sample of the preparation thus obtained was inoculated on 3 different bacterial nutrient solutions and incubated at S / ° C. Within 6 days there was no Bacterial growth can be detected.

Another sample of this was injected intraperitoneally into 20 mice in 0.1 ml doses. After 24 and 48 For hours the animals showed no symptoms of the disease, however, during 20 hours with the same Untreated bacteria suspension injected animals 15 within 16 hours and 5 within 24

^w hours died.

Another sample of this was mixed with the same volume of adjuvant. With 1.5 ml each of this Vaccines were vaccinated subcutaneously in 5 rabbits. The vaccination was repeated after 14 days. at

■> "'none of these animals showed bacteremia Serum from these animals could be determined with the help of the aggluination test an antibody titer against the same bacterium can be determined, which is then determined after the booster vaccination clearly drowned.

1 sheet of drawings

Claims (2)

Claims; 1, Use of neutralized ethyleneimine solutions to inactivate infectious virus and Bacterial suspensions for the production of vaccines, the ethyleneimine solutions Suspensions up to a final concentration of ethyleneimine of 0.01 to 0.5% (vol / vol!) Added will. _ ι ο 2. Use of neutralized ethyleneimine solutions according to claim 1 for the preparation of Vaccines made from IBR viruses, FMD viruses or Pasteurelia haemolytica bacteria.