HRP20010300A2 - Pharmaceutically active composition and dispensing device - Google Patents

Abstract

The present invention relates to an improved pharmaceutical substance in addition to an improved dispensing device that allows for more effective nasal administration of a substance.

Description

Various printed works are cited in this application. By referring to them, their content is included in the application.

The proposed invention relates to the composition of a pharmaceutically effective preparation, especially to antigens, which are mixed with a mucosal adjuvant and can be applied to the nasal mucosa with a dosing device, designed as an applicator-sprayer, so that excellent effectiveness is achieved.

In particular, the application includes a vaccine for intranasal administration consisting of:

(a) surface proteins of influenza, liposomally formulated (virosomes);

(b) mucosal adjuvant of bacterial origin;

(c) a specific applicator-sprayer, designed so that almost 100% of the volume of the preparation, injected with one stroke of the sprayer, can be applied to the mucous membrane of the nose, which is essential for its effectiveness.

Antigens are primarily influenza surface glycoproteins, liposomally formulated (so-called virosomes). These virosomes are mixed with a mucosal adjuvant of bacterial origin. Ideally, they come from a class of active or inactive toxins, such as Heat Labile Toxin (HLT), Cholera Toxin (CT) or Procholeragenoid (PCG). The nasal spray-applicator is designed in such a way that most of the volume of the effective preparation can be applied to the nasal mucosa. The formula on which the invention is based can be used for various medical indications, such as vaccination against influenza or other infectious diseases, as well as for therapeutic treatment of blocked nose, reconstitution of damaged nasal mucosa or, in general, for mucosal localized diseases.

Various authors have described the immunostimulating effect of liposomes (artificial membranes) (Gregoriadis, G.: Immunological adjuvants: The role of liposomes. Immunol. Today 11 (1990) 89-97). This immunostimulatory effect is achieved by antigen binding to the liposome surface (Glück R, Mischler R, Finkel B, Que JU, Scarpa B, Cryz SJ Jr: Immunogenicity of a new virosomal influenza vaccine in the elderly. Lancet 344 (1994) 160-163), by inclusion in the interior of liposomes (Gregoriadis G, Davis D, Davis A: Liposomes as immune adjuvants: Studies on antigen incorporation. Vaccine 5 (1987) 145 – 151) or only by mixing with liposomes (De Haan A, Geerligs HJ, Huckshorn JP, van Scharrenburg GJM, Palache AM, Wilshut J: Mucosal immunoadjuvant activity of liposomes: Induction of systemic IgG-.a and secretory IgA responses in mice by intranasal immunization with subunit influenza vaccine and coadministration of liposomes.Vaccine 13 (1995) 613-616). An additional, immunostimulating effect was observed if such liposomes were "spiked" with transmembrane and fusogenic glycoproteins, (e.g. Influenza glycoproteins (Glück, R., Mischler, R., Brantschen, S., Just, M., Althaus, B,. Cryz, S. J., Jr.: A System for the Production of an Immunopotentiating, Reconstituted Influenza-Virosomal (RIV) Vaccine for Hepatitis Immunization. A. J. Clin. Invest. 90 (1992) 2491-2495) or Sendaiglycoproteins (Gould-Fogerite, S., Mazurkiewicz, S. J. E., Bhisitkul, D., Mannino, R. J.: Reconstitution of Biologically Active Glycoproteins into Large Liposomes. Use as Delivery to Animal Cells.Retrieved from C. H. Kim, J. Diwan, H. Tedeschi, and J. J. Salerno (Eds.), Advances in Membrane Biochemistry and Bioenergetics, Plenum Publishing Corp., New York (1987) p. 569).

Until now, this effect has been described in most cases after parenteral administration (i.m., s.c. or i.p.). Some authors have found that such formulations can be successfully administered by conventional methods (a dropper or a conventional nasal spray). However, the results most often refer to experiments with laboratory animals, usually mice, which, compared to their body weight, have a much larger nasal mucosa than humans. In addition, the applied amount of antigen was so high that for economic reasons alone, as well as product safety, application in humans would not be considered. It could also be proven that with a reasonable dosage of the antigen (influenza), despite the liposomal formulation and correct nasal application, a satisfactory effect could not be achieved.

For this reason, other authors tried to develop an effective vaccine for nasal administration, by mixing antigens with a mucosal adjuvant of bacterial origin instead of a liposomal formulation. HLT, CT or non-toxic derivatives of HLT or CT were especially used (Elson CO, Ealding W: General systemic and mucosal immunity of mice after mucosal stimulation with cholera toxin. J. Immunol. 132 (1984) 2736-2741).

After nasal application, the results in the literature really show very encouraging effects, which, again, were primarily achieved on laboratory animals. However, some few human clinical trials have confirmed some efficacy (Tamura S-J, Ishihira K, Miyata K, Aizawa C, Kurata T: Mechanism of enhancing immune responses to influenza vaccine, with cholera toxin B subunit and trace holotoxin. Vaccine 13 ( 1995) 339-341). But again, the amount of antigen and adjuvant used was very large, so that again there was an unsolved reserve regarding the safety of the product and the possibility of its commercialization.

Further disadvantages of the so far described attempts to develop a nasally applicable, preventively or therapeutically effective vaccine are the unsatisfactory devices for administration, which are available today.

Nowadays, there are nasal sprays in the form of mono, di and multi-dose applicators. Extensive research and testing of previous nasal sprays using dye dosing (methylene blue) and nasal endoscopy have shown that only a maximum of 25% of the sprayed preparation (vaccine, pharmaceutical solution) reaches the mucosa of the immune system associated with the mucous turbinalia.

The mucous membrane of the mucosa-associated immune system is located on the lateral nasal wall in the nasal cavity, in the area of the turbinals (nasal turbinates), as, for example, Figure 1 shows the turbinals of humans and deer. Our tests have shown that with the use of nasal sprays to date, due to significant losses in the nasal vestibule (vestibulum nasi) (see Figure 2), as well as on the nasal septum, an insufficient amount of pharmaceutical active preparation reaches the main nasal cavities. These nasal structures are lined with immunologically inactive, squamous epithelium and sebaceous glands (Walter Becker, Hans Heinz Naumann, Carl Rudolf Pfaltz: Hals-Nasen-Ohren-Heilkunde, Thieme Verlag, Stuttgart, New York 1992).

The causes of the inability of the previous nasal applicators to disperse a sufficient dose on the respiratory mucosa (site of specific and non-specific defense) are various:

(1) Due to insufficient knowledge of anatomy, the main vector of the sprayed jet, when applied with a sprayer, is directed in the wrong direction. At the same time, the pharmaceutical active preparation barely reaches the main nasal cavity. The sprayed liquid partially flows out through the upper lip.

(2) The diameter of the part of the nozzle that enters the nose is not adjusted by anatomical conditions. The natural shape of the inner nostril is a crack and has the function of a nozzle (see picture 2). Conventional, wide parts of nebulizers that enter the nose cannot overcome this anatomical narrowing. When spraying in front of the lumen nasi, only that sector of the cone of sprayed liquid that corresponds to the width of the lumen nasi reaches the main nasal cavity.

(3) The optimum scattering angle has not been scientifically investigated so far. With common applicators-sprayers, it arbitrarily varies between 30° and 80° to the horizontal, with a normal head posture, that is, to the perpendicular to the longitudinal axis of the human body. This also explains considerable losses, up to 90%, of the applied volume. Our systematic tests have shown that the optimum scattering angle is between 50° and 80°.

(4) Conventional nasal sprays have too short a part that enters the nose. The distance between the finger cuff and the spray opening is too short. With the current nebulizers, it doesn't even reach the inner nostril. The part that enters the nose should be at least 0.7 cm longer, while a special advantage is achieved at a length of 1 to 1.5 cm.

Therefore, the task of the proposed invention is to prepare an improved composition of a pharmaceutically active preparation as well as to improve a dosing device, especially for the prevention and treatment of infectious diseases. This task is solved by the features of the patent claims.

The most effective composition for preventive and therapeutic action consists of a formulation (formula), a surprising co-stimulating effect of liposomes, mixed with a mucosal adjuvant and applied with a new dosing device, i.e. a new nasal sprayer.

Therefore, the invented complex consists of the following components:

(a) active substances (antigen);

(b) balanced mixtures of liposomes and mucosal adjuvant; and

(c) a new, medical device, namely, a specific dosing device or nasal spray.

The invention specifically relates to a vaccine for intranasal application, which consists of:

(a) liposomally formulated surface proteins of influenza (virosomes);

(b) mucosal adjuvant of bacterial origin; and

(c) a specific applicator-sprayer, designed so that almost 100% of the sprayed volume, created by one stroke of the sprayer, can be applied to the nasal mucosa, which is important for the effectiveness of the vaccine.

Therefore, the active preparation refers to antigens, especially influenza glycoproteins, which, due to their transmembrane domains, can be easily incorporated into artificial membranes (liposomes). However, other antigens can also bind to the liposome surface, alone or in combination with influenza antigens. Binding takes place depending on the chemical composition of the antigen, spontaneously or via a chemical cross-linking molecule, as previously described (Martin F J, Papahadiopoulos D: Irreversible binding of immunoglobulin fragments to previously formed vesicles. J. Biol. Chem. 257 (1982) 286- 288).

It can also be a DNS-plasmid, or an RNS-plasmid, which can code for an antigen and be included in liposomes.

The term "antigen", in the sense of this invention, includes both complete molecules and their fragments that have antigenic properties and/or can be used for immunization. Furthermore, the term "antigen" includes molecules and/or fragments of molecules that have an immunostimulating effect.

The surface protein of influenza, inserted into the vaccine, according to the proposed invention, contains primarily hemagglutinin (HA). Furthermore, the surface protein influenza hemagglutinin (HA) may contain a compound/combination with neuroaminidase (NA).

In a special embodiment, the invention relates to a vaccine that also contains other antigens. In particular, these antigens can be attached to the surface of virosomes.

In a further, preferred embodiment, the Application relates to a vaccine according to this invention, which, in addition to antigens consisting of surface protein(s) of influenza, contains further antigens obtained primarily from pathogenic organisms. The pathogenic organism can be a virus, bacteria, fungus or parasite. These organisms include various pathogens, such as hepatitis A virus, hepatitis B virus, respiratory syncytial virus (pneumovirus), para-influenza virus, mumps virus, Mobili virus, HIV, diphtheria bacterium (Corynebacterium diphtheriae), tetanus bacillus (Clostridium tetani ), pneumococci, haemophilus influenzae, E. coli, Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida krusei, Aspergillus species, Trichomonas species, Trypanosoma species, Leishmania species, Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax , Plasmodium ovale, Plasmodium malariae, species of Trematodes and Nematodes. Trematode species in particular include Schistoma haematobium, S. mansoni, S. japoniaum and Nematode species Tricharis trichiura, Ascaris lumbricoides and Trichinella spiralis.

Furthermore, the invention includes an influenza vaccine. In particular, the invention includes a vaccine that can be used for the prevention and/or treatment of general infections/infectious diseases. Furthermore, the vaccine is used for the prevention and/or treatment of influenza, for the prevention and/or treatment of nasal congestion, and the treatment of injuries to the nasal mucosa. The invention further preferably includes a vaccine in which the hemagglutinin content in a dose (100 μl) is between 1-30 μg, with the preferred concentration being 3-10 μg, and the most preferred concentration being 3.75 μg.

In a preferred embodiment, the invention relates to a vaccine in which the ratio of liposomes-phospholipid to HA is between 1:10 and 20:1, more preferably a ratio of 1:1 to 10:1, and most preferably a ratio of 3:1.

Furthermore, the invention relates primarily to a vaccine in which the phospholipid of the liposome is selected from the group of neutral, cationic and/or anionic phospholipids.

In a preferred embodiment, the invention relates to a vaccine for which the mucosal adjuvant is an active toxin, an inactive toxin and/or a non-toxic toxin. In particular, the mucosal adjuvant comprises Heat Labile Toxin (HTL), Cholera Toxin (CT) and/or Procholeragenoid (PCG). In another preferred embodiment, the mucosal adjuvant is Heat Labile Toxin (HLT) from Escerichia coli. In a further preferred embodiment, the toxins may be inactivated. This deactivation can be achieved by recombination technology.

In a further embodiment, the invention includes a vaccine containing Heat Labile Toxin (HLT) and/or Cholera Toxin (CT) in a ratio between 1:2 and 20:1, better between 1:1 and 1:10 and best 7.5:1 .

The previously mentioned term "balanced mixture of liposomes and mucosal adjuvant" refers, on the one hand, to the correct ratio of antigens and phospholipids, as well as to the mucosal adjuvant, which is required for a satisfactory effect. Clinical tests have shown the following: the ideal ratio of phospholipids (eg phosphatidylcholine, phosphatidylethanolamine, neutral, anionic or cationic phospholipids) to influenza antigen lies between 1:1 and 20:1. The ideal ratio is 3:1. The ratio of influenza antigen and active mucosal adjuvant (toxin) HLT or CT lies between 1:2 and 20:1. The optimal ratio is 7.5:1.

As mentioned before, the ratio of influenza antigen and inactive, mucosal adjuvant (inactive toxin) PCG or non-toxic derivative of HLT and CZ lies between 3:1 and 1:20, ideally 1:2.

Measurements of the immunostimulating mucosal effect in in-vitro experiments unexpectedly showed that the mixture of adjuvant liposomes and mucosal adjuvant does not cause the sum of the effects but an increase of the sum by a factor of at least five.

The term "new medical device" refers to an applicator-nebulizer that is specially adjusted for intranasal immunization (vaccination). It is not enough to put together a mucosally effective preparation if the application is poor or unsatisfactory.

The new device is distinguished by the fact that it fully respects the anatomical features of the nose:

(1) The distance between the finger cuff and the spray head is preferably at least 4.0 cm.

(2) the front part of the attachment, which enters the nose, consists essentially of a cylindrical attachment, which is, for example, at least 5 mm long and at most 5 mm in diameter.

(3) the injection angle of the described dosing device is primarily 50° to 70° to the horizontal.

(4) the axis of the main direction of the dosing device, i.e. the applicator-sprayer, is primarily determined by a special cuff: it can primarily be put on the front part of the dosing device, which is placed in the nose, it is designed so that during nasal spray application it can be relied on the upper lip, so the direction of injection essentially hits the lateral wall of the nasal cavity.

The construction of the cuff for resting on the upper lip is more favorable because the lip is relatively close to the nose, but the distance is large enough to be an effective lever for creating a useful alignment. In addition, sensitive touch sensors on the user's lips enable correct (centered or parallel) resting of the cuff, without checking by looking in the mirror. In addition, the cuff can be designed to rest on other parts of the body, i.e. the head.

The narrow part, which is inserted into the nose, preferably has a minimum length of 0.5 cm, preferably 1 cm, while the next, wide part, which is inserted into the nose, is preferably at least 1 cm long, while a length of 2 cm is preferred. The distance between the dorsal surface for the fingers and the tip of the sprayer should be at least 2 cm, preferably 2.5 to 3 cm, that is, the distance between the finger rest (cuff) and the tip of the sprayer should be at least 3 cm, preferably 4 to 5 cm. The spray angle of the atomizer should preferably be 50o to 70o to the horizontal, if the head is held in a normal, vertical position.

Furthermore, the main stroke vector of the nebulizer should be directed between the middle and lower nasal concha. This means that it must be aimed at the middle, nasal passage. As a rule, this assumes an almost horizontal direction of the applicator.

Such a direction, as described in the proposed invention, can be fixed by attaching a special cuff to the applicator. The cuff and the applicator can also be made as a separate part or integrally. In this case, the attachment/connection device is firmly connected to the applicator or is made in one piece with it.

In a particular embodiment of the applicator-sprayer, it is a device whose part that enters the nose together with the spray head has primarily a maximum width of 5 mm and a length of at least 5 mm, preferably 7 mm, a distance between the finger cuff and the tip of the sprayer of at least 3 cm and mounting cuff, which allows introduction into the nostril so that the main sector of the nasal spray makes an angle of about 15 to 20o with the horizontal.

In a further, preferred embodiment, the antigen is a mixture of influenza surface antigens that is present on the surface of liposomes. This agent can be combined with antigens of other pathogenic microorganisms. As already mentioned above, the vaccine that is the subject of this invention may contain further antigens, primarily antigens of other pathogenic organisms.

In a further embodiment, the aforementioned procedure is used for the prophylaxis of infectious diseases, for the treatment of blocked nose and various disorders of the nasal mucosa.

Next, preferred embodiments of the invention will be described with reference to the images. Shown on:

Figure 1: a comparison of human and doe turbinals;

Figure 2 is a schematic view of the anatomical internal structure of the nostril;

Figure 3 is a schematic representation of the principle of construction of an embodiment of the dosing device according to this invention, which is preferred;

Figure 4 shows the correct angle of spraying during effective application;

Fig. 5 presentation of nasal cytology: quantification of different populations of cells obtained by nasal swab technique from individuals (20 from each group) up to 29 days after three different types of intranasal vaccination. Pay attention to the clear increase in centroblasts, as a sign of a local, immune response in group a, against a significantly weaker increase in groups B and C (p< 0.005);

Fig. 6 is an embodiment of a cuffless dispensing device according to the present invention, which is preferred i

Figure 7a and 7b are two projections of the preferred embodiment of the cuff, on the dosing device, in Figure 6, which is the subject of the proposed invention

Example 1

Preparation of a mucosal virosomal vaccine against influenza with HLT as an additional mucosal adjuvant

The preparation of a virosomal vaccine against influenza is described by Glück R, Wegmann A: Liposomal production of influenza antigen. In eds Nicholson KG, Webster RG, Hay AJ, handbook of influenza (1998) p. 400-409. London, Blackwell. Briefly, it covers the preparation of: H1N1 A/Singapore/6/86, H3N2 A/Wuhan/359/95 and B/Beijing/184/93 strains of influenza virus cultured in hen's eggs, laid by hens, and supplied by National Institute of Biological Standards and Control, London, Great Britain. Intact virions were isolated from chorioallantois fluid by zone centrifugation and inactivated with β-propiolactone. Cleaned virions were placed in a buffer containing 0.1 m octaethyleneglycol mono(N-dodecyl) ether (OEG) (nikko chenmicals, Japan) in PBS-NaCl and incubated for 20 min at 21°C, to allow complete degradation of the components viruses.

The mixture was centrifuged for 60 min. At 100000 x g due to hemagglutinin (HA) and neuraminidase (NA) extraction. The residue, which contained HA, NA and viral phospholipids (PL), was used to prepare various intranasal vaccine formulations. Supplemental phospholipids (phosphatidylcholine [Lipoid, Germany]) were added and converted to a soluble form. Virosomes were created spontaneously, during the removal of OEG-detergent by chromatography. In one mucosal dose of the vaccine (100 μl), which contains 3.75 μg of HA from each of the three influenza virus strains, as recommended by the WHO and 35 μg of lecithin, 0.5 μg of heat-labile toxin was added as a mucosal adjuvant. (HLT) from E. coli, obtained from the production strain E. coli HE22VK (Vogel FR, Powell MF: compendium of vaccine adjuvants and adjuvants. In Vaccine Design: the subunit and adjuvant approach (ed. M.F. Powell, M.J. Newmann) Plenum Press , New York (1995) p. 141).

Example 2

E. coli cells were sedimented in a flow centrifuge (Westfalia AG) and suspended in "saline" buffered with phosphate buffer (PBS, 6.06 g/l Na2HPO4 1.46 g/l KH2PO4, 2.4 g/l NaCl, pH 7.4)

Release of intracellular heat labile toxin (HLT) occurs by cell disruption in a ball mill (eg Dyna-Mill, W. Bachofen AG) or in a French press. For example: 10 l of cell suspension is pumped through a ball mill at a flow rate of 33 ml/min. It is filled with 500 ml of glass beads, rotates at a speed of 3000 rpm and the size of the opening, in the form of a slit, is 0.05 mm.

The solid components of the cell in the solution of the destroyed cells are separated by tangential microfiltration. For example: 10 l of cell lysate is evaporated to about 4 l in a simple system (Millipore AG), using a filter with a pore size of 0.2 μ. After that, it is washed with 24 l of PBS. The permeate is filtered through a sterile filter, pore size 0.2 μ (eg Gelman Supor DCF, Pall).

HLT is isolated from the permeate using affinity chromatography. Immobilized galactose (Galactose-Gel, e.g. from Pierce) or immobilized lactose (Lactosyl-Gel, e.g. from Pharmacia) is used as a stationary phase. The mobile phase consists of PBS (buffer a) and 5% (w/v) lactose in semi-concentrated PBS (buffer b). For example: a sterile filtered cell lysate is applied to a column, preconditioned with buffer a. The column is then washed with buffer a until the amount of UV absorbance at 280 nm reaches the baseline. After that, HLT is eluted from the column using buffer b.

Example 3

Preparation of a mucosal, virosomal vaccine against influenza with pcg as an additional, mucosal adjuvant

The preparation of the virosomal influenza vaccine is described in Example 1. To each mucosal dose of the vaccine (100 μl), containing 3.75 μg ha of each of the three strains of the influenza virus, as recommended by the WHO and 35 μg lecithin, 6 μg procholeragenoid was added, prepared by heating purified CT, which originated from V. cholerae (Inaba 569b), as a mucosal adjuvant (Pierce NF, Cray WC, Sacci JB, Craig JP, Germanier R, Fürer E: Procholeragenoid, a safe and effective antigen for oral immunization against experimental cholera. Inf. Immunity 40, 3 (1983) 1112 – 1118).

Example 4

Preparation of mucosal vaccine against hepatitis B-(HB)

Phosphatidylethanolamine (R. Berchtold, Biochemisches Labor, Bern, Switzerland) was dissolved in methanol and 0.1% (vol/vol) triethylamine was added. Subsequently, the solution was mixed with γ-maleimidobutyric acid N-hydroxysuccinimide ester (gmbs) (Pierce Chemical Company, Rockford, IL) (phosphatidylethanolamine : gmbs = 2:1 ratio) previously dissolved in dimethylsulfoxide (dmso). After a 15-minute incubation at room temperature, the solvents were evaporated for 1 hour in a vacuum, in a Speedvac centrifuge.

Recombined HB particles were prepared in CHO cell lines and purified according to known procedures.

In order to obtain a reduced HB particle with free cysteine residues, the particles were treated for 5 minutes at room temperature with 40 mmol/l dl-dithiothreitol (dtt). Dtt was removed on a Sephadex-g10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) and octaethylene glycol (Fluka Chemicals, Switzerland) (OEG) was added at a final concentration of 100 mmol/l. Then the evaporated phosphatidylethanolamine-gmbs was mixed for 1 hour with the HB solution (ratio of HB : gmbs = 1:2). Free gmbs is bound by cysteine. The reaction was monitored by thin layer chromatography.

32 mg of phosphatidylcholine (Lipoid BmbH, Ludwigshafen, Germany) and 6 mg of phosphatidylethanolamine were added to the previously cross-linked phosphatidylethanolamine-gmbs-HB and this mixture was dissolved in a total volume of 2.66 ml of PBS containing 100 mm of dissolved oeg (pbs -oeg).

Influenza A/Singapore hemagglutinin was purified as previously described in example (1). The solution, which contained 4 mg of hemagglutinin, was centrifuged for 30 minutes at 100,000 g and the sediment was dissolved in 1.33 ml of PBS-oeg.

Phospholipids and hemagglutinin solution were mixed and sonicated for 1 minute. The mixture was then centrifuged for 1 hour at 100,000 g and the upper layer was sterile filtered (0.22 μm).

Virosomes with absorbed HB were removed by detergent removal using BioRad-SM-Bio beads, as in example 1. To the stock solution, which contained 50 μg of HB-antigen/ml, 10 μl/ml of HLT from example 2 was added and filled into applicators-sprayers according to example 6.

Example 5

In vitro test for testing mucosal adjuvant activity

The biological activities of mucosal adjuvants were measured with influenza virosomes, HLT, pcg, a mixture of virosomes and HLT, according to example (1) and a mixture of virosomes and pcg, according to example (2). The following solutions were added to Y-1 adrenal cells ATCC: CCL 79 Y -1- Adrenal tumor, mouse steroid secretion) in miniculture according to Sack, da and Sack, RB (Sack DA and Sack RB: enterotoxigenic assay Escerichia coli using Y-1 adrenal cells in miniculture. Infect. Immun. 11 (1974) 334-336):

(a) HLT (5 μg/ml), (b) pcg (60 μg/ml), (c) influenza virosomes and HLT: 37 μg/ml influenza hemagglutinin from each of the 3 strains (H1N1, H3N2, b), 100 μg/ml phosphatidylcholine and 5 μg/ml as (d) influenza virosomes and pcg: 37 μg/ml influenza hemagglutinin from each of the 3 strains, 100 μg/ml phosphatidylcholine and 60 mg/ml pcg.

The following adjuvant activities were determined (expressed in units):

(a) 15 units, (b) 6 units, (c) 80 units, (d) 36 units. The highest biological activity of the adjuvant was achieved with a mixture of virosomes and HLT or virosomes and pcg. The effect of the adjuvant is determined by the parameters of cell toxicity.

Example 6

Clinical evaluation of different applicators-nebulizers

The basic performance of the dosing device, according to this invention, is essentially comparable to conventional applicators-sprayers, but is particularly well adapted to the anatomical data for nasal application of any, but especially the pharmaceutically effective preparations described here.

The particular embodiment of the proposed dosing device 2 is shown in Figures 3 and 6. On the side view of the dispenser according to this invention, shown in Figure 3, the essential component parts of that device (applicator-sprayer) 2 are recognizable. The dosing device 2 essentially contains a container 4 with (not shown here) a pharmaceutical active substance and a sprayer, i.e. a pumping mechanism 6. The pumping mechanism 6 is connected, impervious to liquids, to the container 4 by a connecting element 8. Pumping and spraying by the dosing device 2, according to this invention, takes place according to the usual way, by pressing the cuff 10 with a finger. The pharmaceutical preparation from the container 4 passes through the channel 12, which extends through the pump mechanism, and is dispensed at the outlet 14.

The dosing device 2 differs from known applicators-sprayers in particular by the design of the shape of the nose element 16, which plays an essential role in the effective application of the pharmaceutical preparation. The nose element 16 is primarily constructed as an integral part of the pumping mechanism 6 and the finger cuff 10. This element 16 is particularly divided into two sections 18 and 20, whereby the section 18, which lies in the direction of the cuff 10, has a larger diameter than the section 20, which is located next to the outlet opening 14. The transitional part between both sections, 18 and 20, forms a stop 22 for the cuff 24, which can be attached in one preferred embodiment of the proposed invention - to the section 20 of the nasal element, due to its reliance on the user's upper lip.

The section 20 of the nose element 16, which lies up to the exit opening 14, is primarily made essentially cylindrical, with a diameter of d20 max. 5 mm, and preferably with a diameter between 2 and 4 mm.

The length I20 of the front section 20 of the nose element 16 is, for example, at least 5 mm, in particular it has a length of at least 10 mm, and it is particularly preferred that it has a length of 10 to 20 mm. The cuff 24 can be moved up via the section 20 of the nose element 16, primarily from the outlet opening 14, and has the advantage that it is firmly connected to the dosing device 2 by means of the unscrewing block. The unscrewing block can be, for example, realized in the form of a polygonal profile, e.g. Elliptical section, on the front section 20 of the nose element 16 and the corresponding protrusion 26 on the cuff 24.

According to another embodiment of the dosing device 2, according to this invention, the section of the applicator-sprayer, which enters the patient's nose, can also be performed on the cuff 24. Such an embodiment is shown, for example, in Figure 7b. As this picture shows, a tubular extension 28 is essentially constructed on the cuff 24, which can be pushed onto the section 20, next to the exit opening 14, of the nose element 16. The dimensions of the tubular extension 28 correspond primarily, in this case, in essential dimensions before of the described embodiment (d20 and I20).

The section 18 of the nose element 16 primarily has a length I18 of at least 10 mm, a length of about 20 mm is preferred. The dimensions of both sections 18 and 20 are primarily chosen so that the distance between the dorsal surface for the user's fingers and the nozzle of the sprayer, i.e. the exit opening 14, is at least 2 cm, preferably 2.5 to 3 cm. This is especially guaranteed when the distance between the cuff 24 and the tip of the sprayer 14 is at least 3 cm, and especially 4 to 5 cm.

According to this invention, using a dosing device 2 of these dimensions, the pharmaceutical active preparation can be injected more precisely into the main nasal cavity, i.e. into the inner nostril, so that a significantly higher effectiveness is achieved than with known systems.

The dosing device 2, according to this invention, has the good side that it is provided with a cuff 24, so that, in addition to more favorable dimensions, the optimal injection angle α can be set. However, the cuff 24 can, independently of the previously described applicator-sprayer 2, be applied with conventional dosing devices, so that even with such applicators-sprayers, significantly better effectiveness can be achieved on the basis of the precisely adjustable spraying angle α.

With the dosing device 2, according to the present invention, an improved efficiency can be achieved, either by choosing the dimensions of the nose element 16 or by means of a suitable cuff 24 to adjust the optimal spraying angle α. In order to achieve the best results of the treatment, preference is given to the combination of a new type of dosing device 2, with the previously mentioned dimensions, and a cuff 24 for setting the optimal spray angle α.

The cuff 24 will be, as already briefly explained, put on the section 20 of the nose element 16 with a tubular extension 28. When mounted, the cuff 24 has the good feature of being connected, securely against unscrewing, to the dispensing device 2. For example, the unscrewing block can be, at least on one side, a flattened circular profile on the nose element 16, with which the cuff 24 can, through a suitably shaped opening, connect securely against unscrewing. The cuff 24 has a resting section 30, which can rest against the user's upper lip, in order to define, together with the section 20 of the part of the nasal element 16, and which is directed into the nasal opening of the patient, the dispersion angle α. The optimal scattering angle lies primarily between 50° and 70°.

The resting section 30 of the cuff 24 is primarily slightly bent, corresponding to the shape of the upper lip, in order to have an optimal resting and supporting surface. The cuff 24 can be made from any suitable material, but preferably from plastic materials, which can be molded by injection molding.

Different dosing devices were tested to evaluate the effectiveness. For this purpose, a 1% solution of methylene blue was filled in five different types of nasal applicators:

(a) Applicator-sprayer according to the present invention with a directional cuff that fixes the direction of travel of the sprayer;

(b) An applicator-dispenser according to the present invention, without a guiding cuff;

(c) A conventional commercial spray applicator without a narrow part, inserted into the nose, 2 cm in length, 4 mm in diameter and a spray angle of 50°;

(d) Applicator-sprayer according to this invention but with a spray angle of 50°;

(e) Applicator-spray according to the present invention but with finger cuffs, which are only 3 cm away from the spray head.

All applicator-sprayers were so-called double-dose applicators. Each applicator-sprayer was tested on 5 subjects under medical supervision. Subjects injected one dose of methylene blue solution into their right and left nostrils. Using nasal endoscopy, the mucous turbinalia (mucous shells) were illuminated and photographed. The intensity of the blue coloration, as well as the blue-colored surface of the turbinalia, was evaluated on a scale from 1 to 4. The following result was obtained (geometric mean):

Group (a): 19 points; group (b): 16 points; group (c): 7 points; group (d): 9 points; group (e): 12 points.

The sprayed liquid was almost completely applied to the nasal mucosa only in group (a). In group (b) too, most of the sprayed content reached the mucous membrane. In the other groups, a large proportion of the sprayed content reached the vestibule of the nose. The liquid sprayed there, which needs to be applied, cannot be effective.

Example 7

Comparison of the clinical efficacy of an intranasal, virosomal influenza vaccine, with and without HLT, administered by the novel nebulizer device described in the proposed invention, with a parenteral, commercial influenza vaccine in volunteers

An open, randomized clinical trial was conducted in full compliance with the principles of the Declaration of Helsinki and with local laws and guidelines that apply to this type of trial. After the protocol was approved by the Ethics Committee of the Canton of Lucerne and notified to the Swiss Federal Health Authority (Schweizer Bundesgesundheitsbehörde), 80 volunteers (18–64 years of age) gave their written, informed consent to participate. Volunteers with an acute or chronic disease at the time of immunization were excluded, if they were simultaneously treated with immunosuppressive drugs or had a known immune weakness.

Intranasal vaccine formulations were administered to each of three groups of 20 volunteers (Table 1). Groups A and B received 2 doses of formulation A and B, respectively, in each nostril on day 1, and 2 doses one week later. Group C received two doses with twice the concentration of formulation A on day 1. Group D was vaccinated intramuscularly, in the area of the deltoid muscle, with a parenteral formulation. Blood and saliva samples (Omnisal®, UK) were taken immediately before the first vaccination and one month after the first immunization (29 ± 2 days). Due to the technical problem, only the saliva samples of the first 47 people could be evaluated. Cytology with a brush of the nasal cavity was performed before immunization and on the 4th and 8th day and one month after the first immunization.

Blood and saliva samples were coded for analysis.

The serum immune response to the HA component of the vaccine was determined by a standard test, with the use of four hemagglutinin units of the corresponding antigens. Before the test, the sera were heated for 30 minutes at 56°C. Titers are expressed as the reciprocal of the highest diluted serum, which completely prevented hemagglutination. A titer ≥ 1:40 was considered protective.

Total and influenza-specific IgA antibodies were determined by the known ELISA method (Tamura SI, Ito Y, Asanuma H et al., Cross-protection against influenza viruses achieved by intranasal vaccination with trivalent, inactivated vaccines, inoculated with cholera toxin B subunit. J. Immunol. 149 (1992) 981-987). Virus-specific IgA values are expressed as ELISA units of specific IgA per μg of total IgA concentration.

Epithelial cells from the nose were collected exclusively from the maxillary turbinates of the nasal shells of each person with the same type of small, nylon brush, which is used for cytopathological examinations during bronchoscopy (Glück U, Gebbers J-O, Nasal cytopathology of smokers; possible biological markers of air pollution? Am. J. Rhinol. 10 (1996) 55-57). The samples were taken by the same examiner (U.G.), under rhinoscopic control, with a circular and translational movement along the lower turbinate socket. The cells were transferred to a glass slide and immediately fixed with a solution containing 200 ml of ethanol + 100 ml of acetone + 6 drops of trichloroacetic acid.

Pathologists in training at the Institute of Pathology and Cytopathology of the Cantonal Hospital in Lucerne, who were not informed about the vaccination status, examined the object slides, stained according to Papanicolaou.

The average number of ciliated and goblet cells, lymphocytes, centroblasts, neutrophils, eosinophils and squamous epithelial cells was determined on 25 representative areas, per one slide, at magnification

100 times.

Significance between baseline and post-immunization titers was determined by paired T-test. The differences between the 4 vaccinations in the ability to induce the formation of anti-HA protective antibodies in the studied groups was determined by the χ2 test.

It was necessary to write down all the adverse events that were observed during the clinical research. An adverse event was defined as an adverse change in the basic features of the person's condition (before vaccination), regardless of whether the event was considered related to vaccination or not. Adverse events (local or systemic reaction) that occurred after vaccination were recorded by clinicians in a special report form created for this purpose. The base rate of adverse effects was determined before immunization.

80 people, average age 40, of comparable social status were taken for the research. 27.5% of the participants were women. All 3 nasal preparations for vaccination, as well as the parenteral vaccine, were well tolerated. Considering the anamnesis, there were no significant differences between the 3 vaccinated groups with nasal vaccines and all 3 formulations were well tolerated. In some cases, the following possible side effects have been reported: fever, fatigue, nausea, rhinitis, nasal congestion and nasopharyngitis. The parenteral, virosomal vaccine is also remarkably well tolerated.

The serological immune response is shown in Table 2. Significant titer increases were measured in group A (2 nasal vaccinations 7 days apart), group C (1 nasal vaccination with a double dose) as well as in group D (parenteral vaccination against all 3 virus strains) . Higher geometric means of antibody titers (GMT) were found in groups A and D. Group D had significantly the highest GMT values according to the H1N1 strain (p ≤ 0.05). In the case of the H3N2 strain, there were no significant differences between groups A and D.

These groups responded significantly better than groups B and C. With strain B, there were no significant differences between groups A, C and D. But they showed significantly higher titers than group B. Serum conversion rates were highest in groups A and D. They were usually significantly higher than the rates in groups B and C and met for all three strains the serological requirements for parenteral influenza vaccine, according to the requirements of the European Community (Commission of the European Community, Guidelines for Medicinal Products in the European Community. Harmonization of Requirements for Influenza Vaccine (1992) pp. 93-98 Luxemburg: Publications Office of the European Community).

Humoral, mucosal, immune response (saliva) is shown in table 3. The highest increases in I-gA titer were measured in group A. They were significantly higher than in other groups. Taking into account the total I-gA, GMTs in group A were also the highest. The mucoconversion rate (multiple increase in IgA titer) was again extremely highest in the A group. In the case of intramuscular vaccination, mucoconversion rates were very low.

In groups A, B and C, cytology was performed with a brush. The results are summarized in Figure 5. We determined the number of cells in the epithelium of the nasal mucosa (cylindrical cells with and without cilia, goblet cells and squamous epithelial cells) and the number of myelo/mono and lymphopoietic cells (lymphocytes, eosinophils, neutrophils and centroblasts). In group A, goblet cell hyperplasia could be clearly demonstrated on the 4th and 8th day after the first vaccination. In addition, on the same days, we observed a strong increase in lymphocytes and centroblasts. An increase in eosinophils and neutrophils was found on the 8th day after the initial vaccination. The number of cylindrical cells remained unchanged.

In group B, there was only a small increase in the number of lymphocytes and neutrophil and eosinophilic granulocytes. In this group there was no sign of the presence of activated lymphocytes.

In group C, goblet cell hyperplasia was even stronger than in group A. In addition, eosinophilic and neutral granulocytes increased the most in this group on the 4th and 8th days. The increase of lymphoblasts in this group was weaker than in group A. p = ≤ 0.05. One month after vaccination, the previous composition of cells was restored in all groups.

Figure 5 refers to nasal cytology: quantification of different populations of cells, which were obtained by taking nasal swabs from individuals (20 in each group) up to 29 days after three different types of intranasal vaccination. It is worth paying attention to the marked increase in centroblasts, as an indication of the local immune response of group A, in contrast to the significantly weaker increase in groups B and C (p = ≤ 0.005).

Example 8

Comparison of the clinical efficacy of intranasal virosomal influenza vaccines with HLT or PCG in volunteers, administered by the novel nebulizer device described in the proposed invention

The immune response and tolerance to two dispersed, anti-influenza vaccines was investigated in a double-blind study conducted on a total of 158 healthy Swiss volunteers, aged 18 to 67 years.

Trivalent virosomal influenza vaccine (purified HA, formulated with phosphatidylcholine) is combined with Heat Labile Toxin (HLT) from E. coli in a format suitable for intranasal administration. The human dose contained 7.5 μg of HA from each influenza strain and 2 μg of HLT. One dose was injected into each nostril on the 1st and 8th day to groups of individuals aged 18-59 years or ≥60 years. Serum samples were taken approximately 4 weeks after immunization. Reactions were rare and weak. The rates of the percentage of persons (adults and elderly) who reached the protective titer of serum-anti-HA-antibodies (≥40) were as follows: A/Bayern (92 %, 91 %), A/Wuhan (92 %, 78 %) and B/ Beijing) 59 %, 50 %). Post-immunization GMT and multiple-fold increase in GMT were comparable in the two age groups. The percentage rate of persons (adults and elderly respectively), who did not have a protective titer at the baseline level, but reached a protective level after immunization, was as follows: A/Bayern (79 %, 85 %), A/Wuhan (86 %, 56 % ) and B/ Beijing (48%, 41%).

The second, mucosal preparation of the vaccine contained 7.5 μg of HA and 12 μg of procholeragenoid (PCG). This preparation was also well tolerated and proved to be slightly less immunogenic than the HLT preparation. The percentage rate of individuals achieving a protective serum anti-HA antibody titer was as follows: A/Bayern 94.2%, A/Wuhan 80.8% and B/Beijing 36.5%. Serum conversion rates in all groups are shown in Table 4.

These results show that virosomal influenza vaccine, taken intranasally, is safe and highly immunogenic in adults, including the elderly.

Example 9

Mucosal immune response of mice after intranasal administration of virosomal influenza vaccine with HLT, PCG or without additional mucosal adjuvant

Groups of 10 adult female Balb/c mice were vaccinated intranasally with 30 μl of the influenza vaccine described in example (1) or in example (2). A control group of 10 mice received the virosome preparation of the vaccine without additional adjuvant, but with the same composition of virosomes. Half of each group received a second intranasal dose one week later. Nasal lavage (NW) and bronchial alveolar lavage (BAL) were performed after 3 weeks. The results of example 9 are shown in table 5. Determination of specific IgA was carried out by the known ELISA procedure. The results (GMT) are summarized in Table 6.

In the group, which was vaccinated twice with the vaccine according to the example in (1), the highest GMTs could be demonstrated for all three vaccine strains (A/Johannesburg, A/Nanching, G/Harbin). This group responded with even the highest levels of IgA in BAL, after being vaccinated only once.

A satisfactory IgA response was also obtained in a group of mice that was vaccinated twice intranasally with the preparation according to example (2). The control group, in which the virosome preparation was administered without added mucosal adjuvant, showed a very weak mucosal immune response.

The results show that in mice, intranasal administration of a viral influenza vaccine containing a mucosal adjuvant can induce a high mucosal antibody response.

Preclinical testing of a virosomal influenza vaccine in mice: intranasal administration

(Table 5 for example 9)

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Influenza IgA antibodies in mice

Reciprocal titer GMT (ELISA)

(Table 6 with example 9)

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Example 10

Clinical evaluation of mucosal vaccine against hepatitis B-(HB)

The vaccine was prepared according to example 4 and filled into a device for nasal application, according to example 5. The product was tested on 10 volunteers: 100 μl was applied in each nostril on the 1st day and repeated one week later. Blood samples were taken on the 1st day (before vaccination) and on the 29th day. Anti-HB-antibodies were determined by RIA (Abott). The geometric mean of the titer before vaccination was 7 IU/ml, and on the 29th day it was 159 IU/ml.

Example 11

Mucosal immune response of mice after intranasal administration of a DNA plasmid, which codes for the HN antigen of the mumps virus, introduced into influenza virosomes, containing 10% cationic phospholipids, and mixed with HLT (5μg/ml)

We intranasally immunized groups of mice with a) native DNA, which is coded for the HN antigen of mumps virus (group C) or b) DNA included in virosomes after preimmunization with virosomes (group A) or c) without preimmunization (group B). The control group (H) was immunized i.n. with live Urabe - mumsa virus (Priorix, SKB-Rix). As shown in Table 7, the geometric mean titer (GMT) of IgG in the group of preimmunized mice (A) was higher than that described in the groups of mice B and C (Lovell GH: Proteosomes, hydrophobic anchors, iscomes and liposomes for improved efficacy of peptide and protein vaccines In "New Generation Vaccines (1990) (G.C. Woodrow and M.M. Levine, eds. Dekker, New York, pp. 141-168; Cusi MG and Glück R: Intranasal immunization of mice with DNA mums, embedded in influenza virosome. IBC 4th Annual Conference on Genetic Vaccines, 25-27 October 1998, Washington D.C.).A group of mice immunized i.n. with native DNA developed very low levels of IgG, whereas, in contrast, mice immunized with mumps virus (group H) showed a good IgG response. When analyzing mucosal immunity, we found that all groups of mice, except those immunized with native DNA, developed IgA. We could demonstrate an elevated IgA titer (group H) only in with nasal eluates (NW) of mice immunized i.n. with mumps virus.

Cytokine measurements were performed using primary spleen cells from the spleen of mice, taken twelve days after immunization. Table 8 summarizes 8 representative measurement results obtained in two separate experiments. The cells of mice, stimulated with mumps virus antigen, which were previously (i.n.) vaccinated with DNA-virosomes, induced the production of IL-2 and IFN-γ. In addition, mice infected with influenza induced the production of IL-4. Cells taken from animals immunized with mumps virus produced IFN-γ, IL-2, IL-4 and IL-10 after in-vitro stimulation with mumps antigen. Immunization with DNA-virosomes, as well as control immunization with purified mumps antigen, correlated with the Th1 phenotype. In addition, when considering the ratio of total IgG and virus-specific IgG1 or IgG2a levels, the amount of IgG2a—isotype in group A predominated, indicating a Th2 response.

Table 7. Geometric mean titer (GMT) of humoral IgG, IgA in the eluate of bronchial alveoli (BAL) and IgA in the eluate of the nose of mice

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Table 8. Representative measurements of cytokine production in mice, obtained in two separate experiments

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Example 12

Treatment of nasal congestion in volunteers using influenza virosomes, with and without HLT, in a new applicator-nebulizer

In the clinic, 30 volunteers with cold symptoms and acute nasal congestion were selected. In all volunteers, breathing intensity (in Pascals) through each nostril was measured rhinometrically. After that, they were randomly divided into 3 groups of 10. Group A received one dose of vaccine (100 μl in each nostril) according to example (1). Group B received the same dose, but without mucosal adjuvant HLT. Group C received 100 μl of 0.9% NaCl solution (physiological table salt solution) in each nostril. Air flows were measured after 15 min, 30 min, 1 hour and 2 hours.

The measured data are collected in table 9. In groups A and B, the values were significantly better than in group C. In both groups, a markedly improved breathing function was established.

Table 9. Geometric means of rhinometric measurements on volunteers before and after nebulizer treatment

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Example 13

Treatment of mucosal lesions in volunteers using influenza virosomes and HLT

Volunteers were vaccinated intranasally, as described in example (7). As stated there, the average goblet cells were determined on the 3rd, 7th and 28th day after intranasal vaccination. Epithelial changes could be determined using hyperplasia of goblet cells in cytological smears (Glück U, Gebber J-O: Nasal cytopathology of smokers: a possible biological marker of air pollution? Am. J. Rhinol. 10 (1996) 55-57). Goblet cells have a protective function for the mucous layer. One month after the first nasal vaccination, the cytological condition of the mucosa, compared to the previous condition, was significantly better.

Thus, the new vaccine can be used as a therapeutic for the treatment of damaged nasal mucosa.

Example 14

Prevention of enterotoxic diarrhea caused by E. coli by nasal application of HLT with and without influenza virosomes

The group of travelers who visited Tunis consisted of 38 people. After informed consent, all agreed to participate in the study. By random selection, 19 volunteers were vaccinated twice, 1 week apart, with the preparation according to example (1), while 19 people were not vaccinated. 28 days after the first intranasal vaccination, blood samples were taken from all volunteers. 19 vaccinated persons showed a high level of IgG in serum according to the mucosal adjuvant (HLT). The control group remained anti-HLT negative. One month later, before leaving Tunisia, a special form for entering health events was distributed to all participants. 20 days later, the group returned from Tunisia and the completed forms were evaluated in relation to diarrheal diseases. In the vaccinated group, only two people reported problems related to diarrhea, while in the unvaccinated group, 9 people had symptoms of diarrhea during their stay in Tunisia. These data show that the vaccine is also effective in preventing enterotoxic diarrhea caused by E. coli.

Example 7. Table 1. Clinical protocol

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Claims (42)

1. Vaccine for intranasal application, characterized by the fact that it consists of: (a) liposomally formulated surface proteins of influenza (virosomes); (b) mucosal adjuvant of bacterial origin; (c) a specific applicator-sprayer, designed so that almost 100% of the sprayed volume, created by one stroke of the sprayer, can be applied to the nasal mucosa, which is important for the effectiveness of the vaccine. 2. Vaccine according to claim 1, characterized in that the surface protein is influenza hemagglutinin HA. 3. Vaccine according to claim 1 or 2, characterized in that the surface proteins are influenza hemagglutinin HA and neuraminidase NA. 4. The vaccine according to one of the claims between 1 and 3, characterized in that it contains further antigens. 5. Vaccine according to claim 4, characterized in that further antigens are bound on the surface of virosomes. 6. Vaccine according to claim 4 or 5, characterized in that the antigens are antigens of pathogenic organisms. 7. Vaccine according to claim 6, characterized in that the pathogenic organism is a virus, a bacterium, a fungus or a parasite. 8. Vaccine according to claim 7, characterized in that the virus, bacterium or parasite is selected from the group consisting of: hepatitis A virus, hepatitis B virus, respiratory syncytial virus (/Pneumovirus), parainfluenza virus, mumps virus, Morbilivirus, HIV -a, diphtheria bacteria (Corynebacterium diphtheriae), tetanus bacillus (Clostridium tetani), pneumococcus, Haemophilus influenzae, E. coli, Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida krusei, Aspergillus species, Trichomonas species , Trypanosoma species, Leishmania species, Toxoplasma gondii, Plasmodium falciparum, Plasmodium vavax, Plasmodium ovale, Plasmodium malariae, Trematoda species and Nematoda species. 9. Vaccine according to one of the claims between 1 and 8, characterized in that it is a vaccine against influenza. 10. The vaccine according to one of the claims between 1 and 9, characterized in that it can be applied for the prevention and/or treatment of general infections/infectious diseases. 11. Vaccine according to one of the claims between 1 and 10, characterized in that it serves for the prevention and/or treatment of influenza. 12. Vaccine according to claim 10 or 11, characterized by the fact that it serves for the prevention and/or treatment of blocked nose. 13. Vaccine according to one of claims 10, 11 or 12, characterized in that it serves for the treatment of injuries of the nasal mucosa. 14. Vaccine according to one of the claims between 1 and 13, characterized in that the hemagglutinin content per dose (100 μl) is 1-30 μl, preferably between 3-10 μl and most preferably 3.75 μl. 15. Vaccine according to one of the claims between 2 and 14, characterized in that the ratio of liposome phospholipids and HA is between 1:10 and 20:1. 16. The vaccine according to claim 15, characterized in that the ratio of liposomal phospholipids to HA is between 1:1 and 1:1. 17. Vaccine according to one of claims 15 and/or 16, characterized in that the ratio is 3:1. 18. Vaccine according to one of claims 15 and 17, characterized in that the phospholipid of the liposome is selected from the group of neutral, cationic and anionic phospholipids. 19. Vaccine according to one of the claims between 1 and 18, characterized in that the mucosal adjuvant is an active toxin, an inactive toxin and/or its non-toxic derivative 20. Vaccine according to claim 19, characterized in that the toxin and/or its non-toxic derivative is Heat Labile Toxin (HLT), cholera toxin (CT) and/or procholeragenoid (PCG). 21. Vaccine according to one of the claims between 1 and 20, characterized in that it contains Heat Labile Toxin (HLT) and/or cholera toxin (CT) in ratios between 1:2 and 20:1, preferably between 1:1 and 1:10 and preferably 7.5:1. 22. Vaccine according to one of claims 19 and 20 characterized by the fact that as a mucosal adjuvant it contains heat-inactivated procholeragenoid (PCG), through recombination technology inactive HLT and/or cholera toxin (CT), whose ratio is HA:PCG (and/or rHLT and /or rCT) is between 3:1 to 1:20. 23. The vaccine according to claim 22, characterized in that the HA:PCG (and/or rHLT and/or rCT) ratio is from 1:1 to 1:10. 24. Vaccine according to claim 23, characterized in that the HA:PCG (and/or rHLT, and/or rCT) ratio is 1:2. 25. The vaccine according to one of the claims between 1 and 24, characterized in that it further contains DNS and/or RNS plasmids, attached to and/or in virosomes. 26. The vaccine according to one of the claims between 1 and 25, characterized in that it is a vaccine against influenza. 27. Mucosal adjuvant according to one of the claims between 1 and 26, characterized in that the heat-labile toxin (HLT) which, according to example 2, is eluted with lactose buffer and purified on a Lactosyl-gel chromatographic column (Pharmacia). 28. A cuff (24), characterized in that it has a connecting element (28) for putting on a dosing device (2), in particular for dosing by spraying a pharmaceutically active preparation, according to one of the claims between 1 and 27, and a resting section (30 ). 29. Cuff (24) according to claim 28, characterized in that the spraying angle (α) lies between 50o and 80o to the horizontal. 30. Cuff (24) according to claim 28 or 29, characterized in that the connecting element (28) is essentially a cylindrical section of pipe. 31. Cuff (24) according to claim 30, characterized in that the tubular section (28) can be moved over at least one part of the nose element (20) of the dosing device (2). 32. A cuff (24) according to one of the claims between 28 and 31, characterized in that the end of the section for resting (30), on the user's side, is bent in such a way that it essentially corresponds to the shape of the outer part of the upper lip. 33. Cuff (24) according to one of the claims between 28 and 32, characterized in that the cuff (24) can be fitted to the dosing device (2) securely against unscrewing. 34. Cuff (24) according to claim 33, characterized in that the tubular section (28) and the part of the nose element (20), over which the tubular section (28) can be moved, are at least partially polygonal constructed to form a security against unscrewing. 35. Dosing device (2), characterized in that it serves in particular for dosing a pharmaceutically active preparation in a jet, according to one of the claims between 1 and 27, and consists of a pumping mechanism (6), a finger cuff (10) for starting pumping mechanism (6), connecting element (8), for tight connection with the container (4) and the nose element (16), which has a first section (18) and a second section (20), where both sections (18, 20 ) of the nasal element (16) so dimensioned that the outlet opening (14) of the nasal element (16) essentially reaches all the way to the main nasal cavity of the patient. 36. Dosing device (2) according to claim 35, characterized in that at least the second section (20) of the nose element (16) is substantially cylindrical. 37. Dosing device (2) according to claim 38, characterized in that the diameter of the second section (20) is between 3 mm and 10 mm, preferably around 7 mm. 38. Dosing device (2) according to one of the claims between 35 and 37, characterized in that the second section (20) of the nose element (16) is between 5 and 50 mm long, preferably around 20 mm. 39. Dosing device (2) according to one of the claims between 35 and 38, characterized in that the first section (18) of the nose element (16) is between 10 and 40 mm long, preferably around 30 mm. 40. The dosing device (2) according to one of the claims between 35 and 39, characterized in that the distance between the finger cuff (10) and the dispensing opening (14), provided on the tip, is at least 30 mm, preferably 45 mm. 41. Dosing device (2) according to one of the claims between 35 and 40, characterized in that it has a cuff (24) according to one of the claims between 28 and 34. 42. Dosing device (2) according to claim 41, characterized in that it is made in one part, i.e. in one piece with a cuff (24).