US 3755557 A
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United States Patent 3,755,557 SPRAY VACCINES Jan Jacobs, Weesp, Netherlands, assignor to US. Philips Corporation, New York, N.Y. No Drawing. Filed Aug. 27, 1971, Ser. No. 175,778 Claims priority, application Netherlands, Aug. 29, 1970, 7012832 Int. Cl. A61k 9/00 US. Cl. 424-46 ABSTRACT OF THE DISCLOSURE The invention relates to spray vaccines in which the antigen material in a dry form is dispersed in a liquid propellant. The dispersing agent used is not a liquid and at the same time non-ionic compound, but lecithin which is solid and ionic.
The invention relates to spray vaccines for medical and veterinary use.
British patent specifications Nos. 837,465 and 994,734 describe pharmaceutical sprays in which a powdered medicament is dispersed in a liquid propellant. A medicament formulated in this manner may be introduced into the upper respiratory passages through the pharynx in the form of an aerosol by means of an atomizer.
According to both patents, a liquid non-ionic surfaceactive substance is used to form and maintain the dispersion.
Surprisingly we have now discovered that lecithin, which is neither liquid nor non-ionic, may be used for dispersing dry prophylactics (antigens) in a liquid propellant.
The invention relates to spray vaccines for medical and veterinary use which are characterized in that dry antigens are dispersed in a liquid propellant by means of lecithin.
The term antigen is used herein to mean both viral and bacterial antigens. It includes both killed and attenuated living viruses and bacteria and also toxoids.
Bacteria, microplasmata and viruses of various natures may be worked up into vaccines according to the invention. We may mention various influenza strains, such as A Aichi, A Japan, A Hong Kong, A England, B Johannesburg, B Massachusetts, B Netherlands, para-influenza strains such as types 1, 2 and 3. Adenovirus strains, for example types 3, 4 and 7, meashes virus, Poliovirus, tetanus bacteria, diphteria bacteria and whooping cough bacteria, Reovirus, infectious bronchitis virus, bovine viral diarrhea virus, horse influenza virus, Rhino-viruses, rhinopneumonitis virus, Newcastle disease virus, infectious laryngotracheitis virus, canine herpes virus, feline herpes virus, Miyagawanella virus, panleukopenia virus, distemper virus, rabies virus, pseudorabies virus, hogcholera virus, footand mouth disease vircus, vaccinia virus, bue tongue virus, Pasteurella multicida, cocci, such as Staphylococcus aureus, Staphylococcus albus, Streptococcus, Diplococcus pneumoniae, and further Escherichia, for example Escherichia coli, Salmonella, Corynebacteria, Actinobacillus, Hemophilus, Neisseria, Proteus, Pseudomonas, and the like.
The term vaccine as used in this specification includes both monovalent and polyvalent vaccines.
The antigens to be used in the vaccines may be obtained by the methods known for each particular species.
The viruses may be obtained by multiplication on incubated eggs or in tissue cultures, possibly after previous attenuation in similar media or in animals. In general the bacteria are obtained from artificial culture media.
Virulent antigen material may be killed by known means, such as formaldehyde, fi-propiolactone, ultraviolet irradiation and heat treatment.
6 Claims 3,755,557 Patented Aug. 28, 1973 If desired, the antigen material may be purified by conventional techniques.
The antigens must be Worked up in to a vaccine in the dry condition. For this puropse, they are subjected to a freeze-drying treatment, which may be succeeded by a second drying treatment. The second treatment, however, is not always necessary, its use depending upon the conditions in which the material is processed after the freezedrying treatment. If the processing is such as to preclude 0 the absorption of moisture, the second drying treatment may be dispensed with.
The second drying treatment may be eifected by storing the antigens several days in a vacuum over a strongly hygroscopic agent such as, for example, concentrated sulphuric acid, CaCO Na SO silica gel, P 0 and the like. The treatment may be shortened by slightly raising the temperature, for example, to the range of about 35 to 50 C.
The spray vaccines according to the invention may be obtained by dispersing dry antigen material in a propellant by means of lecithin.
Efiiciently the antigen suspension is mixed with lecithin before the freeze-drying process. Thus, intimate mixing is simply achieved. As an alternative, however, the lecithin may be added to the antigen material after the freeze-drying process.
The amount of lecithin required to disperse the antigen material as a rule is at least 1 mg. per ml. of vaccine liquid. To ensure a higher stability, the amount of lecithin is preferably increased to -10 mg. per ml. However, even greater amounts of, for example, mg. per ml. may be used. The upper limit is determined by the solubility in the liquid propellant. For practical purposes the amount of lecithin may be chosen between 1 and 100 mg. per ml. and preferably between about 10 and 100 mg. per ml. Obviously, in achieving a satisfactory suspension stability the amount of antigen material per ml. also is significant. In any case, the amount of lecithin required may simply be determined. For this purpose the antigen material is mixed with the liquid propellant in a ratio which yields the desired concentration whilst adding, for example, 10 mg. of lecithin per ml. of suspension. When a homogenous suspension has been obtained, the time which elapses before the suspended material has deposited is determined. If this time is too short, the stability may be increased by adding more lecithin.
Suitable propellants are the gases which generally are used in pharmacy and which are liquid under pressure at room temperature. Examples of such propellants are: halogenated hydrocarbons, such as dichloro-difluoromethane, dichlorotetrafluoroethane, trichloro-mono fluoromethane, dichloromonofiuoromethane, monochlorodifluoromethane, trichlorotrifiuoro ethane, difluoro ethane, mono-chlorotrifluoro-ethane, hydrocarbons, such as butane, isobutane, propane and the like, or mixtures of these substances. Alternatively, mixtures of gases and gases which are liquid at room temperature under pressure may also be used.
The invention, although of importance for vaccines in general, is of particular importance for vaccines which protect againt infections of the respiratory organs, since especially for this category the use of a vaccine according to the invention enables the antigens to be conveyed to the area of attack of the infection which is to be controlled by the vaccine.
More particularly, the invention is of importance for influenza vaccines, since these aim at the most frequent infection of the respiratory organs.
The invention will be described more fully with reference to the following examples.
V v 3 (l MONOVALENT VACCINE 4,000 embryonated eggs were inoculated by the allantois route with influenza virus strain A Hongkong which was adapted to eggs and mice according to the formula MK2E3M12E'1 (MK'=monkey kidney, E=eggsallantois route, M=mouse). After being incubated at 35 C. for 2 days the eggs were cooled to 4' C. (16 hours) and the allantoic fluid was separated. The volume obtained was 30 litres.
The virus was purified by means of low-speed and highspeed centrifugation at 1,300 and 50,000 g respectively, the latter process being performed in a Sharples centrifuge with a throughput of 1.5 litres per hour. The sediment of the high-speed centrifugation was re-suspended in an isotonic phosphate buffer having a pH of 8.0. At 1,300 g some impurities were still removed by centrifugation. For the purpose of inactivation, 0.03% by weight and subsequently 0.02% by weight of fl-propiolactone was added. The volume, 5 litres, was dialysed against water for three days with the use of a 10-fold volume per litre. The dialysis liquid was renewed once.
To the dialysate soya lecithin in an amount of 4 mg. per 100 CCA of virus (CCA=chicken cell agglutination) was added. The assembly was freeze-dried in a Leyboldt type 904 freeze-drier. The obtained dry material was subjected to a second drying treatment over P in a vacuum, after which the dry substance was suspended in 40% by volume of dichlorodifiuoromethane and 60% by volume of trichloromonofiuoromethane. The resulting product contained 1,500 CCA virus per ml.
(2) BIVALENT VACCINE 4,000 embryonated eggs were inoculated by the allantois route with influenda virus of the strain A Ainchi which had been adapted to eggs according to the formula E After being incubated at 35 C. for 2 days, the eggs were cooled at 4 C. (16 hours) and the allantoic fluid was separated. The volume obtained was 30 litres. The virus was purified by means of low-speed and high-speed centrifugation at 1,300 and 50,000 g, respectively, the latter centrifugation being carried out in a Sharples centrifuge having a throughput of 1.5 litres per hour. The sediment of the high-speed centrifugation was re-suspended in an isotonic phosphate buffer having a pH of 8.0 and containing 0.01 M of citrate. At 1,300 g some impurities were still removed by centrifugation. For the purpose of inactivation first 0.03% by weight and then 0.02% by weight of 8- propiolactone was added. The volume, 5 litres, was dialysed against water for three days with the use of a fold volume of water. The dialysis liquid was renewed once.
3,000 embryonated eggs were inoculated by the allantois route with influenza virus strain B Massachusetts which had been adapted to eggs by the Formula E After being incubated at 33 C. for 3 days the eggs were cooled and the allantoic fluid was separated. The volume was 22 litres. The virus was purified by means of lowspeed and high-speed centrifugation at 1,300 and 50,000 g respectively, the latter centrifugation being carried out in a Sharples centrifuge having a throughput of 1.5 litres per hour. The sediment of the high-speed centrifugation was resuspended in an isotonic phosphate buffer having a pH of 8.0. At 1,300 g some impurities were still removed by centrifugation. For the purpose of inactivation first 0.03% by weight and then 0.02% by weight of B-propiolactone was added. The volume, 3.5 litres, was dialysed against water for three days with the use of a 10-fold volume of water. The dialysis liquid was renewed once.
After the dialysis the two pools were mixed. The ratio of the amounts of virus of strain A and strain B was 3:2.
Per 100 CCA virus 1 mg. of lecithin was added. The assembly was freeze-dried in a Leyboldt type G04 freezedrier, after which the dry substance was suspended in 40% by volume of dichlorodifluoromethane and by volume of trichloromonofluoromethane. The resulting product contained 1,500 CCA of A virus and 1,000 CCA of B virus per ml.
What is claimed is:
1. A spray vaccine for medical and veterinary use consisting essentially of a dry antigen dispersed as a stable suspension in a pressurized liquid form of a propellant gas with lecithin as suspension stabilizer.
2. The spray vaccine of claim 1 wherein the antigen is useful against infections of the respiratory organs.
3. The spray vaccine of claim 2 wherein the antigen is an influenza antigen.
4. The spray vaccine of claim 2 wherein at least 1 mg. of lecithin per ml. is present.
5. The spray vaccine of claim 4 wherein from 1 to mg. of lecithin per ml. is present.
6. The spray vaccine of claim 5 wherein from 10 to 100 mg. of lecithin per ml. is present.
References Cited UNITED STATES PATENTS 3,551,558 12/1970 Takebe et a1. 42446 2,959,325 11/1960 Beard 424-46 3,378,443 4/1968 Cooper et a1 42446 3,594,471 7/1971 Hertzberger et al 424-89 3,038,816 6/ 1962 Drell et al. 252-305 SHEP K. ROSE, Primary Examiner U.S. Cl. X.R.